International Symposium on Cell Signaling—from Disease to Drug Discovery

Cell Biology International 2001, Vol. 25, No. 10, 1037–1077 doi:10.1006/cbir.2001.0808, available online at http://www.idealibrary.com on

MEETING ABSTRACTS

International Symposium on Cell Signaling—from Disease to Drug Discovery Hong Kong, 15–17 October 2001 ORGANIZERS: Hsiao Chang Chan, The Chinese University of Hong Kong, Epithelial Cell Biology Research Center Jie Ying Gao, The Academy of Military Medical Sciences, Epithelial Cell Biology Research Center Ting Lei, Biotechnology and Pharmacology, Beijing (Center) SPONSORED BY: Innovation & Technology Commission, Hong Kong Special Administration Region Beijing Science & Technology Commission Natural Science Foundation of China International Federation for Cell Biology Eu Yan Sang (HK) Limited Innomed Group Limited Venue: The Chinese University of Hong Kong, Shatin, Hong Kong Section 1. Invited Speakers Section 2. Session Speakers Section 3. Posters Note: Abstracts in each section are arranged in alphabetical order according to the family name of the presenting (underlined) author.

1065–6995/01/101037+41 $35.00/0

 2001 Academic Press

1038

Cell Biology International, Vol. 25, No. 10, 2001

Section 1. INVITED SPEAKERS THE NITRIC OXIDE/CYCLIC GMP PATHWAY: TARGETS FOR DRUG DEVELOPMENT Ferid Murad Department of Integrative Biology and Pharmacology, Institute of Molecular Medicine, University of Texas Medical School, Houston, Texas, U.S.A.

The role of nitric oxide in cellular signalling in the past two decades has become one of the most rapidly growing areas in biology. Nitric oxide is a gas and free radical with an unshared electron that can regulate an ever-growing list of biological processes. Nitric oxide is formed from L-arginine by a family of enzymes called nitric oxide synthases. These enzymes have a complex requirement for a number of cofactors and regulators including NADPH, tetrahydrabiopterin, flavins, calmodulin and heme. The enzymes are present in most cells and tissues. In many instances, nitric oxide mediates its biological effects by activating the soluble isoform of guanylyl cyclase and increasing cyclic GMP synthesis from GTP. Cyclic GMP, in turn, can activate cyclic GMP-dependent protein kinase (PKG) and can cause smooth muscles and blood vessels to relax, decrease platelet aggregation, alter neuron function, etc. These effects can decrease blood pressure, increase blood flow to tissues, alter memory and behavior, decrease blood clotting, etc. The list of effects of nitric oxide that are independent of cyclic GMP formation is also growing at a rapid rate. For example, nitric oxide can interact with transition metals such as iron, thiol groups, other free radicals, oxygen, superoxide anion, unsaturated fatty acids, and other molecules. Some of these reactions result in the oxidation of nitric oxide to nitrite and nitrate to terminate the effect, while other reactions can lead to altered protein structure function and/or catalytic capacity. These effects probably regulate bacterial infections, inflammation of tissues, tumour growth, and other disorders. These diverse effects of nitric oxide that are cyclic GMP dependent or independent can alter and regulate numerous important physiological events in cell regulation and function. Nitric oxide can function as an intracellular messenger, an antacoid, a paracrine substance, a neurotransmitter, or as a hormone that can be carried to distant sites for effects. Thus, it is a unique molecule with an array of signalling functions. However, as with any messenger molecule, there can be too little or too much of the substance, resulting in pathological events. Some of the methods to regulate either nitric oxide formation, metabolism, or function have been in clinical use for more than a century, as with the use of organic nitrates and nitroglycerin in angina pectoris that was initiated in the 1870s. Inhalation of low concentrations of nitric oxide can be beneficial in premature infants with pulmonary hypertension and increase survival rates. Ongoing clinical trials with nitric oxide synthase inhibitors and nitric oxide scavengers are examining the effects of those agents in septic shock, hypotension with dialysis, inflammatory disorders, cancer therapy, etc. Recognition of additional molecular targets in the areas of nitric oxide and cyclic GMP research will continue to promote drug discovery and development programs in this field. Current and future research will undoubtedly expand the clinician’s therapeutic armamentarium to manage a number of important diseases by perturbing nitric oxide formation and metabolism. Such promise and expectations have obviously fueled the interests in nitric oxide research for a growing list of potential therapeutic applications. There have been and will continue to be many opportunities from nitric oxide and cyclic GMP research to develop novel and important therapeutic agents. There are presently more than 40,000 publications in the area of nitric oxide research. The lecture will discuss our discovery of the first biological effects of nitric oxide and how the field has evolved since our original reports in 1977.

ENDOCRINE/PARACRINE REGULATION OF EARLY EMBRYO-ENDOMETRIAL INTERACTIONS Stanley R. Glasser and 1Christopher R. Murphy Department of Molecular and Cellular Biology, Baylor College of Medicine; Institute for Biosciences and Technology, Texas A&M University, Houston, TX; 1 Department of Anatomy and Histology, University of Sydney, NSW 2006, Australia

Compared with other epithelia, the biology of the uterine luminal epithelial (LE) cell is unique. The developing embryo (blastocyst) is able to attach without discrimination, without need for ovarian hormone preconditioning, to a wide array of natural and artificial sites. This does not apply to the interrelationship between the blastocyst and the LE. The intact uterine LE is a barrier to ubiquitous attachment and signal transduction. Attachment requires a specific reexpression of LE cell barrier function. For LE to acquire receptivity it must be redirected by a stringent steroid hormone controlled program of endometrial cell growth and differentiation that redefines the cell signalling networks that sequentially direct embryo recognition, apposition, adhesion and initiation of placentation. Historically, embryo receptivity has been defined in terms of hormone regulated transformation of the non-receptive apical plasma membrane of the uterine LE cell. Elevated progesterone (P) titers provide the environment for the LE, the initial site of contact with the blastocyst, to launch a program of structural and functional plasma membrane alterations that allow contact and initiate the events by which pregnancy is established. This has been termed ‘the plasma membrane transformation’. Apical plasma membrane transformation details the involution of microvilli and the subepithelial actin cytoskeleton, the decrease in net surface charge and the increase in apical surface adhesivity which is central to blastocyst attachment. However receptivity cannot be analyzed solely in terms of the apical plasma membrane. The LE cell is anisotropic. Its two domains, apical (A) and basolateral (BL) represent distinct compartments. Compositional heterogeneity of the LE cell (A vs BL) is real and critical to the structural and functional mechanisms which establish pregnancy. The barrier function of the LE is not fixed. A and BL plasma membrane transformation, cytoskeletal restructuring and correlative vectorial transport exploit the plasticity of the polarized phenotype and allows the LE to differentiate in concert with changes in other endometrial compartments and the developing conceptus. Alterations in the A and BL plasma membrane are coupled with the activation and inactivation of species of protein and glycoprotein remodeling enzymes. This results in changes in endocytosis, exocytosis, the profile of LE cell proteins and the composition of the glycocalyx. Epithelial architecture has a profound effect on cell type determination. Regulation of LE polarity determines the compostion of different compartments which proves to be a fundamental aspect of function. Polarity is controlled by the transmembrane proteins localized in the apical, lateral or basal plasma membrane domains. Recent studies have defined the relationship of E-cadherin/catenins of the lateral junctions relative to the role of cytoskeleton and associated cytoplasmic filaments in determining the unique surface domain specific cargoes (selective transport of vesicles). These hormone/growth factor dependent expressions initiate and modulate signal transduction and gene transcription. Redistribution and accumulation of constitutive and specific induced proteins generates the cascade of endometrial cell and embryo processes that directs the structurally and functionally distinct epithelial cell phenotypes that express shifts in the rate and location of cell proliferation and differentiation of the conceptus. Domain specific biotinylation has served to characterize the specific patterns of protein and glycoprotein expression which define the planar polarity by which the apical LE surface acquires adhesivity. H-type 1 antigen, either directly or cooperatively with other molecules, facilitates adhesion during the early stages of trophoblast-endometrial interaction. The date suggest that if the expression of these selectintype carbohydrate antigens is spatio-temporally correct these interactions will induce a series of downstream processes, involving all the LE cell domains, i.e., specific integrin heterodomain expression, cadherin shifting, that will result in higher affinity adhesion with the developing trophoblast. This evolution is not restricted solely to transformation of the apical plasma membrane but integrates relocation changes in all the LE surface domains. Apical shifts in BL hemidesmosomal and desmosomal plectin and plakoglobin, BL cadherins and integrins progressively alter the individual domains of the LE cell and their role in the intercellular communication (endometrial epithelial and stromal cells, trophectoderm) by which pregnancy is established and maintained. The enigma of these studies is that all these P regulated changes in the LE cell occur in an epithelial cell with no steroid hormone

Cell Biology International, Vol. 25, No. 10, 2001 receptors; neither the progesterone receptor (PR) nor the estrogen receptor (ER) is detectable. The first assumption is that the pregnant endometrial cells have reacquired their fetal phenotype, i.e., P induced growth and differentiation of the receptor deficient epithelia is being directed via stimulation of the stromal (mesenchymal) cell PR. This is not the case. Based on preliminary evidence, we hypothesize that P induces the stromal cell to synthesize and secrete paracrine factors, one of which compromises the stability of the LE cell receptor mRNA, whereas the other directed through the stromal cell PR acts on the epithelium, via the basal domain, as a progestamedin.

FLOW MEDIATED DILATION IN THE HUMAN CORONARY MICROCIRCULATION: PATHOPHYSIOLOGICAL SIGNALING MECHANISMS David D. Gutterman Medical College of Wisconsin

Arguably the most prominent physiological mechanism of endothelial dependent vasodilation is flow-mediated dilation (FMD) that occurs in response to increased laminar shear stress exerted on vascular endothelial cells. The mechanism of FMD involves mechanical transduction of shear stress from the cell surface to focal adhesion sites where the cell attaches to the basement membrane. The signalling process involves activation of integrins and tyrosine kinases ultimately leading to activation of enzymes systems that produce endothelial derived factors that migrate to the underlying vascular smooth muscle and elicit dilation. Of the three major such endothelial derived factors, nitric oxide has been identified as the most important factor especially in FMD of conduit arteries. In the microcirculation, FMD is even more prominent, and appears to be mediated by either nitric oxide or prostacyclin, depending on the vascular bed and species. We have examined the mechanism of FMD in human coronary arterioles from patients with coronary artery disease. In contrast to the porcine coronary microcirculation, nitric oxide does not play a role in FMD. Rather endothelial derived hyperpolarization factor (EDHF) is involved. This EDHF is derived from arachidonic acid through metabolism by cytochrome P450. Interestingly this mechanism is in part compensatory since FMD in children without CAD is partially mediated by nitric oxide. Furthermore the signalling pathway involves tyrosine kinase and g-proteins similar to that observed in porcine arteries where nitric oxide exclusively mediates FMD. In summary FMD is a prominent mechanism of endothelial dependent dilation in the human coronary microcirculation. The mechanism involves production and release of an endothelial derived hyperpolarization factor that is in part compensatory for loss of NO production, but involves a similar signalling pathway.

CFTR PHOSPHATASES: POTENTIAL TARGETS FOR CYSTIC FIBROSIS PHARMACOTHERAPY J. W. Hanrahan, J. Luo, D. Dahan and T. Zhu Department of Physiology, McGill Univ., 3655 Sir-William-Osler St., Montre´ al, Que´ bec, Canada H3G 1Y6

The CFTR chloride channel is tightly regulated by protein kinasemediated phosphorylation at multiple sites and by a membrane-bound phosphatase. Rapid deactivation of the channels in excised patches suggested that CFTR and its phosphatase might be associated within a regulatory complex. We tested that possibility using coimmunoprecipitation and crosslinking experiments. A monoclonal anti-CFTR antibody co-precipitated PP2C from baby hamster kidney (BHK) cells stably expressing CFTR, but not PP1, PP2A or PP2B. Conversely, a polyclonal anti-PP2C antibody co-precipitated CFTR from BHK membrane extracts. When cell lysates were exposed to the bifunctional crosslinking reagent dithiobis [sulfosuccinimidyl propionate] (DTSSP), histidine-tagged CFTR (CFTRHis10) and PP2C became crosslinked into high molecular weight complexes that could be isolated by chromatography on Ni2+ -NTA agarose. PP1, PP2A and PP2B did not co-purify with CFTRHis10 after DTSSP exposure. Thus CFTR and PP2C exist in a stable complex which may facilitate regulation of the channel. More recently we have found that 1 pmole of CFTR-associated PP2C can be purified from 50 large plates of

1039 BHK cells without crosslinking. This has enabled the application of a direct proteomics approach (2-D gel electrophoresis and mass spectrometry) for characterization and sequencing of the phosphatase. A major goal of CF research remains to identify drugs that increase CFTR activity. The CFTR phosphatase has been proposed as the site of action for two of the most potent CFTR channel activators known; genistein and bromotetramisole. We have examined the possible effects of these drugs on phosphatases using patch clamp and biochemical methods. Genistein inhibited spontaneous rundown of CFTR channel activity in excised patches but had no effect on the activities of purified PP1, PP2A, PP2B, PP2C or endogenous phosphatases when assayed by measuring [32P]PO4 release from pre-labelled casein, a recombinant glutathione-s-transferase-R domain fusion protein, or immunoprecipitated full-length CFTR. Bromotetramisole (Br-t) also slowed rundown of CFTR channels, but in marked contrast to genistein, it did inhibit all four protein phosphatases tested. Half-maximal inhibition of PP2A and PP2C was observed with 0.5 mM and 1.5 mM Br-t, respectively. Br-t seemed to act exclusively through phosphatase inhibition, since it did not affect CFTR channels in patches with low apparent endogenous phosphatase activity (i.e. those lacking spontaneous rundown). Thus genistein stimulates CFTR without affecting phosphatases whereas bromotetramisole acts by inhibiting a membrane-associated protein phosphatase (probably PP2C), which may elevate basal phosphorylation sufficiently to cause channel activation. Once the CFTR-associated phosphatase is characterized at the molecular level, a high throughput screen will be established to identify inhibitors that could serve as therapeutics in CF patients with mutations that do not impair CFTR trafficking. Phosphatase inhibitors will also provide a useful adjunct therapy when attempts to correct the folding defect or deliver the CFTR gene are only partially successful. Supported by the NIH (NIDDK), Canadian Cystic Fibrosis Foundation, and Canadian Institutes of Health Research.

NON-GENOMIC CONVERGENT AND DIVERGENT SIGNALLING OF ALDOSTERONE AND ESTRADIOL IN MAMMALIAN DISTAL COLON Brian J. Harvey, Christina M. Doolan, Steven B. Condliffe, Celine Reanrd, and Rodrigo Alzamora Wellcome Trust Cellular Physiology Research Unit, Department of Physiology, University College Cork, Ireland

Studies from our laboratory have demonstrated rapid (<1 min) nongenomic activation of Na + -H + exchange, K + recycling, PKC activity and a PKC-dependent Ca2+ entry through L-type Ca2+ channels specifically by mineralocorticoids in distal colon. Aldosterone directly stimulates the activity of PKC (but not PKC , PKC and PKC ) in a cell-free assay system containing only purified commercially available enzyme, appropriate substrate peptide, co-factors and lipid vesicles. The primary ion transport target of the non-genomic signal transduction cascade elicited by aldosterone in epithelia is the Na + -H + exchanger. In isolated colonic crypts, aldosterone produced a PKC sensitive intracellular alkalinization within 1 min of hormone addition. Intracellular alkalinization upregulates an ATP-dependent K + channel channel, which is involved in K + recycling to maintain an electrical driving force for Na + absorption, while inhibiting a Ca2+ dependent K + channel, which generates the charge balance for Cl
secretion. The non-genomic response to aldosterone in distal colon appears to enhance the capacity for absorption while down-regulating the potential for secretion. We have also demonstrated rapid (<1 min) non-genomic activation of Na + -H + exchange, K + recycling, PKC activity and a PKC & PKA -dependent Ca2+ entry through L-type Ca2+ channels specifically by 17 -estradiol in distal colon. These rapid effects are steroid hormone and gender specific and are insensitive to inhibitors of the classical ER. 17 -estradiol directly stimulated the activity of both PKC and PKC but not PKC and PKC in a cell-free assay system. E2 rapidly inhibited basolateral KCa channel activity which would be expected to result in an acute inhibition of Cl
secretion. Physiological concentrations of E2 (0.1–10 nM) reduced both basal and secretagogue-induced Cl
secretion. This antisecretory effect of E2 is sensitive to PKC inhibition, intracellular Ca2+ chelation, is

1040 gender specific and insensitive to inhibitors of the classical ER. These observations link rapid non-genomic activation of second messengers with a rapid gender-specific effect in the whole tissue. Aldosterone and E2 differ in their protein kinase signal transduction and both hormones stimulate specific PKC isoforms indicating both common and divergent signalling systems for salt-retaining steroid hormones. The physiological function of non-genomic effects of aldosterone and E2 is to shift the balance from net secretion to net absorption in a pluripotential epithelium.

INTRACRINE HEPATOPOIETIN POTENTIATES AP-1 ACTIVITY THROUGH JAB1 INDEPENDENT OF MAPK PATHWAY Chengrong Lu1, Yong Li1, Yanlin Zhao1, Guichun Xing1, Fei Tang2, Qingming Wang1, Yuhui Sun3, Handong Wei1, Xiaoming Yang1, Huipeng Chen1, Chutse Wu1, Jianguo Chen3, Chenggang Zhang1, Fuchu He1 1 Department of Genomics and Proteomics, Beijing Institute of Radiation Medicine, Chinese National Human Genome Center at Beijing, 27 Taiping Road, Beijing 100850, P. R. China; 2National Laboratory of Biomacromolecules, Institute of Biophysics, Academia Sinica, Beijing 100101, China; 3Peking University School of Life Sciences, Beijing 100871, China

Many growth factors and cytokines are involved in liver regeneration (FASEB J, 1995; Science, 1997). Of them, only hepatopoietin (HPO)/ ALR (augmenter of liver regeneration) is a specifically hepatotrophic factor originally identified from the cytosol of regenerating or hyperplastic hepatic cells (Hagiya et al., 1994; He et al., 1993). The previous reports indicate that extracellular HPO triggers the MAPK signal transduction pathway by binding its specific receptor on the cell surface. However, its function in the cytosol of hepatocytes is unclear. Here we have identified JAB1 (Jun activation domain-binding protein 1), an AP-1 coactivator, which specifically interacts with HPO. The endogenous JAB1 mainly distributes in nucleus and colocalizes with endogenous HPO in hepatic cells. As an intracrine factor, the intracellular function of HPO is to increase c-Jun phosphorylation independent of c-JUN amino-terminal kinase(JNK), ERK-1 and –2, and consequently leads to potentiation of AP-1 activity under mediation of JAB1. Amino acids 1-63 of HPO molecule are sufficient to bind JAB1, but its whole molecule is necessary for its intracellular signalling. Taken together, these results elucidate a novel mechanism of HPO in cytokine signalling by specifically modulating the AP-1 pathway through JAB1, in a MAP kinase-independent fashion, which possibly underlies the triggering of immediate/early response transcription factors during initiation of liver regeneration. References 1995. FASEB J 9: 1527–1536. 1997. Science 276: 60. H M, et al., 1994. PNAS 91: 8142. H FC, et al., 1993. Hepatology 17: 225.

cAMP-MEDIATED VESICLE TRAFFICKING AND REGULATION OF FUNCTIONAL APICAL MEMBRANE ENaCs Sandy I. Helman Univ. of Illinois, Urbana, IL 61801, U.S.A.

Diverse hormones including aldosterone, insulin and ADH upregulate Na + transport in tight epithelia by increased apical membrane expression of functional epithelial sodium channels (ENaCs). In continuously short-circuited cell-cultured A6 epithelia, blocker-induced noise analysis has indicated, despite changes of single channel currents and channel open probabilities, that stimulation of apical Na+ entry into the cells is due to increase of the density of functional ENaCs regardless of the time course of activation of transport by these hormones. Major questions remain to be resolved with respect to the origin of functional channels which for example could include surface membrane activation/deactivation of channels and/or cycling/recycling

Cell Biology International, Vol. 25, No. 10, 2001 of channels by way of vesicle trafficking of subunits and/or functional/ nonfunctional channels to and from the apical membranes of the cells. In this regard, it is to be emphasized that surface expression of the
, , and  subunits of ENaC, at least as studied in oocytes bathed with a high, 90 mM Na + Ringer solution1, greatly exceeds expression of functional channels directly involved in transport according to our calculations with the reported data. Functional channels account for only about 3% or less of the total surface expression of subunits. It is of particular interest to note that when oocytes are bathed with a low 12 mM Na + Ringer solution, surface expression of channels is nearly identical to the expression of functional channels (assuming a subunit stoichiometry of four) indicating that extracellular Na + concentration plays an important but an unknown role in surface expression of functional and nonfunctional ENaCs. To begin to address questions related to vesicle trafficking of ENaCs, we turned to studies using impedance analysis to measure changes of apical membrane capacitance2 and to real time confocal microscopy of intact Na + transporting A6 epithelia to visualize specific apical membrane cAMPmediated vesicle trafficking3. Apical membranes were stained with the fluorescent marker FM 4-64 in control tissues and in tissues pretreated with forskolin to stimulate transport and expression of functional ENaC density. From the steady-state rates of appearance of endocytosed vesicles with average diameters in the range of 0.3 m it became apparent that forskolin/cAMP markedly increased the rate of cycling of vesicles (endocytosis and exocytosis) by five fold or more and that each vesicle could traffic on average about 10 functional channels/vesicle with some vesicles possibly containing no channels and others containing considerably more than 10 functional channels/ vesicle. From the changes of apical membrane capacitance measured at frequencies above their audio-frequency relaxation frequencies, it also became apparent that cAMP (forskolin, PGE2) caused average increases of capacitance of about 10–15% that paralleled the time course of surface expression of functional ENaCs. However, it must be noted that packaging of channels in vesicles is very likely not constant. If tissues are pretreated overnight with the PI-3 kinase inhibitor, LY294002, that almost completely but reversibly inhibits Na + transport, the transport response to cAMP is completely inhibited but the capacitance response to cAMP remains intact and normal indicating that despite exocytosis of vesicles, the vesicles trafficked to the apical membranes do not carry or express functional ENaCs. Accordingly, among the complex issues involved in understanding regulation of Na + transport, we are compelled to the additional idea that variable packaging of channels and/or subunits within vesicles is yet another major factor to be considered in understanding how epithelial tissues regulate Na + transport at their apical membranes. Supported by NIH DK 30824.

CO-ORDINATED ADAPTATION OF INTESTINAL NUTRIENT TRANSPORTERS Barry H. Hirst Department of Physiological Sciences, Univ. Medical School, Newcastle upon Tyne NE2 4HH, U.K.

Dietary intake of nutrients is recognized as influencing absorptive mechanisms. The principles of synthesis and maintenance costs, calorific payoff, fixed requirements and toxicity may all influence the patterns of regulation. Regulation of transporter activity may be specific, such as effects on transporter number and/or affinity, or non-specific changes in surface are for absorption or electrochemical gradients. Regulation may also reflect short-term, hormonal and/or neural mechanisms, or longer-term mechanisms involving changes in gene transcription. The transport of the products of dietary protein, amino acids and small peptides, illustrate a complexity of interaction, both to maximize dietary amino-nitrogen assimilation, while also allowing for the metabolic demands of the enterocytes in the fasting state. Experimental paradigms, which illustrate the co-ordinated regulation of the various transporters, include the rat total parental nutrition model and human intestinal Caco-2 cells to investigate the underlying cellular and molecular mechanisms. Total parental nutrition is a model for longer-term adaptive responses, where the gut lumen is relatively starved of nutrient input. In rats this leads to increased expression in the ileum of transcripts for the

Cell Biology International, Vol. 25, No. 10, 2001 intestinal peptide transporter, PepT1, the neutral amino acid transporter, ASCT2 and the glycine-specific transporter, GLYT1. Such adaptive responses in intestinal enterocytes may reflect adaptational responses to maintaining the metabolic requirements in the absence of luminal nutrition. These changes may involve complex hormonal interactions, but can be revered by administration of glucagons-like peptide-2. Human Caco-2 cells provide a model system more amenable to uncovering molecular mechanisms of the adaptive responses. Supplementation of normal culture growth media with dipeptide, glycylglutamine, resulted increased peptide transporter mRNA, a result of both increased mRNA stability and transcription, yielding increased PepT1 protein and an increase in PepT1-mediated transport maximum, with no change in transporter affinity. These changes are not observed with dipeptides which are not substrates for PepT1, nor for stable substrates, indicating degradable substrates are required to initiate the response. Co-ordinated changes in free amino acid transport are also observed. Adaptation of the GLYT1 transporter is an example an amino acid transporter. GLYT1 is a basolateral transporter in the intestine, which mediates Na + - and Cl
-dependant glycine uptake, with a restrictive inhibition profile, sensitive to sarcosine, but not other amino acids. In contrast to PepT1, GLYT1 is down-regulated by excess glycine. Down-regulation of GLYT1 is also seen upon protein kinase C activation, suggesting a putative mechanism. The maintenance of GLYT1 in response to reduced luminal glycine availability made indicate an important role of glycine in maintaining normal enterocyte function.

ROLE OF FREE RADICALS IN THE ACTIONS OF INHIBITING APOPTOSIS OF HEPATOCYTES AND INDUCING LIVER NF-kB ACTIVATION BY BICYCLOL, A NOVEL ANTI-VIRAL HEAPTITIS DRUGS, IN MICE Geng Tao Liu Department of Pharmacology, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China

Chronic viral hepatitis is a worldwide disease. The incidence of chronic viral hepatitis B (HBV) in China is at the top in the world. Although many drugs have been used in the treatment of HBV and viral hepatitis C (HCV), no satisfied drug is available. Even interferon 2-
, its efficacy is limited and very expensive. In order to develop safe, effective and cheaper anti-hepatitis drug, we synthesized a number of compounds with biphenyl structures based on our previous study on the active components from Fructus schizandrae, a traditional Chinese herb. Pharmacological screening found that bicyclol, one of the synthesized compounds, has significant hepato-protective action in experimental liver injury models induced by CCl4, D-galactosamine, acetaminophen and B.C.G plus LPS (lipo-polysaccharides). Bicyclol markedly reduced the elevated serum transaminases and liver pathological damage. Bicyclol also has antiviral action in duck viral hepatitis B and in 2.215 cell lines. Clinical trials on 269 cases of chronic HBV patients proved that bicyclol 25 mg given orally once daily for 6 months markedly reduced serum transaminses (ALT, AST) and also inhibited HBV replication. The returning rate of HBeAg and HBVDNA from positive to negative by bicyclol is corresponding to that by interferon 2-
, particularly after withdrawal of drug therapy, the sustained efficacy of bicyclol is superior to that of interferon 2-
. No noticeable side effect was observed. The results of mechanistic study showed that bicyclol can scavenge free radicals including trichloromethyl radical (CCl3) converted from CCl4 and oxygen free radicals formed in activated neutrophils. Moreover, the treatment of mice with bicyclol reduced apoptosis of hepatocytes and liver nuclear DNA fragmentation induced by Con A

1041 injection in mice. Bicyclol was further found to induce liver nuclear factor NF-kB activation in Con A injected mice. In brief, free radicals are involved in the liver injury which might play a signal role in the apoptosis of hepatocytes and nuclear NF-kB activation, while bicyclol exerting its protection against liver injury might be through inhibiting apoptosis of hepatocytes and induces NF-kB activation. Further study on the active mechanism of bicyclol is still in progress.

TWO STEP ACTIVATION OF SOLUBLE EPIDERMAL GROWTH FACTOR (EGF) RECEPTOR Qi-shui Lin, Gao-xiang Ge and Jing Wu Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai, 200031, China

Epidermal Growth Factor Receptor (EGFR) belongs to a receptor tyrosine kinase superfamily. EGF initiates a series of cellular responses upon binding to the receptor. A number of mechanisms were proposed for the activation process of EGFR tyrosine kinase. One widely accepted mechanism is the dimerization mechanism. Although considerable efforts had been made, it was not clear how the kinase domain inside the cell was activated after EGF bound to the extracellular domain of the EGF receptor. Our studies indicated that dimerization of EGFR was not only important to the activation of EGFR tyrosine kinase, but also important for the maintenance of the active form. Based on studies on the activation status of wild-type EGFR in the heterodimer with a number of kinase deficient mutant EGFR, we investigated the activation mechanism of EGFR tyrosine kinase. sEGFR is a naturally existed mutant EGFR, which does not possess transmembrane domain and intracellular tyrosine kinase domain. Lys721 involves in the binding with ATP. Mutant receptor K721A cannot bind with ATP and thus lose kinase activity. In the heterodimer of wild type EGFR with sEGFR or K721A mutant receptor, the wild type part in the heterodimer can be partly activated. However, the wild-type part in the heterodimer with D813N mutant, can be fully activated by EGF. Asp813 is responsible to the -phosphate transfer of ATP, although the D813N mutant can bind ATP but does not possess kinase activity as it no longer able to transfer phosphate group. Kinetic analysis of EGFR tyrosine kinase showed that after activated by EGF, both the affinity for ATP and Vmax increased, while if EGFR was pretreated with excess amount of sEGFR, although there was similar increase of the affinity for ATP, the Vmax was unchanged. In the light of the above findings, we proposed a two-step activation mechanism for detergent-solubilized EGFR tyrosine kinase. (1) EGFR formed dimer after binding with EGF. The conformational changes of the extracellular EGF-binding domain was transmitted to intracellular part and led to the conformational change of the kinase domain and the affinity for ATP increased; (2) Binding of ATP to the kinase domain led to the interaction of intracellular kinase domain, activation of receptor and the Vmax increased; (3) The increase of ATP affinity is a fast process and the increase of Vmax is the rate limiting step of the activation process. EGFR is a membrane protein, it might subject to changes upon solubilization. To evaluate whether the above two-step mechanism also works in membrane bound form of EGFR, we reconstituted EGFR into liposome. In our hand, 10–20% of the total amount of EGFR incorporated into liposome membrane and more than 80% of the reconstituted EGFR had a right-side-out orientation. The tyrosine kinase activity could be determined in the presence of Alamethicin. Preliminary results indicated that the membrane fluidity had obvious effect on the receptor tyrosine kinase activity.

ENDOTHELIAL CELL SIGNALLING AND ANGIOGENESIS: FROM VEGF TO NOTCH Zhao-Jun Liu The Wistar Institute, Philadelphia, PA, U.S.A.

Angiogenesis, a process of generating new capillary blood vessels, has important functions under both physiological and pathological conditions. Physiological angiogenesis is fundamental to wound healing, reproduction and embryonic development. Abnormally enhanced angiogenic response is observed in rheumatoid arthritis, diabetic retinopathy, and during tumour development. Inhibition of the

1042 neovasculature was shown to abolish or slow tumour growth in various experimental models, while promotion of the angiogenic response can prove beneficial in the treatment of ischemic conditions, such as myocardial ischemia/infarction. Vascular endothelial growth factor (VEGF) plays a pivotal role in the regulation of both physiological and pathological angiogenesis. Our laboratory has devised a novel in vitro three-dimensional collagen matrices culture system in which human endothelial cells cultured as a monolayer under collagen matrices can be induced to develop a microvascular-like network in the collagen matrices. Stimulation of endothelial cells with VEGF, which is mediated by adenovirus-transfected fibroblasts, drastically facilitates this process. To study differential expression patterns that implicate a potential role for particular genes involved in the distinct developmental stages of formation of microvascular structures and to identify VEGF specific signalling pathway(s), we have employed cDNA microarray analyses to define the transcript levels found in the process of endothelial development under VEGF stimulation. Around 8000 genes have been investigated. Of up-regulated population of genes, we are particularly interested in Notch-1 gene because it has been shown crucial for vessel formation. By RT-PCR analysis we have confirmed that Notch-1 and some of its ligand, can be induced by VEGF stimulation in endothelial cells. Using a CBF-1 promotor luciferase construct as a reporter for Notch activity we have shown that this pathway is activated upon VEGF stimulation. Stimulatory signalling of VEGF is mediated by two tyrosine kinase receptors, Flt-1 (VEGFR-1) and KDR (VEGFR-2). By comparison with PLGF and VEGF-C/D, which only bind to Flt-1 and KDR respectively, we demonstrated that both Flt-1 and KDR are involved in the delivery of VEGF signalling for the induction of Notch-1 and its ligand. Although VEGF is able to activate both PI3-K and MAP-K pathways, only PI3-K pathway is responsible for the induction of Notch and Notch ligand. Functional significance of induction of Notch and Notch ligand in endothelial cells is under investigation.

PHOSPHOINOSITIDE 3-KINASE AND SMALL GTPase Rab5 IN PROTEIN TARGETING AND SORTING Toshiaki Katada Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033, Japan

A family of phosphatidylinositol 3-kinases (PI 3-kinase), comprising three major classes (I–III) in terms of substrate specificity and regulation, plays important roles in a variety of cell functions. We previously reported that the class-I heterodimeric PI 3-kinase consisting of p110-catalytic and p85-regulatory subunits is synergistically activated by two different types of membrane receptors, one possessing tyrosine kinase activity and the other activating trimeric G proteins. In this symposium, I would like to report an additional unique feature of the p110/p85 PI 3-kinase. The small GTPase Rab5, which is involved in early endocytic pathway, was identified as a binding protein for the p110-catalytic subunit in a yeast two-hybrid screening system. The interaction appears to require at least two separated amino-acid sequences present specifically in the  isoform of p110 and the GTP-bound form of Rab5. The expressions of constitutively active and dominant negative mutants of Rab5 in THP-1 cells induce the stimulation and inhibition, respectively, of protein kinase B, which is a downstream target for the PI 3-kinase product phosphatidylinositol 3,4,5-triphosphate. These results suggest that there is a specific interaction between GTP-bound Rab5 and the p110/p85 PI 3-kinase, leading to efficient coupling of the lipid kinase product to its downstream target, protein kinase B. We also searched for Rab5-interacting proteins by screening a cDNA library in the yeast two-hybrid system with a GTPase-deficient form of Rab5b as bait. This screening led us to identify a novel Rab5-binding protein (Rab5-bp), which contained many functional domains including Src-homology 2, proline-rich, Vps9, and Rasassociation domains. Rab5-bp was characterized as a unique guaninenucleotide exchange factor (GEF) for Rab5, since it enhanced GDPGTP exchange reaction on Rab5 but preferentially interacted with GTP form of Rab5 rather than its GDP form. Confocal microscopic analysis of HeLa cells revealed that Rab5-bp is mostly localized in the intracellular endosomes containing Rab5. Quite interestingly, Rab5-bp

Cell Biology International, Vol. 25, No. 10, 2001 was, but Rab5 was not, translocated into nucleus in mitotic phase of HeLa cells. These results indicate that the newly identified Rab5-bp functions as an upstream activator and/or a downstream effector for Rab5 and that the association is selectively prevented by their different localization in mitotic phase of the cells. References K H, et al., 1997. Heterodimeric phosphoinositide 3-kinase consisting of p85 and p110 is synergistically activated by the subunits of G proteins and phosphotyrosyl peptide. J Biol Chem 272: 24252–24256. K T, et al., 1999. Synergistic activation of a family of phosphoinositide 3-kinase via G-protein coupled and tyrosine kinaserelated receptors [Review]. Chemistry and Physics of Lipids 98: 79–86. T S, et al., 1999. Enhancement by adenosine of insulininduced activation of phosphoinositide 3-kinase and protein kinase B in rat adipocytes. J Biol Chem 274: 19545–19550. K H, K T, 2001. Association of phosphatidylinositol 3-kinase composed of p110-catalytic and p85-regulatory subunits with the small GTPase Rab5. J Biochem 130: 73–78.

PHYSIOLOGICAL ROLES OF BMP-4 AND ITS SIGNALLING PATHWAYS Hsiang-fu Kung Institute of Molecular Biology, The University of Hong Kong, Pokfulam, Hong Kong, China

We demonstrated that bone morphogenetic protein 4 (BMP-4) plays important roles in the hematopoiesis, neurogenesis and dorsal-ventral patterning during Xenopus embryonic development. Using dominant negative mutants, we elucidated the involvement of Ras, Raf, and activator protein 1 (AP-1) in BMP-4 signalling leading to the biological responses. The AP-1 activity was determined by injecting an AP-1 dependent luciferase reporter gene into two-cell stage Xenopus embryos and measuring the luciferase activity at various developmental stages. We found that injection of BMP-4 mRNA increased AP-1 activity, whereas injection of dominant negative BMP-4 receptor (DN-BR) mRNA inhibited AP-1 activity. Similar inhibitory effects were seen with injection of mRNAs encoding dominant negative mutants of c-Ha-Ras, c-Raf, or c-Jun. These results suggest that the endogenous AP-1 activity is regulated by BMP-4/Ras/Raf/Jun signals. We next investigated the effects of Ras/Raf/AP-1 signals on the biological functions of BMP-4. DN-BR-induced dorsalization of the embryo, revealed by the formation of a secondary body axis or dorsalization of the ventral mesoderm explant analyzed by histological and molecular criteria, was significantly reversed by coinjection of [Val12]Ha-Ras, c-Raf, or c-Jun mRNA. Furthermore, the BMP-4 stimulated erythroid differentiation in the ventral mesoderm was substantially inhibited by coinjection with the dominant negative c-Ha-Ras, c-Raf, or c-Jun mutant. Our results suggest the involvement of Ras/Raf/AP-1 in the BMP-4 signalling pathway.

PLASTICITY AND ADAPTATION OF Ca2+ SIGNALLING AND Ca2+ -REGULATED EXOCYTOSIS IN SERCA2 +/
MICE, A MOUSE MODEL OF DARIER’S DISEASE Xiao-Song Zhao1, Dong Min Shin1, Lynne H. Liu, Gary E. Shull2 and Shmuel Muallem1 1 The Department of Physiology, The University of Texas Southwestern Medical Center at Dallas, 2The Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, OH, U.S.A.

The importance of understanding plasticity and adaptation of Ca2+ signalling and Ca2+ -dependent cell functions in vivo is highlighted by the recent diagnosis of Darier’s Disease (DD) as a high penetrance, autosomal dominant mutation in the ATP2A2 gene, which encodes the SERCA2 Ca2+ pump. Here we have used a mouse model of DD, a SERCA2 +/
mouse, to define the adaptation of Ca2+ signalling and Ca2+ -dependent exocytosis to a deletion of one copy of the SERCA2 gene. Measurement of [Ca2+ ]i in two secretory cell types and with three

Cell Biology International, Vol. 25, No. 10, 2001 different agonists showed that the [Ca2+ ]i transient evoked by maximal agonist stimulation had the same initial level but lasted for a much shorter time in cells from SERCA2 +/
mice than in cells from wild-type mice. The shorter transient was due to up-regulation of specific isoforms of the plasma membrane Ca2+ pump, rather than a compensative up-regulation of Ca2+ uptake into stores, an abnormal IP3-mediated Ca2+ release or a reduced capacitative Ca2+ influx. The change in cellular Ca2+ handling caused about 50% reduction in [Ca2+ ]I oscillation frequency evoked by physiological agonist concentrations. Nonetheless and most notably, the dose response for agoniststimulated exocytosis was identical in cells from wild-type and SERCA2 +/
mice, resulting in a normal secretory phenotype. This was due to adaptation of the Ca2+ sensors for exocytosis in cells from SERCA2 +/
mice. While the level of proteins forming the core complex for exocytosis, VAMP and syntaxin 1, did not change, the level of the low affinity Ca2+ sensor synaptotagmin I was decreased and the level of the high affinity Ca2+ sensor synaptotagmin III was increased in cells from SERCA2 +/
mice compared to cells from WT mice. As a result, Ca2+ -dependent exocytosis was about 10 fold more sensitive to Ca2+ in cells from SERCA2 +/
mice. These findings reveal a remarkable plasticity and adaptability of Ca2+ signalling and Ca2+ -dependent cellular functions in vivo and can explain well the normal function of most physiological systems in DD patients.

MECHANISMS OF CFTR REGULATION BY SYNTAXIN 1A AND PKA Steven Y. Chang1, Anke Di2, Anjaparavanda P. Naren3, H. Clive Palfrey2, Kevin L. Kirk3, and Deborah J. Nelson2 1 Department of Medicine, Section of Pulmonary & Critical Care Medicine, University of Chicago Hospitals, 5841 S. Maryland Ave, MC 6026, Chicago, IL 60637, U.S.A.; 2 Department of Neurobiology, Pharmacology and Physiology, University of Chicago, 947 East 58th St., MC 0926, Chicago, IL 60637, U.S.A.; 3Department of Physiology and Biophysics, Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, 1918 University Blvd., MCLM 985, Birmingham, AL 35294, U.S.A.

Cystic Fibrosis (CF) is caused by defects in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), a PKA-dependent chloride (Cl
) channel. Some controversy exists concerning whether PKA activates CFTR-mediated Cl
currents (ICFTR) by increasing the number of channels in the plasma membrane and/or by stimulating membraneresident channels. Recently, we established that CFTR is regulated by the target membrane t-SNARE Syntaxin 1A. This protein plays a well-known role in exocytosis and has recently been implicated in the regulation of ion channels. This functional dualism lead us to investigate two related issues: (a) whether Syntaxin 1A modulates CFTR via direct effects on the gating of channels residing in the plasma membrane versus alterations in membrane traffic and (b) whether CFTR current activation by PKA is linked to changes in membrane turnover. Our data demonstrate, for the first time, that Syntaxin 1A inhibits CFTR as a result of direct protein-protein interactions that decrease channel open probability (Po), and serves as a model for other SNARE protein-ion channel interactions. We also show that PKA activation can enhance membrane trafficking in some epithelial cell types, independent of CFTR activation or Syntaxin 1A association. Our observations highlight t-SNAREs as novel drug targets in the modulation of ion channel activity as well as membrane trafficking.

RESCUE FROM APOPTOSIS BY BLOCKERS OF VOLUME-REGULATORY ION CHANNELS Yasunobu Okada1,2, Emi Maeno1,2, Yasuki Ishizaki3, Takeshi Tsubata4 and Toku Kanaseki2 1 Department of Cell Physiology, National Institute for Physiological Sciences; 2CREST, JST, Okazaki 444-8585; 3 Department of Hygiene, Kobe University School of Medicine, Kobe 650-0017; and 4Department of Immunology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo 113-8510, Japan

1043 Under physiological and pathological conditions, apoptosis is induced by a variety of stimuli. A major hallmark of apoptosis is normotonic cell shrinkage. In fact, we found that the apoptotic volume decrease (AVD) is induced in lymphoid U937, epithelial HeLa, neuronal NG108-15 and PC12 cells shortly (0.5–4 h) after apoptotic induction by staurosporine (Maeno et al., 2000), TNF
(Maeno et al., 2000) or Fas ligand (Okada et al., 2001). The AVD induction was found to be always coupled to facilitation of the regulatory volume decrease (RVD) (Maeno et al., 2000; Okada et al., 2001) which is accomplished by operations of volume-regulatory K + and Cl
channels (Okada, 1997), observed after osmotic swelling. Therefore, there is a possibility that AVD is also induced by KCl release from the cells via volumeregulatory K + and Cl
channels. Actually, we found that both the AVD induction and the RVD facilitation were abolished by conventional blockers of volume-regulatory K + channels (Ba2+ and quinine) and Cl
channels (DIDS, SITS, niflumic acid, glibenclamide and NPPB) (Maeno et al., 2000; Okada et al., 2001). Similar effects were also observed with a low concentration of phloretin (Maeno et al., 2000), which has recently been shown to block potently volumesensitive outwardly rectifying Cl
channel, but not Ca2+ - and cAMPactivated Cl
channels (Fan et al., 2001). These blockers of volumeregulatory K + and Cl
channels were found to inhibit cytochrome c release, activation of caspase-3, -8 and -9, DNA laddering, and chromatin condensation (Maeno et al., 2000). Also, cell death was prevented by these channel blockers (Maeno et al., 2000). The AVD induction was not prevented by application of broad-spectrum caspase inhibitors (zVAD-fmk and zD-dcb) (Okada and Maeno, 2001). The AVD was found to be induced by apoptotic stimuli even in WEHI/ Bcl-2 cells, to which a VDAC closer protein, Bcl-2, had been introduced, indicating that the AVD is independent of mitochondrial anion channel, VDAC. Taken together, it is concluded that blockers of volume-regulatory K + and Cl
channels prevent the AVD induction, which is an event prerequisite to apoptosis and upstream to cytochrome c release and caspase activation, thereby rescuing the cells from apoptotic death. References M E, I Y, Y T, H A, O Y, 2000. Normotonic cell shrinkage due to disordered volume regulation is an early prerequisite to apoptosis. Proc Natl Acad Sci USA 97: 9487–9492. O Y, M E, S T, D K, W J, M S, 2001. Receptor-mediated control of regulatory volume decrease (RVD) and apoptotic volume decrease (AVD). J Physiol (London) 532: 3–16. O Y, 1997. Volume expansion-sensing outward-rectifier Cl- channel: fresh start to the molecular identity and volume sensor. Am J Physiol 273: C757–C789. F H-T, M S, K H, O Y, 2001. Phloretin differentially inhibits, volume-sensitive and cyclic AMP-activated, but not Ca-activated, Cl- channels. Br J Pharmacol (in press). O Y, M E, 2001. Apoptosis, cell volume regulation and volume-regulatory chloride channels. Comp Biochem Physiol (in press).

TRIMERIC G-PROTEIN CONTROL OF FLUID ABSORPTION M. M. Reddy1 and P. M. Quinton1,2 1 Department of Pediatrics, School of Medicine, University of California, San Diego; 2Division of Biomedical Sciences, University of California, Riverside, CA, U.S.A.

Since it is well established that CFTR (Cystic Fibrosis Transmembrane Regulator) is highly expressed and functional in the human sweat duct, we investigated the potential role of this anion channel in controlling electrolyte transport in this purely absorptive tissue. We have previously established that the channel can be activated in this tissue by Protein Kinase A and ATP. We recently discovered that the universal activator of G-proteins, GTP- -S, activated a Cl
conductance (GCl
) in the apical membrane of the isolated sweat duct, which we take to be that of CFTR. Most intriguingly, this activation was independent of cAMP and only required the presence of ATP. To

1044 show the specificity of the activation, we attempted to activate CFTR with GTP alone. We found that GTP could not activate the GCl
, unless phosphatase inhibitors were present (e.g., vanadate). In order to insure that the effect was due to the stimulation of a G-protein and not due to phosphatase resistant thio-ester phosphorylation of a kinase activated product or inhibition of phosphatase action on the same product, we showed that the GTP- -S activation could be competitively blocked by GDP. We then established that the nature of the G-proteins was most likely that of the trimeric class by showing that a similar activation could be achieved by adding AlF44
, which is generally taken as a selective agonist for trimeric (vs. monomeric) forms. We then proceeded to characterize the nature of the G-Protein stimulated activation of the CFTR GCl
. In the presence of the promiscuous kinase inhibitor, staurosporine, we were not able to effect activation of GCl
. However, staurosporine did not interfere with the activation of the G-protein since GCl
was activated after removing the inhibitor even though it had been present during the application of GTP- -S. Likewise, if we removed Mg + + from the cytoplasmic media, only CFTR activation was inhibited and not the G-protein activation. We can therefore conclude that the direct action of GTP- -S is independent of phosphorylation, but that a kinase is probably required to effect the G-protein signal that activates CFTR and controls absorption. The process is apparently independent of intracellular Ca + + , since chelating free cytoplasmic Ca + + to ca. 80 nM had no effect on the G-Protein activation of CFTR GCl
. By using immunocytochemistry to localize antibodies to G-protein subunits, we also found that trimeric G-proteins are potentially expressed at the apical membrane. The data to this point seems consistent with a G s subclass of trimeric G-protein. However, several additional pieces of evidence suggest that in this tissue, the CFTR GCl
may not be activated physiologically via adenylyl cyclase and Protein Kinase A, the normal effectors of the G s subclass. Moreover, we have recently acquired data to show that simply raising the concentration of the neurotransmitter, glutamate, induces activation of GCl
, even without exogenous cAMP or ATP added to the cytoplasm. We have shown that G-proteins in the duct can effectively activate CFTR in the presence of ATP to regulate fluid absorption. However, we are at a loss as to what ligand or receptor might physiologically stimulate the G-protein. By exclusion, we are almost forced to conclude that the G-protein is most likely activated by mechanical stretch of the apical membrane. The hypothesis is difficult to prove because we cannot make electrical measurements without perfusing the duct and we cannot peruse the duct without stretching it.

THE MOLECULAR ARCHITECTURE OF NEURONAL KINASE/PHOSPHATASE SIGNALLING COMPLEXES John D. Scott, Kimberly L. Dodge, Dario E. M. Diviani and Lorene K. Langeberg Howard Hughes Medical Institute, Vollum Institute, 3181 S.W. Sam Jackson Park Road, Portland OR 97201-3098, U.S.A.

Subcellular location is a critical factor in determining the specificity of protein kinases and phosphatases. At the molecular level this is achieved through the interaction of these enzymes with scaffolding, anchoring and adapter proteins. Through this mechanism individual kinases and phosphatases are positioned where they can optimally respond to activation signals and close to preferred substrates. For example, the cAMP dependent protein kinase (PKA) is localized thorough the association of the regulatory subunit with a growing family of A-Kinase Anchoring Proteins (AKAPs). The AKAPs have emerged as a functionally related family of signalling proteins that contain two distinct domains: a conserved anchoring surface that binds the R subunit dimmer of the PKA holenzyme and a unique targeting domain that directs the anchored kinase complex to specific cellular compartments. Structural information from both domains have been exploited to define the function of anchored PKA in the control of cAMP responsive events inside cells. Another prominent feature of AKAPs is their ability to bind other signalling enzymes and there by maintain multi-component signalling complexes. Recent examples include evidence that mAKAP coordinates a cAMP signal-

Cell Biology International, Vol. 25, No. 10, 2001 ling module of type 4 phosphodiesterase and PKA. Likewise, WAVE-1 and AKAP-lbc maintain anchored complexes of PKA and the GTPases, Rho and Rac respectively.

FROM DISEASE TO DRUG DISCOVERY—A GLOBAL PROSPECTIVE Wang Yifei Department of Reproductive Health and Research, World Health Organization, Geneva, Switzerland

To combat 1000 different types of diseases, some 5000 kinds of existing drugs are far from enough. The current status of drug development is far from satisfactory. The fact is: 60% of the prescription drugs are based on Nature’s compounds; Drug development is largely done by ‘trial and error’; Doctors are intervening at the level of symptoms using drugs, instead of actually fighting illness; Less than 10% of global spending on health research is devoted to diseases that account for 90% of the global disease burden. To meet these challenges, the following actions are recommended: (1) Setting priority areas in health research and drug development Step 1: Calculate the burden attributable to each disease; Step 2: Identify the main determinants of the disease; Step 3: Judge the adequacy of the current knowledge base, including the cost-effectiveness of current interventions; Step 4: Assess the promise of the proposed research and development effort in drug development; Step 5: What are the present resource-flows for the disease? (2) Taking an innovated approach for drug development To link the information systems including hospital records, pharmacy records and outpatient records of academic medical centres, managed care-organizations and pharmaceutical firms; To establish institutional arrangements to promote dialogue and cooperation among relevant entities leading to better interdisciplinary communication; To coordinate basic, translational and clinical studies and make them complementarity. (3) Seizing the unprecedented opportunities provided by the new biology, especially cell signalling and Genomics to accelerate drug development. With the advances in the study on Cell Signalling, Genomic Medicine and Bioinformatics, now the drug development is in the fast lane. There are experiments that we could only dream of but could never do before the Genome, Cell Signalling and Bioinformatics. In terms of economic benefits, investing in medical research including drug development yields a spectacular return on investment.

SIGNALLING: SENSITIVE RECEPTORS ON A SENSITIVE ORGANELLE Denys N. Wheatley Department of Cell Pathology, University of Aberdeen, MacRobert Building, 581 King Street, Aberdeen AB24 5UA, U.K.

Eukaryotic cells have through antiquity carried a considerable number of signalling molecules and the complex receptor mechanism, entraining secondary messenger pathways that have a strong commonality. This has been shown from ciliated protozoa to mammalian cells. The origin and nature of these signalling systems have been discussed by Christensen et al. (1998; Wheatley and Christensen, 0000). It follows that studies on ‘primitive’ organisms can quickly lead us to basic understanding of the role of hormone and growth factors in signalling in all eukaryotes. Using insulin as the paradigm, we have studied its action on ciliates as well as mammalian cells. It has receptors exclusively on the ciliary membranes of ciliates, whose homology with mammalian insulin receptor proteins (both the alpha and beta chains) are remarkable. Cilia in mammalian cells that seem to be particularly devoted to sensory function are the primary cilia, which issue from the centrosomal cilia. A life-long study of these was mostly anecdotal, but more recent evidence has gradually accumulated a picture of them as mechanochemical receptors. Although this was surmised much earlier,

Cell Biology International, Vol. 25, No. 10, 2001 evidence was lacking. Showing a wide range of modifications, primary cilia seem to be highly adapted in many circumstances to particular functional roles, and a selection will be described. But more importantly, recent evidence relates to the malformation or dysfunction of primary cilia, since this can result in major pathological conditions. This was predicted 6 years ago (Wheatley, 1995), and some these conditions will be described. References C ST, et al., 1998. Signalling in unicellular eukaryotes: Regulation of cell survival, proliferation, differentiation, mating, chemosensory behaviour, and programmed cell death. Int Rev Cytol 177: 181–253. W DN, C ST, 1999. Origins of signalling and memory: matters of life versus death. Acta Biol Hung 50: 441–462. W DN, 1995. Primary cilia in normal and pathological conditions: a review. Pathobiology 63: 222–238.

Section 2. SECTION SPEAKERS STUDYING SIGNALLING MECHANISMS IN APOPTOSIS IN AN INTACT LIVING CELL Donald C. Chang and Kathy Q. Luo Department of Biology, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong

Apoptosis is an induced cell suicidal process that allows the biological organisms to destroy damaged or unwanted cells in an orderly manner. It can be activated by many stimuli including growth factor withdrawal, UV-irradiation, activation of the ‘death domain’ via the TNF (tumour necrosis factor) receptor, treatment of hormones (e.g. glucocorticoid) or chemotherapy drugs (e.g. camptothecin). Those death signals can induce apoptosis via two major pathways, the mitochondria pathway or the death receptor pathway, both of which require the activation of a set of cysteine proteases termed caspases, particularly, caspase-3. The activated caspase-3 is known to be capable of cleaving many important cellular substrates, including actin, fodrin, lamin and the inhibitor of CAD (caspase-activated deoxyribonuclease). Thus, the active caspase-3 can cause disassembly of the cell structure as well as DNA fragmentation and eventually leads to cell death. Because activation of caspase-3 plays such a central role in apoptosis, we have developed a GFP-based FRET (fluorescence resonance energy transfer) probe that can accurately assay the activation of caspase-3 in intact living cells. This probe was constructed by fusing a CFP (cyan fluorescent protein) and a YFP (yellow fluorescent protein) with a specialized linker containing the caspase-3 cleavage sequence: DEVD. The linker design was optimized to produce a large FRET effect. Using purified protein, we observed a 5-fold change in the fluorescence emission ratio when the probe was cleaved by caspase-3. To demonstrate the usefulness of this method, we introduced this FRET probe into HeLa cells by both transient and stable transfection. We observed that during UV-induced apoptosis, the activation of caspase-3 varied significantly between different cells; but once the caspase was activated, the enzyme within the cell became fully active within a few minutes. This technique will be highly useful for correlating the caspase-3 activation with other apoptotic events and for rapid-screening of potential drugs that may target the apoptotic process. (Work supported by RGC grants HKUST6619/97M and HKUST6109/01M from the Hong Kong SAR Government).

CYCLIC GMP PROLONGS THE SURVIVAL AND SPECIFICALLY PREVENTS THE ONSET OF APOPTOTIC DNA FRAGMENTATION IN NEURAL CELLS: ASSESSMENT USING THE NEW QUANTITATIVE ULTRASENSITIVE CE-LIF TECHNOLOGY Ronald R. Fiscus, Jessie P. S. Yuen, Catherine P. C. Leung and Siew Boon Cheng Chew

1045

Department of Physiology, Faculty of Medicine, Epithelial Cell Biology Research Center, and The Center for Gerontology & Geriatrics, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong

Neurodegenerative diseases, such as Alzheimer’s disease and Parkinson’s disease, are associated with apoptotic cell death of neurons and a resulting loss of neuronal functions, leading to loss of memory or motor functions. Recently, many studies have investigated agents that cause neuronal apoptosis, especially focusing on four endogenous neurotoxic substances, -amyloid peptide (A ), nitric oxide (NO), presenilin and reactive oxygen species (ROSs). However, very little is known about endogenous substances and intracellular signalling mechanisms that could protect against the onset of apoptosis and thereby increase the survival of these neurons. In 1987, we showed that two neural cell lines, PC12 and C6 cells, responded to added NO or atrial natriuretic peptide (ANP) with increased production of cyclic GMP (cGMP), resulting in increased intracellular (and extracellular) accumulation of cGMP (Fiscus et al., 1987). However, at the time, the biological function of this signalling pathway in neural cells was unknown. Later, in collaboration with Mark Mattson’s laboratory at the University of Kentucky, we found that elevation of cGMP levels in hippocampal neurons increases their survival, specifically blocking cell death induced by stress, such as glutamate toxicity (Barger et al., 1995). Recently, we showed that cGMP inhibits the onset of apoptosis and prolongs the survival of stressed PC12 cells, a cell culture model of catecholaminergic neurons (Fiscus et al., 2001). To accurately quantify the onset of apoptosis in neural cells, we have developed a new technology involving capillary electrophoresis with laser-induced fluorescence detector (CE-LIF). This technology provides a much better sensitivity (>1000-fold improvement) compared to conventional agarose gels for assessing apoptotic DNA fragmentation and, by using DNA standard curves, has been developed into a truly quantitative technology. Using CE-LIF, we found that ANP and a related natriuretic peptide, BNP, inhibit serumdeprivation-induced apoptotic DNA fragmentation by more than 80% in PC12 cells. Elevation of cGMP levels inhibits a key step in the signalling pathway leading to DNA fragmentation and apoptosis in stressed neural cells. Furthermore, we have shown that blocking soluble guanylyl cyclase with ODC in NG108-15 cells (a cholinergic neuronal cell line) exaggerates that DNA fragmentation caused by high (toxic) concentrations of NO. These data indicate that preventing cGMP synthesis in response to NO leads to even greater NO-induced apoptosis, suggesting the cGMP response to NO is playing an important physiological role by limiting the toxic pro-apoptotic actions of NO. (Supported by a Direct Grant for Research.) References F, R, W, M, 1987. J Neurochem 48: 522–528. B, F, R, H, M, 1995. J Neurochem 64: 2087–2096. F, T, C C, 2001. NeuroReport (Neurochemistry) 12: 185–189.

PHARMACOLOGICAL CHARACTERISTICS OF THE NOVEL PUTATIVE PURINOCEPTORS IN VASCULAR ENDOTHELIUM Hai Wang, Shu-Hong Liu and Li-Mei Shan Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences, Beijing 100850, China

Many protein entities of purinoceptor subtypes have been identified according to the differences in the molecular sequences, but the functions remain to be investigated. In the isolated rat aorta with intact endothelium, precontracted with norepinephrine at the concentration of 0.62 mol/L, the maximal endothelium-dependent relaxation was obtained in the presence of acetylcholine, the additional administrations of ATP could induce contraction, which was mediated by a novel subtype of putative purinoceptors. This endothelial membrane receptor could be activated by the P1 receptor agonist adenosine as well as P2 receptor agonists ATP and ADP in the presence of endogenous nitric oxide; coupled with PTX-sensitive G proteins;

1046 stimulated the arachidonic acid turnover and the prostaglandin synthesis, resulting in the intracellular calcium mobilization. This type of putative purinoceptors was different in the pharmacological properties from P1 as well as P2 purinocepors reported previously. In addition, the changes of this receptor-effector systems were determined in hypertension, which could be characterized by as the following properties: insensitive to adensonine; uncoupled with PTX-sensitive G protein; unable to stimulate the protaglandin synthesis. It is reasonable to suggest that the novel putative endothelial purinoceptors play important roles in control of vascular tones in physiological or hypertensively pathophysiological states.

CONNEXIN-INDUCED TUMOR GROWTH SUPPRESSION IN VIVO AND REDUCED CELL MIGRATION IN VITRO IN THE ABSENCE OF APPRECIABLE GAP JUNCTION FORMATION Hong Qin, Qing Shao, Taiqi Wang1, Daniel J. Belliveau, Moulay A. Alaoui-Jamali1 and Dale W. Laird Department of Anatomy and Cell Biology, University of Western Ontario, London, Ontario, Canada N6A-5C1; 1 Lady Davis Research Institute, McGill University, Montreal, Quebec, Canada

There are at least four possible mechanisms for how connexins exhibit their tumour suppressing properties. The most obvious is that GJIC allows for the passage of undefined growth suppressing molecules. Secondly, connexin over-expression may induce the secretion of molecules that exhibit growth suppression properties from the surrounding milieu. Thirdly, assembled gap junctions may promote or enhance cell-cell adhesion. Finally, connexin over expression may indirectly reduce the growth of tumour cells by overloading the secretory pathway. In the present study we examined a communication-deficient human breast tumour cell line (MDA-MB-231) in order to determine whether the formation of functional gap junctions is necessary for tumour suppression. Wild type MDA-MB-231 cells were found to express moderate levels of Cx43 but few gap junction plaques were assembled and only 6% of the cells were dye coupled to neighboring cells. Interestingly, retroviral infection of MDA-MB-231 cells with cDNAs encoding either Cx43 or Cx26 resulted in high protein expression of both connexins but neither connexin was efficiently assembled into gap junctions and dye coupling remained low with <60% of the cells exhibiting dye coupling to 1st order cells only. Although forced expression of Cx43 or Cx26 did not lead to a significant difference in cell growth in vitro, a dramatic 7-fold reduction of tumour growth was achieved when connexin-expressing MDA-MB231 cells were implanted into the mammary fat pads of nude mice. Immunohistochemical studies on the excised tumour xenoplants revealed that Cx43 was localized to intracellular compartments and little evidence of gap junctions was observed. Moreover, over-expression of Cx43 in MDA-MB-231 cells inhibited cell migration in vitro by over 70% suggesting that connexin expression in the absence of efficient rescue of GJIC is altering the transformed phenotype of these cells. Together, these data suggest that passage of undefined growth suppressing molecules amongst tumour cells may not be the mechanism by which connexins exhibit their tumour suppressing properties in at least some cell types. Current studies are underway where assembled or unassembled loss-of-function connexins, dominant-negative connexins and non-connexin polytopic integral membrane proteins are overexpressed in these and other tumour cells in order to further dissect how connexins exhibit their tumour suppressing properties. Supported by the Canadian Breast Cancer Research Initiative.

NEW SIGNALLING PATHWAY FOR THE TRANSCRIPTIONAL REGULATION OF THE LUTROPIN RECEPTOR GENE IN GRANULOSA CELLS Xuebo Liu Department of Physiology & Biophysics, Department of Pharmacology, The University of Iowa College of Medicine

Luteinizing hormone (LH) and Follicle-stimulating hormone (FSH) are the central hormones of mammalian reproduction. LH and FSH

Cell Biology International, Vol. 25, No. 10, 2001 play a key role for the regulatory of pituitary-gonadal axis. They act by binding to specific receptors, localized exclusively the target cell in the gonads. The differentiation, growth and mature of ovarian granulosa cells is a complex process that involves induction and expression of many genes. The induction of the LHR gene is essential for the ability of the granulosa cells of the growing follicle to respond to pituitary LH with increased steroidogenesis and ovulation and for the luteal cells of the ruptured follicle to respond to placental human CG with increased steroid production. Earlier studies have shown that the induction of the LHR in differentiating granulosa cells is mediated by concerted actions of both FSH and estradiol. It has been known for a long time that FSH induces an increase of intracellular cAMP levels in granulosa cells and the effects of FSH on LHR induction are apparently due to its ability to increase intracellular cAMP levels. It has been shown that the FSHR or 8-bromo-cAMP mediated increase in LHR and LHR mRNA in granulosa cells obtained from diethylstilbestrol (DES)-treated rats is associated with a marked increase in LHR gene transcription. In spite of the role of cAMP in the induction of LHR gene transcription, interestingly, a search of the 2 Kb sequence of 5 -flanking region of the rat LHR gene does not contain any classical cAMP-responsive elements. However granulosa cells transfected with a luciferase reporter gene driven by 2.1 Kb of the 5 -flanking region of the rLHR gene respond to exogenously added 8-Br-cAMP. A number of cis-regulatory elements and trans-acting factors are involved in the transcriptional regulation of the rLHR gene in rat granulasa cells. In the promoter region, there are three Sp1 sites, which are functionally important as determined by luciferase assay, and mutation of each of these sites results in both diminished basal as well as cAMP-induced transcriptional activity of the rLHR gene. Mobility gel shift assays have shown the marked increase in the abundance of a complex formed between the promoter region of the rLHR gene and nuclear extracts from 8-Br-cAMP-induced rat granulosa cells as opposed to extracts from control cells. By 8-Br-cAMP-treated rat granulosa cells revealed two complexes for Sp1 but there is another complex that depends on 8-Br-cAMP induced and is not for Sp1 cis elements. We have designated complex A. To identify the DNA element involved in this complex. We found the sequence (G/T)GGGG in promoter region that are have termed this cis element SAS for Sp 1 adjacent sequence and an additional site, located in the distal region of the rLHR gene at nt-933/-924, Both of them appear to recognize the same transcription factor(s)-complex A. The core sequence of the later AGTGG(A)GGGG is also G-rich, but it is distinct from that of the SAS site. We designated the distal site as being an SAS-like site. Using reporter gene assays, we have shown that the selective mutation of SAS and SAS-like sites have marked inhibitory effect on the cAMP increases the abundance of complex A formation at both of the sites, it is reasonable to postulate that the increase in complex A formation plays a role in the cAMP-mediated transcription of the rLHR gene, although the classic cAMP-responsive element (CRE) is not present in the 5 -flank region of the rat LHR gene. It has shown that CREB is not the only transcription factor activated by elevated levels of intracellular cAMP. Is the complex A on rLHR gene transcription the new transcription factors?, a new signal pathway?, and SAS, SAS-like are novel cAMPresponsive elements? These are clearly important issues that will need to be addressed in future experiments.

ELEVATED GLUCOSE IMPAIRS cAMP MEDIATED DILATION BY REDUCING VOLTAGE-DEPENDENT K + (Kv) CHANNEL ACTIVITY IN CORONARY VASCULAR SMOOTH MUSCLE CELLS Y. Liu, Qiang Chai and D. D. Gutterman Cardiovascular Research Center, Medical College of Wisconsin, Milwaukee, WI 53226, U.S.A.

We have shown that elevation of extracellular glucose reduces Kv channel activity in rat coronary artery (RCA) VSMCs. Previous studies indicate that cAMP-induced coronary vasodilation is mediated by opening of Kv channels. We tested the hypothesis that dilation to isoproterenol (ISO) and forskolin (both mediated by cAMP) is impaired in RCA exposed to high glucose (HG) as a result of reduced opening of Kv channels. RCA segments from Sprague-Dawley rats were either freshly isolated or incubated for 24 hours in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum with either

Cell Biology International, Vol. 25, No. 10, 2001 normal glucose (NG, 5.5 mM) or HG (23 mM). Videomicroscopy and patch clamp methods were used to examine vasomotor function and K + channel activity in VSMCs in response to ISO and forskolin. ISO (10
10 to 10
5 M) and forskolin (10
10 to 10
5 M) dilated RCA in a dose-dependent fashion and 3 mM 4AP blocked the dilations to ISO and forskolin by 50% and 90%, respectively. Following incubation, dilations to both ISO (598%) and forskolin (604%) were significantly reduced in arteries exposed to HG compared to NG (ISO 775%, forskolin 904%, n=7 each group, P<0.05). ISO increased whole-cell peak K + current density from 398 to 6315 pA/pF (n=6, P<0.05) in cells from RCA incubated in NG, but had little effect on current densities of HG cells. 4-AP reduced 52% and 16% of whole-cell currents in NG and HG cells. These results suggest that vasodilation mediated by cAMP is impaired in RCA exposed to high glucose due to reduced Kv channel activity.

Etk/Bmx, A SIGNALLING MOLECULE OF EGF PATHWAY Hsiang-Yiang Jui, Kai-Yun Chen, Jei-Liang Lin, Li-Ming Huang, Rong-Jeng Tzeng, Hsing-Jien Kung and Hsiu-Ming Shih Division of Molecular and Genomic Medicine, National Health Research Institutes, Taipei, Taiwan, R.O.C.

Tyrosine kinases play important roles in a variety of signalling cascades in many cell types. Tec tyrosine kinases, a new class of nonreceptor tyrosine kinase, are an emerging family of proteins that are expressed in both hematopoietic and non-hematopoietic tissues. This family consists of the Btk, Itk, Tec, and Etk/Bmx tyrosine kinases with closely homologous structures that include an N-terminal PH domain, followed by Tec homology (TH), SH3, SH2, and tyrosine kinase domains. While Tec, Btk and Itk kinases have been implicated in the signalling pathways of a variety of hematopoietic receptor and antigen receptors, thus far upstream regulators and downstream signalling pathways of Etk and their roles in biological functions remain largely unknown. To explore the biological functions of Etk, we have identified its interacting proteins through yeast two-hybrid screens. Several proteins were identified including Eps8. Eps8, EGF receptor substrate no 8, was further verified as Etk binding protein both in vitro and in vivo. We further linked Etk to be a downstream signalling molecule of EGF receptor by following evidence: (1) Etk can be phosphorylated in the cells treated with EGF. (2) EGF receptor can be detected in the immunocomplex of Etk. (3) Kinase dead mutant of Etk can block the EGF-induced cell transformation. (4) Overexpressed Etk can enhance EGF-induced cell death. (5) A conditionally active form of Etk can trigger and potentiate cell death of EGF responsive cells. These findings suggest that Etk can be placed in the EGF signalling pathway.

MOLECULAR MECHANISMS OF HUMAN ESOPHAGEAL CARCINOGENESIS IN HENAN, NORTHERN CHINA Li Dong Wang1, Chang Wei Feng1, Qi Zhou1, Chung S. Yang2, Bin Liu3 and Yan Rui Zhang4 1 Laboratory for Cancer Research, College of Medicine, Zhengzhou University, Zhengzhou, Henan Province, 450052; 2 Laboratory for Cancer Research, College of Pharmacy, Rutgers University, Piscataway, NJ 08854, U.S.A.; 3 Department of Gastroenterology, TongRen Hospital, Capital Medical University, Beijing, 100730; 4Department of Gastroenterology, People’s Hospital of Henan Province, Zhengzhou, Henan, 450003, China

Esophageal Cancer (EC) is one of the six most common malignant diseases worldwide. Linxian and the nearby Huixian and Huojia counties of Henan province in northern China have been recognized as the highest incidence areas for EC in the past decades and EC is still the leading cause of cancer-related deaths in these areas. To further understand the mechanisms of human esophageal carcinogenesis, we characterized the alterations of P53-Rb system, including P53, Rb, Waf1/p21, cyclin D1, P14, P15 and P16, in the normal esophageal epithelia and the epithelia with different severity of lesions, including basal cell hyperplasia (BCH), dysplasia, and carcinoma in situ (CIS),

1047 which could be considered as precancerous lesions of EC. EC and nearby precancerous lesions were from the surgically resected specimens. All the biopsies with normal and precancerous lesions were from the symptom-free subjects during mass survey with endoscopy examination. All the esophageal cancer and precancerous tissues were from the subjects at Linxian and nearby counties. P53 mutation analysis showed that in surgically-resected SCC specimens and the nearby precancerous lesions, all the lesions with dysplasia and CIS had the same mutations as those in the nearby SCC. In SCC with homogeneous P53 staining, the same mutation was observed in different areas of tumour. But this was not true in tumour areas with heterogeneous P53 staining. Waf1/P21-positive cells were mainly located in the interior layers of the cancer nests. Conversely, P53-positive cells were formed mostly in the peripheral layers. In precancerous lesions, the number of Waf1/P21-positive cells were fewer than P53-positive cells, and Waf1/P21-positive cells mostly located at 2-3 layers higher than P53-positive cells. All the P16-positive cancers showed an absence of PRb immunostaining. In the majority of the morphologically normal samples, P21and pRb immunostaining were found in most of the cells in the basal layers, where PCNA-positive cells also resided. LOH of the Rb gene was observed in 30 of the 55 informative tumour samples. There was a strong association between LOH and diminished pRb expression in our samples (P<0.0001). Rb LOH is more frequently in tumours with P53 mutations (P<0.05), when the status of Rb and P53 alterations were evaluated by the combined results of immunostaining and genetic analysis, alteration of Rb and P53 had an even closer association. The present results suggested that Waf1/P21 and pRb may be involved in esophageal epithelial cell proliferation and differentiation, and that defects in the Rb and P53 pathways, and their potential synergistic effect in deregulating cell-cycle and apoptosis, are major mechanisms for esophageal carcinogenesis. Supported in part by National Outstanding Young Scientists Foundation of China 30025016 and NCI CA65817, U.S.A.

ANALYSIS OF INTERFERON-ALPHA EFFECTS ON GENE EXPRESSION PROFILING IN HBV TRANSFECTED CELL LINE Z. H. Yuan1, X. Wang1, L. J. Zheng1 , Y. Feng2, G. X. Hu2 and Y. M. Wen1 1 Department of Molecular Virology, Medical Center of Fudan University, Shanghai; 2Institute of Biochemistry and Cell Biology, CAS, Shanghai, China

Cells do not synthesize IFN unless they are activated, and virus infection is the most common biological cause of IFN induction. IFN-
binds to its cell receptor and induces activation of receptorassociated tyrosine kinases, resulting in tyr-phosphorylation of specific cytoplasmic proteins and their translocation to the nucleus, where they bind to cis-acting sequences of IFN-inducible genes and promote their transcription. Most IFN-
inducible genes contain a variation of consensus sequence (ISRE) and some of the trans-acting factors have also been identified (ISGF, IFN-stimulated gene factors). We have reported previously that, IFN-
production by PBMC from HBV infected patients was much lower than that in other viral infections and IFN-
has been employed as one of the main therapeutic measures for HBV infections for nearly two decades. To further elucidate the roles played by IFN-
in treatment of HBV infection and to explore for unidentified IFN-induced transcriptional changes in HBV infected cells, we used the cDNA microarray technology to study the expression profiling induced by IFN-
in an HBV genome transfected hepatoblastoma cell line. This cell line (HepG2 2215) persistently secretes HBV virions and expresses HBsAg and HbeAg. The mRNAs from HepG2 2215 and its parental cell line (HepG2) were separately isolated after treatment with IFN-
for 6, 24 and 48 hours respectively. These mRNAs were hybridized with the cDNA arrays of 18,000 human genes, and after being scanned, data were analysed using ArrayGauge software. Some of the microarray data were further checked by Northern blotting. At time 0, there were 89 genes showing 3-fold up- and downregulation differences in cellular gene expression between HepG2 and HepG2 2215, which could be related to the integration of HBV DNA into cellular genome, expression of HBV proteins and the active

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Cell Biology International, Vol. 25, No. 10, 2001

replication of HBV. Six hours after treatment with IFN-
, marked changes in mRNA expression were found in both HepG2 and HepG2 2215 cells. There were 83 and 34 genes that showed more than 3 fold up-or down-regulation in these two cells respectively. At 24 hours after IFN-
treatment, the number of genes that showed changes in expression was increased to 103 and 48 respectively, while at 48 hours, the number of genes showing change in expression dropped to 48 and 45 in the two cell lines. Changes in the expression of known genes were consistent throughout the study at different time course. These genes were arbitrarily grouped as cytokine/receptor, extracellular matrix and cell adhesion, GTP binding protien and signal transduction, ligands and receptors, mitochondrial, phosphatase and phosphodiesterase, protein degradation and protooncogenes etc. In addition, around 200 and 400 differentially regulated genes were new ESTs or unknown genes. Interestingly, after treatment with IFN-
, the number of cellular genes that showed changes in their expression was less in HepG2 2215 cells than that in HepG2 cells. In addition, the number of differentially regulated genes between HepG2 and HepG2 2215 decreased after the treatment of IFN-
, which suggested that interferon could partially restore the effects caused by HBV infection through transcriptional regulation of many downstream cellular effector genes.

Go
1 MEDIATES MAP KINASE ACTIVATION AND SERVES AS A TARGET FOR THE FEEDBACK REGULATION BY THE ACTIVATED MAP KINASE IN NEURONAL GPCR SIGNALLING Zhe Zhang, Yan-Xiang Ni, Ya-Lan Wu, Lan Ma1 and Gang Pei Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, 320 YueYang Road, Shanghai 200031; 1 National Laboratory of Medical Neurobiology, medical school, Fudan University, Shanghai 200032, People’s Republic of China

A variety of PTX-sensitive G protein-coupled receptors (GPCR) couple to the mitogen-activated protein kinase (MAP kinase/MAPK, also called ERK), however the signalling pathways and its physiological function are not fully understood. In the current study, using neuroblastomaglioma NG108-15 hybrid cells, we found that activation of MAPK by stimulation of delta opioid receptor (DOR) was PTX-sensitive but independent on G subunits, and that overexpression of a dominant negative form of Go
1 but not Gi
2 completely blocked the MAPK activation, indicating specific coupling of Go
1 to MAPK. The specific inhibitors of MPAK, PD98059 or U0126, significantly reversed the desensitization of DOR/Go
1 signalling. Interestingly, stimulation of muscarinic receptor but not nerve growth factor receptor, though both pathways activate MAPK, could crossly induce the desensitization of DOR. Further evidence from immunofluorescence confocal microscopy showed that the activated MAPK colocalized with Goa on the cytoplasm membrane in an agonistdependent manner, which could be blocked by PD98059. Moreover, when S314 of Go
1, a potential MAPK phosphorylation site located in its GPCR coupling domain, was mutated, either uncoupling of DOR/ Go
1 or desensitization of DOR/Go
1 signalling was greatly reduced. In vitro experiment further revealed that Go
1 was phosphorylated by MAPK at S314. Thus, our data strongly suggested a novel function for Go
1 as a MAPK activator and a target for MAPK in the feedback regulation of neuronal GPCR signalling. K: desensitization; G protein-coupled receptors; heterotrimeric G protein Goa; MAP kinase; NG108-15 cells.

Section 3. POSTER ABSTRACTS ULTRASTRUCTURAL OBSERVATION OF LIVER TISSUE ABLATION INDUCED BY HIGH-INTENSITY FOCUSED ULTRASOUND Bao Su-Su1 and Cheng Shu-Qun2 1 Department of Computer Science, South China Normal University, 2The Orient Surgery Center of the Liver and the Gallbladder in Shanghai, China

To observe the ultrastructural changes of liver tissues on normal rabbit ablated by high-intensity focused ultrasound (HIFU). A single shot of

1.1 MHz focused ultrasound at an intensity of 500 W/cm2 with continuous exposure duration of 20 s was applied intraoperatively in normal rabbit livers. Ultrastructural changes of the sono-ablated lesion, as viewed by light and electron microscopy were observed. Liver cells at the centre of the sono-ablated lesion were irreversible degeneration immediately after HIFU treatment observed by electron microscopy, although it appeared normal histologically, irregularly shaped cavities of about 0.3 m–0.5 m in diameter were found in its cytoplasm. Thermal damages may be the main mechanism of HIFU ablating liver tissues and cavitations effect also plays a role in it. High-intensity focused ultrasound (HIFU) is a new method of ablating non-invasively a selected volume of tissue at a certain depth within the body whilst sparing overlying tissues. This technique was originally developed as a method of selective brain tissue destruction for the neurosurgical research. Recently, it has been used successfully in ophthalmology for the treatment of glaucoma and is undergoing clinical trials in the prostate. We have used this principle to ablate liver tumours in a rabbit model with encouraging results. In this paper the ultrastructural changes of the ultrasonic lesion in normal rabbit livers, as viewed by light and electron microscopy were described.

SIGNAL TRANSDUCTION OF K. PNEUMONIEA LPS MEDIATED -DEFENSIN-4 mRNA EXPRESSION IN LUNGS OF MICE Cai Shaohui1, Cheung Xinnian1, Huang Ning1, Du Jun2, Wang Boyao1 and Takaiki Hascgawa2 1 Research Unit of Infection and Immunity, Department of Pathophysiology, West China Medical Center, Sichuan University, Chengdu 610041 and 2Department of Health Sciences, Scholl of Medicine University of Nagoya, Nagoya, Japan 466

The aim of our study is to investigate in vivo effects of LPS on -defensin expression and the relevant signal transduction pathway. Method: LPS tolerant mice C3H/HeJ with a point mutation at Toll-like receptor 4(TLR4) gene and its wild type strain C3H/HeN were used in this study. C3H/HeJ and C3H/HeN were injected with 4 mg/kg of Klebsiella pneumoniae endotoxin (LPS) intraperitoneally. The trachea, lung and kidney of the C3H/HeJ and C3H/HeN were collected at different LPS-treated time points and total RNA of each sample was extracted. The expression of mice -defensin-3 and/or -defensin-4 mRNA was determined in these tissues by reverse transcriptase-polymerase chain reaction (RT-PCR). The sequence of cDNA amplified from the lung of C3H/HeN was analyzed after 24 h treatment with LPS. By using western blotting, p-I-B (phosphorylated IB
) and IB
were detected in the lung of C3H/HeJ and C3H/HeN at different time points after treatment with LPS or without LPS. Result: (1) -defensin-4 mRNA was detected in the lung of C3H/HeN after 24 h treatment with LPS. In contrast, no signal was determined in both of LPS treated C3H/HeJ mice and C3H/HeN mice without LPS treatment. (2) Compared to the control mice, the content of p-IB
increased in the lung of C3H/HeN at the point of 4 h after treatment with LPS, while the content of p-IB
and IB
tended to be going down at 8 h after treatment, and dramatically decreased at 24 h, whereas there was no change in the content of p-IB
and IB
of the lung in C3H/HeJ under same conditions. Conclusion: K. pneumonica endotoxin could induce expression of -defensin-4 mRNA in the lung of C3H/HeN, and TLR4-medicated NF-B activation signalling pathway may be responsible for K. pneumoniea LPS-induced mRNA expression in the lung tissues.

INCREASED iNOS INDUCTION BY LIPOPOLYSACCHARIDE (LPS, ENDOTOXIN), CHINESE GINSENG AND THE NEUROPEPTIDE CGRP IN VASCULAR SMOOTH MUSCLE CELLS (VSMCS) OF ELDERLY, BUT NOT YOUNG, RATS Gabriel H. H. Chan and R. R. Fiscus Department of Physiology, Faculty of Medicine, Epithelial Cell Biology Research Center, and The Center for Gerontology and Geriatrics, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong

Cell Biology International, Vol. 25, No. 10, 2001 Excessive NO synthesis via the increased gene expression of inducible NO synthase (iNOS) in VSMCs is known to occur during LPS-induced shock, resulting in massive vasodilation and life-threatening hypotension. Ginseng, a traditional Chinese medicine, is thought to possess therapeutic effects, in part, by vasodilating blood vessels. Although the exact mechanism is not understood, ginseng has been shown to stimulate rapid vasorelaxation, presumably via activation of endothelial NO synthase (eNOS). However, the role of iNOS induction in the vascular actions of ginseng is unknown. Also, it is not known whether these responses of LPS and ginseng are altered by the aging process or by the presence of the neuropeptide CGRP (known to synergistically enhance iNOS expression induced by interleukin-1 (IL-1)). The purpose of the present study was to examine the effects of aging and co-incubation with CGRP on the induction of iNOS and production of NO induced by either LPS or an extract of Chinese ginseng. Production of NO was assessed by measuring accumulation of nitrite, the stable metabolite of NO, in the media of VSMCs using a modified Griess reaction. VSMCs were isolated from aorta of young (3-month-old) and elderly (20-month-old) female rats and were used in their 1st passage to avoid phenotypic changes that typically occur in VSMC cultures after 3–10 passages. LPS (10 g/ml) or CGRP (100 nM), alone, did not significantly increase nitrite levels in VSMCs of either young or elderly female rats. However, the combination of LPS and CGRP significantly (P<0.05) increased nitrite levels in elderly, but not young, female VSMCs. These data suggest than the synergistic interaction between LPS and CGRP becomes more apparent during advanced age. Chinese ginseng extract (0.1 mg/ml), by itself, caused large, significant (P<0.05) increases (8-fold above control) of nitrite levels in VSMCs of elderly, but not young, rats. Thus, aging appears to increase the iNOS induction caused by Chinese ginseng. The combination of Chinese ginseng and CGRP, however, did not significantly elevate nitrite levels, indicating that CGRP, in this case, inhibits the iNOS induction caused by the Chinese ginseng. VSMCs of elderly female rats appear to be more sensitive than young female VSMCs to the iNOS-inducing actions of LPS (in combination with CGRP) and of Chinese ginseng. Whereas CGRP synergistically enhanced the iNOS induction of LPS, it completely inhibited the increased NO production induced by Chinese ginseng. This novel response to Chinese ginseng may provide new insight into the therapeutic actions of ginseng as well as indicate a potential hazard (i.e. excessive vasodilation and resulting hypotension) when using ginseng in the aging population. (Supported by a Direct Grant for Research.)

ALTERED VASORELAXATIONS INDUCED BY CGRP, VIP AND ACETYLCHOLINE IN AORTIC RINGS OF eNOS- AND iNOS-KNOCKOUT MICE Siu L. Chan and Ronald R. Fiscus Department of Physiology, Faculty of Medicine, Epithelial Cell Biology Research Centre and Centre for Gerontology and Geriatrics, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China

The objective of this study was to determine if vasorelaxant responses to calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (VIP) and acetylcholine are altered in aortic rings of mice lacking genetic expression of eNOS or iNOS genes (i.e. eNOS- and iNOSknockout mice) as compared to control (wild-type) mice. The eNOSknockout (C57BL/6J-NOS3tmlUnc) and wild-type (C57BL/6J) male mice (20–30 g) were injected intraperitonealy with heparin (800 USP in 0.2 l saline of 140 M). After 15 minutes, mice were euthanized by 100% CO2 and decapitated, and the thoracic aortas excised rapidly and placed in Kreb-Ringer-Bicarbonate (KRB) solution (aerated with 95% O2/5% CO2). The aortas were dissected free of surrounding fat and connective tissue and cut transversely into rings 3- to 4-mm wide and mounted on two triangular-shaped stirrups of tungsten wire (0.15 mm). Aortic rings from eNOS-knockout [eNOS (
/
)] mice did not relax in response to acetylcholine, but rather contracted in some rings. Aortic rings from iNOS knockout [iNOS (
/
)] mice relaxed to acetylcholine in an endothelium-dependent manner. However, maximum relaxations in endothelium-intact rings were significantly (P<0.05) larger than in control mice (85.33.1 versus 67.95.6%). CGRP caused concentration-dependent relaxations in

1049 aortas of all three types of mice: control mice, iNOS (
/
) and eNOS (
/
). Vasorelaxant responses of CGRP in control and iNOS (
/
) mice had identical relationship; both were partially dependent on endothelium. In eNOS (
/
) mice, dose-response curves of CGRP in endothelium-intact and endothelium-denuded rings were not significantly different, indicating loss of the partial dependence on endothelium. The vasorelaxant responses of VIP were completely dependent on endothelium in both control and iNOS (
/
) mice. Maximum relaxations in iNOS (
/
) mice (77.42.7%) were significantly larger than in control mice (64.05.5%). Vasorelaxant responses of VIP in eNOS (
/
) aortic rings were also endothelium dependent, but responses were greatly attenuated compared to wildtype mice. Relaxations induced by VIP (110
7 M) in endotheliumintact aortic rings of eNOS (
/
) mice and control mice were 18.35.4% and 64.05.5%, respectively. These findings demonstrate that in eNOS (
/
) mice, aortic vasorelaxant responses to CGRP were fully present but no longer dependent on the endothelium, responses to VIP were greatly attenuated compared to control and responses to acetylcholine were abolished. In iNOS (
/
) mice, aortic vasorelaxant responses to VIP and acetylcholine were significantly larger than in wild-type control. Acknowledgement: This study was supported by funding for research for the Intercalated Bachelor Medical Science Degree (to Siu Lan Chan) from the Department of Physiology, Faculty of Medicine, The Chinese University of Hong Kong.

INITIAL STUDY ON THE MECHANISM OF NEUROPEPTIDE PACAP’S EFFECTS ON THE CARDIOVASCULAR SYSTEM Chang Qing and Deng Yi-ping Department of Histology & Embryology, Medical College of Jinan University, Guangzhou 510632, China

Pituitary Adenylate cyclase activating polypeptide (PACAP) is a novel neuropeptide discovered by Japan scientists in 1989. Increasing reports show that PACAP is closely related to cardiovascular system. But the action mechanisms still keep unknown. The present article measured the distribution of PACAP receptor on the cell membranes of the cardiovascular tissues and on the vascular endothelial cells (EC) and smooth muscle cells (SMC) as well as the change of cell signal substances (cAMP, [Ca2+ ]i) for discussing the possible mechanisms. The lungs (as positive contrast), hearts and aorta of SD rats were obtained to prepare tissue membranes samples of the PACAP receptor. The cultured porcine artery ECs and SMCs from 3rd generation were collected as cell samples. The PACAP receptor were surveyd by radioligand-receptor binding assay. Changes of cAMP were measured by RIA. [Ca2+ ]i were tested by fluorescent indicator (Fura-2) method. The concentration used is 10
8 Mol/L. It is easy to see that (1) the number of PACAP receptor is scarce and the number of PACAP receptor on ECs is more than that on SMCs significantly. (2) PACAP increases the secretion of SMC’s cAMP. (3) PACAP decreases the [Ca2+ ]i of EC,SMC. This results suggest that it is possible that PACAP in blood play some effects by contacting with EC first in physiological condition and that the relationship between PACAP and SMC get stronger in some pathological situation. The cardiovascular effects of PACAP are remarkable though the receptor is scarce. So the present article consider that PACAP effects on the cardiovascular system may have the aid of the activating of post-receptor way except receptor

Table 1. The receptor analysis of lung, heart and aorta as well as EC, SMC Sample

N

XS (fMol/mg pretein)

XS bite/cell

Lung Heart Aorta EC SMC

5 5 5 4 4

1777.96788.712 88.70746.182 157.34784.440 — —

— — — 6699853.132 2784792.430

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Cell Biology International, Vol. 25, No. 10, 2001 Table 2. The effects of PACAP on the cAMP secreted by EC, SMC

Group

EC

SMC

N

XS (pg/ml)

N

XS (pg/ml)

9 7

0.2750.092 0.2500.104

9 10

1.0780.228 3.7100.921**

Control group PACAP group

**Compare to control group, P<0.01, Table 3 is the same. Table 3. The effects of PACAP on the [Ca2+ ]i of the EC, SMC Group

Control group PACAP group

EC

SMC

N

XS (nM)

N

XS (nM)

10 10

189.57330.332 117.11116.027**

10 10

141.25617.933 68.9039.494**

activated. For example PGI2 may excite directly adenylate cyclase to induce the change of the concentration of cAMP, [Ca2+ ]; thus remarkable biological effects appear.

PARATHYROID HORMONE REGULATION OF GENE EXPRESSION IN HUMAN PROXIMAL TUBULES Tsui-Shan Chau1, Wan-Ping Lai1, Ming-Hui Huang2, Meng-su Yang2 and Man-Sau Wong1 1 The Open Laboratory of Asymmetric Synthesis, Department of Applied Biology and Chemical Technology, Hong Kong Polytechnic University, Kowloon, Hong Kong; 2 Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong

Osteoporosis becomes public health concern in the elderly populations. Age is one of the important risk factors in osteoporosis. The primary cause of senile osteoporosis is related to the decline in renal function with age, which leads to decrease in 1
-hydroxylase activity. The reduced in 1,25(OH)2D3 biosynthesis results in negative calcium balance, subsequent in secondary hyperparathyrodism which increases bone resorption and accelerates bone loss in aging. Besides, 1,25(OH)2D3 responsiveness to parathyroid hormone is impaired with age. Parathyroid hormone (PTH) is important regulators of calcium and phosphate homeostasis through action on kidney. The stimulatory effect of PTH on 1-OHase requires both the cAMP-dependent protein kinase A (PKA) and protein kinase C (PKC) pathways. In aged animal, elevated endogenous PTH or administration of pharmacologic doses of PTH fails to increase circulating 1,25-(OH)2D3 or in vitro renal 1-OHase activity. The acquired resistance to PTH stimulation of 1,25(OH)2D3 production in aging appears not involve the hormonal activation of PKA or PKC. Therefore, it is unclear in mechanisms involved age-induced resistance of PTH-mediated 1,25(OH)2D3 production. Recently, constitutive expression of 1
-hydroxylase in a transformed human proximal tubule cell line (HKC-8) was established for the study of the renal vitamin D and mineral homeostasis in vitro. The characterization of the gene expression of the human proximal tubule cell line in the signalling pathways provides fundamental understanding of regulation of 1,25(OH)2D3 by PTH. In this study, a time course study was carried out in HKC-8 cell line with 10
7 M dose of PTH for 24 hours. Regulation of gene expression by PTH can be accessed by cDNA microarray containing 448 genes involved in the signalling pathways. Each gene on the microarray was printed in duplication. The negative controls for the array were yeast tRNA, salmon sperm, pSPORT plasmid, human genomic DNA and Cot-1. The positive controls for the array were beta actin and ribosomal RNAs. Total

RNA obtained from PTH treated cells and vehicle treated cells were prepared for labelling with cy5 or cy3 through reverse transcription. The resulting fluorescently labelled cDNA mixture was hybridized to an array at high stringency and the fluorescent signal was detected by ScanArray 4000 (GSI Lumonics). According to our preliminary result, approximately 50% of genes were expressed in the array. Only 10% of the genes were altered under PTH treatment in the proximal tubules. These data will be further confirmed by RT-PCR.

CRITICAL ROLE OF THE HEPATIC GAP JUNCTIONAL COMMUNICATION INHIBITION IN LIVER CARCINOGENESIS OF MICE Dao-liang Chen, Long Wan, Bi-hua Chen and Hua-son Wu Department of Cell Biology, Fujian Institute of Traditional Chinese Medicine and Pharmacolgoy, Fuzhou, 350003, China

The aim of this study is to obtain in vivo evidence of the importance of normal gap juctional intercellular communication (GJIC) as a natural defense of tissue against carcinogenesis. New born male ICR mice were each given a single i.p. injection of diethylnitrosamine (DEN) for liver tumour induction (40 mice) or of the solvent without DEN for control (20 mice). After weaning, half of the DEN-injected mice received daily s.c. injections of isoproterenol plus caffeine for liver GJIC activation and all the animals were fed a high protein low carbohydrate feed. Liver GJIC capacity was determined in 8 mice of each group by carboxyfluorescin cell-to-cell transfer assay performed on fresh intact liver lobes. Liver tumourigenesis was analysed with 12 mice of each group at the age of 13 months. The results showed that, in the DEN-only group liver GJIC decreased to below 1/3 of the control level (P<0.001) within half month after weaning and remained low up at least to half year later when growing nodules had emerged on the liver surfaces. In DEN isoprolerenol caffeine group the liver GJIC appeared normal for at least 4.5 months after weaning, resulting in 98% reduction in th mean total nodo volume per liver (P<0.001), 42% and 50% reductions in the incidences of hepatocellular adenomas and carcinomas respectively, as compared to the DEN-only group. Control mice showed no liver abnormality. In conclusion, early inhibition of hepatic GJIC is critical for the advancement of DEN-stimulated mouse liver toward carcinogensis; disinhibition by pharmacological means can suppress this process. K: gap junctions; intercellular communication; mouse liver; carcinogenesis.

A PEROXISOME-PROLIFERATOR ACTIVATED RECEPTOR-GAMMA AND Bcl-2 FEEDBACK LOOP FUNCTIONS TO REGULATE APOPTOSIS IN HUMAN COLON EPITHELIAL CELLS George G. Chen, Janet F. Y. Lee, Ursula P. F. Chan, Ping C. Ip and Wan Y. Lau Department of Surgery, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, China

Peroxisome-proliferator activated receptor-gamma (PPAR) is one of the nuclear receptors for peroxisome proliferator. Studies have shown that expression of PPAR is increased in some human tissues and cells including colon epithelial cells. Though the activation of PPAR is associated with cancer prevention, the molecular mechanism between the activation of PPAR and its consequence in colon epithelium is unknown. The present study is to investigate the expression of PPAR in colon cancer tissues and in colon epethelial cells, and the relationship between the PPAR and apoptotic molecules. We have demonstrated that the expression of PPAR is significantly increased in human colon cancer. Activation of PPAR induces apoptosis in human colon epithelial cells and this action can be, at least partially, blocked y overexpression of Bcl-2. PPAR ligand can also suppress the activity of NF-kB, an important transcription factor in regulation of genes governing inflammation and apoptosis. The time course experiment of indicated that inhibition of NF-kB activity by PPAR ligand appeared a few hours earlier than reduction of Bcl-2 level caused by

Cell Biology International, Vol. 25, No. 10, 2001 PPAR ligand treatment, suggesting that the inhibition of NF-kB is an upstream event of Bcl-2 suppression by PPAR ligand. Therefore, PPAR ligand sequentially inhibits NF-kB activity and Bcl-2 expression. A PPAR-Bcl-2 feedback loop may, therefore, exist in human colon epithelial cells and is functional in controlling the balance of cell growth and death.

ISOLATION OF PLASMODIUM FALCIPARUM cDNA ENCODING DRUG-BINDING PROTEINS TO ARTEMISININ ANTIMALARIALS USING DRUG-WESTERN METHOD Cheng Yuanguo, Fu Zhuoshen, Shan Chengqi, Gu Xibo and Wang Jiaxi Institute of microbiology & Epidemiology, Beijing 100071, China

There is a need for new antimalarials from the widespread resistance to those in current use. New antimalarial targets are required to allow the discovery of chemically diverse, effective drugs. The search for such new targets and new drug chemotypes will likely be helped by the advent of functional genomic and structure-based drug design. After validation of the putative targets as those capable of providing effective and safe drugs, targets can used as the basis for screening compounds in order to identify new leads, which in turn, will qualify for lead optimization work. The combined use of combinational chemistry to generate large numbers of structurally diverse compounds and of high throughout screening systems to speed up the compounds hopefully will help to optimize the process. Artemisinin was originally isolated from Artemisia annua, an herb used as ancient Chinese herbal remedy. All of the artemisinin compounds contain stable endoperoxide bridges. Evidence from a variety of labs suggests that the antimalalarial activity of artemisinin is depending on the cleavage of the endoperoxide by intraparasitic heme. The cleaved endoperoxide ultimately becomes a carbon-centred free radical which then functions as an alkylating agent, reacting with both heme and parasite protein. In this experiment, we used a rapid and convenient new expression cloning method, drug-western, to isolated the genes encoding Plasmodium falciprum artemisinin-binding proteins. This method is based on the use of drug conjugated with a marker molecule as a probe for screening of a cDNA library. Unlike other methods, this method allowed us to identify the genes for trace amounts of cellular drug binding proteins without purification. Currently, we have utilized the unique SMART (Switch mechanism at 5 end of RNA transcript) oligonucleotide in the first strand synthesis, followed by long-distance (LD)) PCR amplification to generate high yields of full-length, double-stranded cDNA. No radioactivity is required to estimate yields or size distribution. The SMART libraries contain a significantly higher percentage of full-length colons than cDNA libraries constructed using conventional cDNA synthesis protocols. The capacity of this TripIEx2 cDNA expression library from erythrocytic Plasmodium falciparum and the size of cDNA is suitable for further study. K: artemisinin; Plasmodium falciparum; cDNA expression library; drug-western and drug-binding protein.

ATRIAL NATRIURETIC PEPTIDE (ANP) PROTECTS AGAINST APOPTOSIS IN SERUM-DEPRIVED PC12 CELLS ASSESSED BY TUNEL METHOD S. B. Cheng Chew, Clara K. H. Chong, P. Y. Leung and Ronald R. Fiscus Department of Physiology, Faculty of Medicine, Chinese University of Hong Kong, Shatin, N.T., Hong Kong

Atrial natriuretic peptide (ANP) was investigated to determine effects on apoptotic DNA fragmentation in serum-deprived PC12 cell using TUNEL (TdT-mediated dUTP-biotin nick end labelling) technique. This method allows in situ visualization of apoptotic cells at single cell level. It provides a direct quantification in terms of the number of cell death. PC12 cells were obtained from the American Type Culture Collection (ATCC) and were grown on poly-L-lysine-coated flasks. Cells were collected at 4.5 h, 16 h and 24 h under the following conditions: (1) with serum, (2) without serum, (3) ANP treatments

1051 (1 nM, 10 nM and 100 nM) at the time of serum removal. Cell death in PC12 cells caused by serum deprivation was associated with apoptotic DNA fragmentation as labelled by the TUNEL method at a time point as early as 4.5 h. Serum deprivation for 4.5 h increased the percentage of labelled cells from 0.84% to 3.36%. Addition of ANP at 1, 10 and 100 nM decrease the percentage of labelled cells to 2.09%, 1.45% and 1.38%, giving rise to a concentration-dependent protection against apoptosis to 50.20%, 75.65% and 78.73% respectively. The data were statistically significant (P<0.05) when analyzed using ANOVA followed by StudentNewman-Keuls test for the comparisons of significant differences between the various mean values. On the other hand, serum-deprivation at 16 h and 24 h did not show much changes as far as apoptosis and ANP protection were concerned. The explanation for the latter cases might involve complete disintegration of cells that were not detectable by TUNEL labelling. ANP has been shown to elevate cyclic GMP (cGMP) levels in neural cell lines, PC12 pheochromocytoma and C6 glioma cells, and to stimulate the export of the cGMP from these cells (Fiscus et al., 1987). Our previous studies also shown that ANP and BNP (brain natriuretic peptide) both prolong the survival of serum-deprived PC12 cells by prolonged cGMP elevations (Fiscus et al., 2001). It is therefore concluded that the protective effect of ANP against apoptotic cell death caused by serum-deprivation as measured by the TUNEL method is through the elevation of cGMP in these cells. This study also support the results obtained by agarose gel and capillary electrophoresis with laser-induced fluorescence detector (CE-LIF) that have been reported previously in abstract form (Fiscus, 2000). References F RR, R BT, W SA, et al., 1987. J Neurochem 48: 522–528. F RR, T AWK, C C SB, 2001. NeuroReport 12(2): 185–189. F RR, 2000. FASEB J 14: A1520.

PROSTACYCLIN RECEPTORS STIMULATE MITOGEN-ACTIVATED PROTEIN KINASES THROUGH COUPLING TO Gq PROTEINS K. M. Chu and H. Wise Department of Pharmacology, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, The People’s Republic of China

G protein-coupled receptors can induce cellular proliferation by stimulating the mitogen-activated protein (MAP) kinase cascade. In addition, activation of extracellular signal-regulated kinase 1 (ERK1) and ERK2 (i.e. the MAP kinase cascade) is important for several neuronal functions such as differentiation and survival. The crosstalk between G proteins and the MAP kinase cascade is highly dependent on the cell type. For example, although cAMP inhibits ERK1/2 in most non-neuronal cells, it activates ERK1/2 in pheochromocytoma 12 (PC12) cells and neurons (Cavanaugh et al., 2001). The prostacyclin (IP) receptor is a G protein-coupled receptor which preferentially stimulates adenylate cyclase through coupling to Gs, but can also directly stimulate phospholipase C through coupling to Gq (Namba et al., 1994). Activation of the phospholipase C pathway will lead to increased protein kinase C activity, which could activate ERK1/2 via Raf. We have therefore examined the ability of IP receptors to stimulate the ERK1/2 pathway, and have attempted to define the crosstalk between Gs and Gq-mediated signalling pathways. Chinese hamster ovary (CHO) cells were transiently transfected with mouse IP receptor cDNA (mIP-CHO cells), and reagents of the PathDetect Trans-Reporting system (Stratagene) for the determination of ERK1/2 activation using a firefly luciferase (Fluc) reporter gene. CHO cells were co-transfected with Renilla luciferase (Rluc) plasmid to account for variations in transfection efficiency using a Dual Luciferase Assay system (Promega). The protein content of cell lysates was determined to account for variations in cell numbers. mIP-CHO cells were assayed 48 h post-transfection following overnight incubation in serum-free F-12 medium. Test compounds were then added 30 min before addition of 10
7 M cicaprost (IP agonist) for 5 h in the presence of 3 M indomethacin. Cicaprost produced a 4.70.9 fold increase in ERK1/2 activity, with pEC50 values of 7.5360.001 (meanSEM, n=2) which was

1052 inhibited by staurosporine (0% control) and GF 109203X (37% control). In contrast to the inhibitory effect of protein kinase C inhibitors, the protein kinase A inhibitors H-89 and Rp-cAMPS had no effect on cicaprost-stimulated ERK1/2 activity (87 and 134% control, respectively). The protein kinase C activator PMA produced a 4.20.7-fold increase in ERK1/2 activity, which was not increased further by cicaprost. Protein kinase C activity inhibits IP agoniststimulated inositol phosphate production (unpublished observations), which may account for the failure of cicaprost to increase ERK1/2 activity following 30 pretreatment with PMA. Cicaprost failed to stimulate ERK1/2 in wild-type CHO cells. In conclusion, prostacyclin receptors appear to stimulate the ERK1/2 pathway through coupling to Gq proteins. The work described in this abstract was fully supported by a grant from the Research Grants Council of the Hong Kong Special Administrative Region (CA99/00.SC01). References C JE, et al., 2001. J Neuroscience 21: 434–443. N T, et al., 1994. J Biol Chem 269: 9986–9992.

ROLE OF NITRIC OXIDE ON PROTEIN KINASE C ACTIVATED BY ANGIOTENSIN II IN CULTURED NEONATAL RAT CARDIOMYOCYTES Fu Shigan, Xie Xieju, Ji Limin, Liu Peiqing and Pan Jingyun Hainan Medical College, Haikou 571101, Sun Yat-sen University of Medical Sciences, Guang Zhou 510089, China

We examined the effect of endogenous and exogenous nitric oxide (NO) on protein kinase (PKC) activity induced by angiotensin II (Ang II) in cultured neonatal rat cardiomyocytes. The results were as follows: Activity of PKC was increased by Ang II (10—810
5 mol/L, 15 min) in a dose-dependent manner, but was decreased by NO precursor L-arginine (L-Arg) (10
510
2 mol/L, 30 min) and NO donor sodium nitroprusside (SNP) (10
610
3 mol/L, 5 min) in cultured neonatal rat cardiomyocytes. Activity of PKC was increased by Ang II and a PKC activator phorbol 1,2-myristate 13 acetate (PMA) (10
5 mol/L, 15 min) but was impaired by a NO precursor L-Arg and the exogenous NO donor SNP. NG-nitro-L-argingie methyl ester (L-NAME), a nitric oxide synthase (NOS) blocker, itself had no effect on the PKC activity of cultured neonatal rat cardiomyocytes, but may inhibit the role of PKC activity induced by Ang II and PMA. These results indicate that PKC is controlled by both NO and Ang II, and that PKC may be ‘cross talking’ between cardiac hypertrophy induced by Ang II and prevented by NO. NO abolished activity of PKC and impaired PKC downstream signalling transduction pathway cascades and finally prevented cardiac hypertrophic response.

SYNERGISTIC POTENTIATION OF ADRENOMEDULLIN-INDUCED VASORELAXATIONS AND cAMP ELEVATIONS BY BRAIN NATRIURETIC PEPTIDE (BNP) IN ENDOTHELIUM-DENUDED RAT THORACIC AORTA Erik Fung and Ronald R. Fiscus Department of Physiology, Faculty of Medicine, The Epithelial Cell Biology Research Centre, and The Centre for Gerontology & Geriatrics, The Chinese University of Hong Kong

Human adrenomedullin (ADM) is a peptide hormone consisting of 52 amino acids, and belongs to the same family as calcitonin gene-related peptide (CGRP) and amylin. Rat ADM is 2 amino acids shorter than human ADM and differs in 6 amino acid residues. ADM is produced mostly by vascular smooth muscle cells (VSMC) as well as by endothelial cells. The endothelium is a critical component in determining the effectiveness of ADM to cause vasodilation because when denuded, the response to ADM is drastically reduced. ADM has been demonstrated to elevate cAMP in VSMC. Brain natriuretic peptide (BNP) is a hypotensive and natriuretic factor that is primarily synthesized by the cardiac ventricles. BNP causes activation of particulate guanylyl cyclase in VSMC, resulting in increased cGMP levels, activation of protein kinase G and further mechanisms of vasodilation.

Cell Biology International, Vol. 25, No. 10, 2001 Inhibition of type-3 phosphodiesterase (PDE3), an enzyme responsible for reducing the intracellular levels of cAMP in VSMC, by elevated levels of cGMP, has been shown to enhance cAMP accumulation and prolong vasorelaxation in vascular smooth muscle. We hypothesized that ADM-induced vasodilations may be synergistically enhanced by addition of BNP via elevation of the intracellular pool of cGMP, thereby inhibiting PDE3 and potentiating the accumulation of cAMP induced by ADM. The extent of endothelium dependency of ADM in inducing vasodilation was first investigated and confirmed to be substantial (with endothelium: 34.14.19%, without endothelium: 3.00.65%; P< 0.001). To uncover interactions between ADM and BNP in vascular smooth muscle without the interference from endothelium-derived agents, rat thoracic aortae were denuded of endothelium. After precontraction with phenylephrine (100 nmol/L), BNP (1 nmol/L) pretreatment of aortic rings resulted in mean vasorelaxations of 20.13.71%, followed by addition of log incremental concentrations of ADM at 1 nmol/L, 10 nmol/L and 100 nmol/L, and resulting in mean values of further vasorelaxations of 5.61.87%, 20.96.13% and 559.44%, respectively (n=6). A specific radioimmunoassay technique demonstrated significant elevation in cAMP levels induced by ADM in endothelium-denuded rings pretreated with BNP (1 nmol/ L), whereas in rings without BNP pretreatment no measurable changes in cAMP levels were detected. BNP was confirmed to elevate cGMP levels by 1.6 folds but did not affect cAMP levels. NO donors (e.g. nitroglycerin) and BNP, agents that are known to elevate cGMP levels in VSMC, have previously been shown to potentiate CGRP-induced vasorelaxations and cAMP elevations. The potentiating effects have been proposed to be attributable to the cGMP-mediated inhibition of PDE3. By inhibiting PDE3, cAMP accumulates to a higher level resulting in enhanced activation of PKA and greater relaxation of vascular smooth muscle; as seen in the present study where BNP is shown to enhance vascular effects induced by ADM. Elevated levels of ADM and BNP in sepsis, leading to severe hypotension, may partly be explained by the mechanism of synergistic vasorelaxation as described above. BNP synergistically enhances ADM-induced vasorelaxation and accumulation of intracellular cAMP, likely mediated via cGMPinhibition of PDE3. Supported by a Direct Grant for Research.

UP-REGULATION BINDING ACTIVITY OF GATA AND AP-1 TRANSCRIPTION FACTORS INITIATED BY PANAXIOL OF GINSENG IN HAEMATOPOIETIC CELLS Ruilan Gao1, Weihong Xu1, Xialhong Cheng1, Beng H. Chong2 and Chaoqun Wu3 1 Afflicted Hospital of Zhenjian College of Traditional Chinese Medicine, 2Hangzhou, 3University of New South Wales, Australia and Fu Dan University, Shanghai, China

Up-regulation of DNA binding activity of AP-1 and GATA family members induced by Panaxdiol of Ginseng (PDS) were observed in hematopoietic cells. To investigate whether the transcription factors of c-Fos, c-Jun, GATA-1 and GATA-2 were involved in the PDSinitiated transcription regulation of the genes controlling cell proliferation and differentiation. Methods: PDS was extracted from total saponins of panax ginseng fourthly. The nuclear extracts were obtained from three lineages of cell lines, including myeloid cell of HL-60, erythroid cells of K562, and megakaryoloid cell of CHRF-288 and Meg-01 treated by PDS. Western blotting analysis was performed with the antibodies against human c-Fos, c-Jun, GATA-1 and GATA-2, AP-1 and GATA. Consensus ds-oligonucleotide affinited was radiolabelled with polynucleotide kinase and [
-32P] ATP for electrophoretic mobility shift assay (EMSA). The components of DNA-transcription factor complex were analyzed with antibody gel supershift assay. Results: (1) Western blot showed that in response to PDS, GATA-2 protein in HK-60, K562, CHRH-288 and Meg-01 cells was increased by 1.3, 1.9, 3.4 and 2.2 folds respectively; while c-Jun protein was elevated to 3.9, 5.0 and 5.5 folds by PDS in K562, CHRH-288 and Meg-01 cells. (2) Not only were 4.5, 5.9 and 2.0 fold increased GTAG-1 protein, but also 2.5, 4.9, and 2.1 fold raised GATA-2 induced by PDA respectively in K562, CHRH-288 and

Cell Biology International, Vol. 25, No. 10, 2001 Meg-01 cells. (3) EMSA results indicated that binding complex of AP-1 and DNA appeared as two bands. The supper bands of binding activity was elevated obviously induced by PDS in K562 and CHRH288 cells, which was identified mainly as c-Fos and c-Jun protein by supershift assay. While low band was increase in Meg-01 cells. (4) The increasing binding activity of GATA protein initiated by PDS was mainly attributable to GATA-1 and GATA-2 acted as a major character in K562, CHRH-288 and Meg-01 cells. In HL-60 cells GATA banding activity was expected at low level and no detectable difference was seen before and after GS treatment. In conclusion, these results suggested that PDS was a effective component for enhancing hematopoiesis within total saponiins of panax gingseng. The intracellular signalling pathway initiated by PDS was involved in transcription factor c-Fos, c-Jun, GATA-1 and GATA-2. These proteins might play thee key role through increasing their quantity and activity caused by PDA to up-regulate gene of proliferation and differentiation in hematopoiesis.

CENTRAL ADMINISTRATION OF NEUROPEPTIDE Y-Y5 RECEPTOR ANTISSENSE OLIGODEOXYNUCLEOTIDES STIMULATES APOPTOSIS AND LIPOLYSIS ON WHITE ADIPOCYTES Xirong Guo, Haixia Gong, Li Fei, Mei Guo, Qianqi Liu, Xiaonan Li and Ronghua Chen Department of Pediatrics, Nanjing Medical University, 210029, Nanjing, P.R. China

The new neuropeptide Y (NPY) Y5 receptor has been proposed to mediate the NPY-induced feeding response and therefore plays a central role in the regulation of food intake. This conclusion was based on studies with Y5 antagonists or antisense oligodeoxynucleotides (ODNs). Nevertheless little is known about the possible mechanisms for body weight loss induced by functional blockage of NPY-Y5 axis. This study which was designed to test the possible effects of Y5 antisense ODNs on white adipose tissue (WAT), revealed that intracerebroventriculary (i.c.v.) injection of Y5 antisense ODNs strongly stimulates adipocyte apoptosis and lipolysis both in high-energy diet induce obese rats an in lean controls. In addition, the Bcl-2/Bax mRNA ratio significantly decreased in WAT, and the metabolism was improved. So we conclude that Y5 receptor antagonistic treatment is a promising new method for obesity therapy. Other methods that induce white adipocyte apoptosis or lipolysis may be useful for obese patients. K: neuropeptide Y; Y5 receptor; antisense ODNs; obesity; apoptosis; lipolysis; adipocyte; Bcl-2; Bax.

EFFECT OF POLYASPARTIC ACID ON GENTAMICIN-INHIBITING INOSITOLDELIPID MESSENGER SYSTEM IN COCHLEAR OF GUINEA PIG Guo Yufen, Jiang Sichang and Yang Weiyan Department of Otorhinolaryngology, General Hospital of PLA, Beijing 100853, China

The objective of this study is to observe the effect of polyaspartic acid (PAA) on gentamicin-inhibiting inositoldelipid messenger system in cochlear of guinea pig and investigate the protective mechanism of polyaspartic acid on gentamicin (GM) toxicity. Incorporation of [3H]-inositol and high performance liquid chromatography (HPLC) was used to determine inositol phosphate (IP), polyphosphatidylinositol (PPI) and PPI phospholipase C (PPI and PLC) activity in cochlear tissue following different drug duration and drug treatments. The results demonstrated that following 1 day treatment with PAA there was no change in IP, PPI or PPI PLC activity (P>0.05). Following 5-days treatment with PAA, there was an increase in the level of PPI and a decrease in IP radioactivity. The activity of PPI PLC was inhibited by using only GM. Compared with control groups there was no change in IP, PPI and PPI PLC by using PAA and GM. Following PAA treatment for 10 days, there was an increase in the level of PPI and IP radioactivity, the activity of PPI PLC was partly activated by using only GM; but when compared with using GM alone for 5-days, there was no change of the level of PPI. Whereas there were no change in IP radioactivity, the level of PPI and the activity of PPI PLC by

1053 using PAA and GM. In conclusion PAA has significant preventative effects on the gentamicin-inhibiting phosphinositol messenger system in cochlear of guinea pig, which shows that PAA has protective effect on gentamicin-toxicity in guinea pig. K: polyaspartic acid (PAA); gentamicin (GM); inositoldelipid messenger system; cochlea; [3H]-inositol High performanceliquid Chromatography (HPLC).

EFFECTS OF POLYSACCHARIDE SULFATE ON IONIC CURRENTS IN GUINEA PIG VENTRICULAR MYOCYTES Han Shengna, Cui JinJuan, Zhang Zhao and Tan Shuben Department of Physiology Henan Medical University, Zhengzhou 450052, China

In previous study, we observed by using of a standard microeletrode technique that polysaccharide sulfate (PSS) could decrease the action potential duration of the fast action potential and the contractile force in the isolated guinea pig papillary muscles. The maximal upstroke velocity, action potential amplitude and action potential duration of the slow action potential were suppressed by PSS. Thus, these effects of PSS could be associated with the selective inhibition of the calcium current. In the present study of the mechanism of ionic channel modulation by PSS, we have investigated its effects on ionic current by using of the whole-cell patch clamp technique. Single ventricular cells of guinea pig were isolated with a collagenase (I-type) dispersion method, and the ionic basis underlying the effect of PSS were studied at different voltage-clamp protocols. The results are shown: (1) PSS increased the time constant of activation and decreased the current amplitude of ICa,L. When ventricular myocytes were voltage-clamped at +10 mV, PSS increased the time constant of activation of ICa,L from 0.920.10 ms to 1.150.11 ms (n=5, P<0.05), and decreased the current amplitude of ICa,L from 16.801.71 pA/pF to 13.86 1.67 pA/pF (n=5, P<0.01); (2) PSS increased not only the inward component but also the outward component of IK1. For the inward component, PSS increased the peak IK1 from 22416.17 pA/pF to 25717.36 pA/pF (n=7, P<0.01) at
170 mV; for the outward component, PSS increased the peak IK1 from 8.890.86 pA/pF to 10.381.18 pA/pF (n=7, P<0.05) at
50 mV; but PSS had no effect on IK1 at potentials (
70 mV
120 mV) near the reverse potential. PSS decreased the time constant of activation of IK1 from 1.050.11 ms to 0.780.09 ms (n=7, P<0.01) at
160 mV. And, PSS increased the maximal slope conductance from 2.530.17 nS/pF to 2.820.20 nS/pF (n=7, P<0.05); and (3) PSS had no effect on the delayed rectifier K + current. In conclusion, PSS decreases L-type Ca2+ current, increases the inward rectifier K + current, but have no effect on the delayed rectifier K + current.

STIMULATING EFFECT OF CHEMOKINE LIKE FACTORS (CKLFs) ON SKELETAL MUSCLE CELLS Wenling Han, Donglan Xia, Yaxin Lou, Min Rui, Quansheng Song, Yingmei Zhang, Chunhui Di and Dalong Ma Peking University Center for Human Disease Genomics, 38, Xueyuan Road, Beijing 100083, China

Human chemokine-like factor 1 (hCKLF1) is a novel cytokine that has been cloned from PHA-stimulated U937 cells in our laboratory (Biochem J. 2001, 357, 127-135). CKLF1 has three slicing isoforms, which have been named as CKLF2, CKLF3 and CKLF4, CKLF1 has potent chemotactic effects on a wide spectrum of cells both in vivo and in vitro. CKLF1 can also stimulate the regeneration of skeletal muscle cells in vivo. Does CKLF1 have direct stimulatory effect on skeletal muscle cells, or it is the roles of cytokines secreted by that attracted cells? Do the isoforms of CKLF1 have direct effect on muscle cells? Which signal transduction pathway plays important roles in CKLFs? In order to answer these questions, we have carried out a series of experiments. The results show that CKLFs have direct enhancing effect on skeletal muscle cell; Ras/Rafl/MAP kinase and PI-3 kinase take part in the signal transduction pathway of CKLF2 during its promoting C2C12 differentiation process. We constructed a serial of

1054 eukaryotic expression vectors pcDI-CKLFs, transfected COS-7 cell and C2C12 cell for functional analysis. Compared with the supernatant of pcDI transfected COS-7 cell, the supernatant of pcDICKLF1 transfected COS-7 cell could stmulate the proliferation of the primary satellite cells of newborn Wistar rat and mouse myoblast C2C12 cells. We obtained the stable C2C12 clones expressing hCKLF2. Compared with control vector-transfected or the wild-type C2C12 cells, hCKLF2 could enhance the differentiation of C2C12 cells, the formation of myotubes and the survival of cells after serum withdrawal. When PD098059, an inhibitor of mitogen-activated protein (MAP) kinase activation, or LY294002, a specific inhibitor of PI-3 kinase was added to the pcDI-CKLF2 transfected C2C12 cells, the differentiation was inhibited obviously, which indicates that Ras/Raf1/ MAP kinase and PI-3 kinase play important roles in CKLF2 signal transduction pathway. We also cloned the CKLF homologue of rat and mouse (rCKLFs and mCKLFs) and constructed the corresponding eukaryotic expression vectors pcDI-rCKLFs and pcDI-mCKLFs. Rat CKLF-1 has the similar stimulating effect of hCKLF1, it can stimulate the proliferation of primary satellite cell derived from the neborn Wistar rat and C2C12 myoblast. When the satellite cells are induced to differentiation, the expression level of rCKLFs is increased. Mouse CKLF2 can also stimulate the differentiation of C2C12 cells and when mouse primary satellite cells and C2C12 mybloast are induced to differentiation, the expression level of mCKLFs is increased. All these results further verify that CKLFs have directly on skeletal muscle cells.

STUDYING THE MECHANICAL PROPERTIES OF CELL MEMBRANE BY QUASI-ELASTIC LASER LIGHT SCATTERING Qi-cai He1 and Yao-xiong Huang2 1 Department of Physics, Sun Yat-sen University of Medical Sciences, Guangzhou, China, 2Institute of Biomedical Engineering, Jinan University, Guangzhou, China

Quasi-elastic laser light scattering technique was developed since 1960s. This is a high resolving-power measuring technique, which can be used to measure small frequency shift about 0.1–104 Hz. The shift maybe result from the Doppler effects by scatters, so it can be used to measure the speed of the scatters, for example, to measure the speed of blood in the rabbit vein (1.3 cm/s). This technique can also be used to measure the diffusion coefficient and hydromechanical radius of macromolecule in solution. Since 1980s, the technique has been used to study the single cell. In 1983, Nishio et al had established a set of micro quasi-elastic laser scattering (MQLS), and performed the microscope quasi-elastic laser scattering measurement on single intact red blood cell. Later in 1987, Blank et al had also established another kind of MQLS, and measured the erythrocytes of several kinds of mammal animals. They obtained similar conclusions and suggested that the light scattering of the erythrocyte come mainly from the motion of its membrane. In our microscope laser light scattering study on single intact red blood cell, there were also evidences suggest that the light scattering of the cell be related to the thermodynamic fluctuation of the cell membrane, and the Brownian motion of the molecules inside the cell. The past studies showed that the bending modulus of cell could be determined by analyzing the fluctuations of membrane of cell, which is caused by the flicker phenomena of the membrane. For the scattering of cell, the fluctuations of the membrane must result in the fluctuations of the intensity of the scattering light. In our present study, the relative fluctuations of scattering light can be get from the auto-correlation function (ACF) by theoretical analysis to the correlation function. Then a suitable scattering model of cell was constructed, and the formula between the relative fluctuation of scattering light and the fluctuations of membrane. It makes it possible to measure the bending modulus of membrane of cell. In our experiment, the bending modulus of erythrocyte has been measured, kc =2.730.93 (10
19 J). The value coincides with those obtained by other methods, such as micropipette and flicker fast image methods. And our method shows advantages in non-invasive, real time and in situ measurements over the other methods. K: micro quasi-elastic laser light scattering; erythrocyte; auto-correlation function; flicker phenomenon; bending modulus.

Cell Biology International, Vol. 25, No. 10, 2001

OXYTOCIN (OT) STIMULATED INTRACELLULAR CALCIUM [Ca2+ ]i OSCILLATIONS IN MOUSE ENDOMETRIUM IS REQUIRED FOR THE SYNTHESIS OF CYCLOOXYGENASE (COX) AND THE RELEASE OF PROSTAGLANDIN F-2ALPHA (PGF2
) L. S. Ho and H. C. Chan Epithelial Cell Biology Research Center, Department of Physiology, Basic Medical Sciences Building, Chinese University of Hong Kong

Oxytocin (OT) induced intracellular calcium [Ca2+ ]i influx in mouse endometrium was observed using Fura-2AM fluorescent dye by spectroflurotometry. The effect of OT was compared in the endometrium of immature (3 weeks) and adult mice (8 weeks). OT stimulation showed similar [Ca2+ ]i responses in the endometrium of immature and adult mice, but these responses were sensitive to the extracellular environment, depending whether the medium were Ca2+ free or that contains Ca2+ . OT was added to the endometrial cells at 50 nM, 200 nM and 600 nM and incubated at 37C for 5 minutes. In immature mice, OT stimulation showed no changes in Ca2+ influx of Ca2+ free medium at 50 nM and 200 nM, but at 600 nM, there was an increase of 6.4% Ca2+ influx. In Ca2+ containing medium, OT at 50 nM showed a 5.5% of Ca2+ influx, at 200 nM (48%), at 600 nM (26%). This showed that endometrial cells respond greatest to OT at 200 nM and that increasing the concentration of OT does not further increase Ca2+ influx in the endometrium of immature mice. In adult mice, 50 nM is sufficient to elicit Ca2+ influx in both Ca2+ free and Ca2+ containing environment, with a greater response in the Ca2+ containing medium. The Ca2+ influx pattern in mouse endometrial cells differ between the Ca2+ free and Ca2+ containing suspension. In Ca2+ free suspension, OT induced a sharp peak that gradually falls close to its base level. In Ca2+ containing suspension, OT induced a sigmoid-like curve that gradually increases before levels off. Preincubation of endometrial cells with verapamil (150 M), an L-type calcium channel antagonist, or nickel chloride (NiCl2) (3 mM), a non-selective Ca2+ channel blocker, attenuated OT response in both Ca2+ free and Ca2+ containing medium. OT is believed to stimulate the release of prostaglandin F-2
(PGF2
) in endometrial cells via the G-protein-dependent inositol (1,4,5)-trisphosphate-diacylglycerol (IP3) second messenger system, which mobilizes [Ca2+ ] and activates protein kinase C (PKC). The synthesis of PGF2
requires the enzyme cyclooxygenase (COX), which exists as COX-1 or COX-2. Exactly how OT stimulates PGF2
secretion is unknown, but the pathway is believed to be dependent on IP3-PKC-stimulated intracellular Ca2+ and the influx of extracelluar Ca2+ . Here we investigated the role of Ca2+ in PGF2
production by using two COX inhibitors, indomethacin and piroxicam, to block the synthesis of PGF2
. Pre-incubation of indomethacin (100 M) or piroxicam (100 M) before the addition of OT (50 nM) and a boost of external Ca2+ (2 mM) did not show any changes to the levels of [Ca2+ ]i compared to controls. This result suggested that OT induced-[Ca2+ ]i influx must be involved prior to the formation of COX and PGF2
because [Ca2+ ]i influx was unaffected.

SELECTED MUTATIONS OF THE
5 HELIX OF G
16 IMPROVES THE COUPLING OF Gi-LINKED RECEPTOR Maurice K. C. Ho, Jasmine H. P. Chan, Cecilia S. S. Wong and Yung H. Wong Department of Biochemistry, Molecular Neuroscience Center and Biotechnology Research Institute, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China

A broad repertory of G protein-coupled receptors (GPCRs) shows effective coupling with the hematopoietic G16 protein. Modification of the carboxyl terminal portions of the
subunit of G16 (G
16) promoted its coupling to a number of GPCRs that originally have negligible interaction with G16. In this study, three particular amino acids (glutamine-350, lysine-357 and leucine-364) on the carboxyl terminal
5 helix of G
16 were selected and examined for their contribution in defining receptor-coupling specificity. These three residues are relatively conserved within but diverse between the

Cell Biology International, Vol. 25, No. 10, 2001

1055

subfamilies of cloned G protein a subunits and on the exposed side of a
helix, which may confer to the specificity of receptor coupling. Mutants with one to these three residues converted into the corresponding ones of Gaz were generated and studied for their coupling to a series of Gi-linked GPCRs. Mutation of leucine-364 of G
16 into isoleucine (L364I) resulted in marginally observable enhancements on receptor coupling, but not when glutamine-350 and lysine-357 were mutated into serine and aspartate, respectively (Q350E and K357D). Cooperative enhancements of the receptor coupling efficiencies were observed by incorporating either Q350E or K357D into the L364I mutant, and further functional improvements were seen when all three residues were mutated simultaneously. Our results suggested that the identities of the residues spreading along the exposed side of the
5 helix of G
16 are essential for the determination of receptor coupling specificity.

HYPOSOMATICALLY ACTIVATED Cl
CHANNELS IN THE RABBIT CORNEAL ENDOTHELIUM Huang Fuchun, Wang Zidong and Fang Ruiqi Department of Physiology, Medical College, Jinan University, Guangzhou 510632, China

Evidence for the Cl
channel in the rabbit corneal endothelial cell is lacking. Isolated rabbit corneal endothelia were mounted in Ussing chambers to study the effect of hyposmotic solution on Cl
transport in the corneal endothelium. The rabbit corneal endothelium was perfused with normal HCO
. Ringer solution had a small transendothelium potential difference (PD) of 0.430.01 mV (apical side negative), a short circuit current (Isc) of 4.450.21 A/cm2, and a transendothelium resistance (R) of 98.993.33 W/cm2. A transient reverse of PD was observed when bathing solution was exchanged with low NaCl (isosmotic solution, 53% NaCl of the normal). After recovered and sustained, the PD and R were both higher than that of before, but the Isc was lower. The corneal endothelium pretreated with hyposmotic solution had a lower PD and Isc than that of the un-pretreated, when both were perfused with the low NaCl solution. A transient increase of Isc (max. at 2–3 min, recovery at 7–10 min) was observed when the isosmotic solution (310 mOsm) was washed with the hyposmotic solution (230 mOsm or 190 mOsm). The 190 mOsmstimulated increase of Isc was more noticeable than that of the 230 mOsm. 9-AC (9-anthracenecarboxylic acid, 0.2 mM), SITS (4acetamide-4 -isothiocyanostilbene-2,2 -disulfonic acid, 1 mM) added to the basolateral side abolished the transient increase in Isc, but amiloride (0.6 mM), bumetainde (0.1 mM), ionomycin (0.5 M) had

no effect. Total replacement Cl
by SO2
in the 190 mOsm solution 4 also produced a transient increase of Isc, but the PD declined to zero gradually. Added 9-AC (0.2 mM) to the basolateral side completely abolished the PD and Isc when isosmotic solution was exchanged with the Cl
free solution. It is concluded that the low NaCl solution can affect the ion transport in the corneal endothelium, and the transient increase of Isc may indicate a transient Cl
(and K + ) current. We propose that hyposmotically activated Cl
channels may exist in the basolateral membrane of the rabbit corneal endothelial cell, and the activation pathway for Cl
conductance does not apparently involve [Ca2+ ]o.

SIMULTANEOUS STRUCTURE AND FUNCTION MEASUREMENT ON SINGLE INTACT CELL Yao-Xiong Huang Institute of Biomedical Engineering, Ji Nan University, Guang Zhou 510632, China

The purpose of this paper is to perform simultaneous structure and function measurement on single intact cell, for investigating the correlative variation of the structure and functions of intracellular molecules, cell membrane and cell morphology with physiological and biochemical conditions, using the technique of microscope laser light scattering spectroscopy and image analyzing. The membrane deformability and the intracellular hemoglobin aggregate as well as cell morphology were determined by a microscope laser light scattering spectroscopy and image analyzing system on single intact red blood cell. The diffusion coefficient, the hydrodynamic radius of the hemoglobin aggregates, and the size distribution of the aggregates; the dynamic parameters of the cell membrane such as its deformability, rigidity, elastic modular; and the cell morphology (such as the cell size, section area, perimeter, circularity, largest axis and width, mean gray, volume, and surface curvature) was measured simultaneously as functions of temperature, osmotic pressure, oxygenated state and age. Table I and Table II list the section area a, height fluctuation d, bending modulus kc, the dynamic parameter  of the cell membrane, and the hydrodynamic radius R of the hemoglobin aggregates, as functions of osmotic pressure and oxygen. Accompanied with others as function of temperature and age show that the three level’s parameters are really correlated with each other in some extent, when changing with physiological and biochemical conditions. As the technique of microscope laser light scattering spectroscopy and image analyzing can determine all the parameters in cellular,

Table 1. The section area a, height fluctuation d and bending modulus kc vs. osmotic pressure  (xs)
mOsm/kg H2O

176

238

288

365

430

d (10
9 m) kc (10
19 J) a (m)2

98.612.3 2.400.33 50.086.09

114.113.8 1.790.46 40.485.28

120.512.8 1.610.38 36.435.63

104.112.5 2.150.30 32.946.28

94.713.2 2.600.43 30.235.37

Table 2. The dynamic parameters for intracellular molecule and membrane, accompanied with the size and regular factor (REF) of normal and anemia RBC as a function of oxygen Normal RBC

Oxygenated Deoxygenated

Anemia

 (Hz)

2R (10
9 m)

 (Hz)

5116 4921

28 7 3210

4419 3521

Normal RBC

Anemia

2R (10
9 m)

Area (10
6 m2)

REF

Area (10
6 m2)

REF

3414

40.133.51 57.706.53

0.800.02 0.830.03

28.097.22 37.215.95

0.770.04 0.780.03

1056

Cell Biology International, Vol. 25, No. 10, 2001

sub-cellular and intracellular levels simultaneously on a living cell, it is believed to be very helpful to reveal the relation between the structure and functions of a cell, and very useful in many aspects such as cell engineering, pharmacology, and epidemiology.

is involved in regulation of NO/cGMP sensitive store-operated Ca2+ entry and metastasis in human hepatoma cells. K: HAb18G/CD147; store-operated Ca2+ entry; nitric oxide; cGMP; hepatoma cell.

HUMAN CEA EXPRESSED BY pcDNA3-CEA PLASMID IN BONE MARROW-DERIVED DENDRITIC CELLS EFFECTIVELY PREVENTS CT26 (hCEA + ) IN MICE Huang Youtian and Dong Ziming Department of Pathophysiology, Zhengzhou University Medical School, Zhengzhou 450052, China

Rac AND Cdc42-DEPENDENT REGULATION OF c-Jun N-TERMINAL KINASE BY THE OPIOID RECEPTORS Angel Y. F. Kam and Yung H. Wong Department of Biochemistry, Molecular Neuroscience Center, and Biotechnology Research Institute, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China

The objective of this study is to prepare the vaccine of DCs transfected with pcDNA3-CEA, and to observe the immunity effects of the DCs(pcDNA3-CEA) inoculate against to CT26(hCEA + ) loaded in BALB/c mice. Method: (1) DCs were generated from bone marrow in the presence of rmGM-CSF and rmIL-4, and then, (2) using lipofectamine, transfected with a recombinant pcDNA3-CEA encoding neo gene, to prepare DCs vaccine. (3) While, CT26 were transfected with pcDNA3-CEA too, to prepare CT26(hCEA + ), the cell line of target tumour. (4) Vaccination with the CEA gene-modified DC, the survival time of the BALB/c mice challenged with CT26(hCEA + ), and compared with that of other DCs vaccinate. Results: G418 test showed that about 10% DCs were transfected with pcDNA3-CEA. Vaccination with those genetically modified DC could increase the expression of CEA and induce CEA specific CTL most significantly. After vaccination with the CEA gene-modified DC, the survival time of the mice challenged with CT26(hCEA + ), 6 weeks later was prolonged more potently than that of the mice vaccinated with other DCs. No protective challenged effect was observed in the mice with wild-type CT26 cells. Conclusion: The results demonstrated that specific antitumour immune responses could be induced efficiently by vaccination with tumour antigen gene modified DCs. K: dendritic cell; carcinoembryonic antigen; plasmid; transfection.

INVOLVEMENT OF HAb18G/CD147 IN REGULATION OF STORE-OPERATED CALCIUM ENTRY AND METASTASIS OF HUMAN HEPATOMA CELLS Jianli Jiang1,2, Qing Zhou1, M. K. Yu1, Zhinan Chen2 and Hsiao Chang Chan1 1 Epithelial Cell Biology Research Center, Department of Physiology, Faculty of Medicine, and Chinese University of Hong Kong, Shatin, NT, Hong Kong; 2Cell Engineering Research Center, The Fourth Military Medical University, Xi’an, China

Hepatoma-associated antigen HAb18G (homologous to CD147) is a potential adhesion molecule that has been implicated in the metastasis of hepatoma cells. However, the intracellular signalling mechanism involved has not been elucidated. The present study examined the effect of HAb18G/CD147 on the nitric oxide (NO)/cGMP-regulated Ca2+ mobilization and investigated the possible involvement of the Ca2+ signalling pathway in metastatic process in human hepatoma cells. HAb18G/CD147 cDNA was transfected into human 7721 hepatoma cells to obtain a cell line stably expressing HAb18G/CD147, T7721, as demonstrated by northern blot and immunocytochemical studies. 8-Bromo-cGMP inhibited the thapsigargin-induced Ca2+ entry in a concentration-dependent manner in 7721 cells with complete abolishment at 2 mM. However, expression of HAb18G/CD147 in T7721 cells decreased the inhibitory response to cGMP with only 25.313.1% inhibition observed at 2 mM. Similar concentration-dependent inhibitory effect on the Ca2+ entry was observed in 7721 cells in response to a NO donor, SNAP, 48.313.0% inhibition at 100 M. The inhibitory effect of SNAP on the thapsigargin-induced Ca2+ entry was significantly reduced in HAb18G/CD147-expressing T7721 cells, 19.84.1% inhibition at 100 M, indicating a role of HAb18G/CD147 in NO/ cGMP-regulated Ca2+ entry. HAb18G/CD147-expressing T7721 cells attached to the matrigel-coated culture plates and invaded through matrigel-coated chambers at rates significantly greater than that observed in 7721 cells, which could be suppressed by SNAP and reversed by NO inhibitor, L-NAME. These results suggest that HAb18G/CD147

Opioids are the most valuable painkiller in contemporary medicine and exert their biological actions by binding to the three opioidreceptor classes, µ, and  Opioid receptors belong to members of Gi-coupled receptors and it has recently been shown that activated opioid receptors can regulate Mitogen-Activated Protein Kinases (MAPKs). MAPKs can be classified into at least three subgroups including Extracellular signal-Regulated Kinases (ERKs), c-Jun NH2terminal Kinases (JNKs) and p38 MAPK. We examined whether -opioid receptors (DOR) can activate JNK using a transient expression system in COS-7 cells. Our results demonstrated that DOR significantly activated JNK upon stimulation with their specific agonist [D-Pen2, D-Pen5]enkephalin (DPDPE). Increased JNK activity was primarily mediated by pertussis toxin-sensitive Gi proteins. This DOR-induced JNK activation was dose-dependent on the concentration of DPDPE. Moreover, the maximal JNK activity was observed at 15 minutes of the drug treatment. On the other hand, DPDPE-induced JNK activation was inhibited by transducin that sequesters endogenous G
subunits. Furthermore, we investigated the involvement of the small GTP-binding proteins in JNK activation mediated by DOR. Expression of dominant negative mutants of Rac and Cdc42 completely blocked this JNK activation. In contrast, DOR still elicited JNK activation after cotransfection of dominant negative mutants of Ras and Rho. Thus, these findings indicate that Rac and Cdc42 might play a more important role in DOR-mediated JNK activation. This work was supported by a grant from the Research Grants Council of Hong Kong (HKUST 6115/00M and 2/99C) to Dr Y. H. Wong.

HELICOBACTER PYLORI-INDUCED APOPTOTIC SIGNALLING IN HUMAN GASTRIC EPITHELIAL CELLS Sang Hui Chu, Joo Weon Lim, Hyeyoung Kim and Kyung Hwan Kim Department of Pharmacology, Brain Korea 21 Project for Medical Sciences, Yonsei University College of Medicine, Seoul 120-752, Korea

Helicobacter pylori (H. pylori) evokes gastric epithelial cell damage. Although clinical meaning of H. pylori-induced cell death is important, its molecular mechanisms are still unknown. The present study examined the role of oxidant-sensitive transcription factor NF-B and signalling pathways in H. pylori-induced cell death in human gastric epithelial cells. Human gastric epithelial cells (AGS) were stimulated with H. pylori (NCTC11637) in antibiotic-free culture medium for 24 h (bacterium:cell—300:1). Cell death was measured as viable cell counting. Apoptosis was determined using Hoechst staining and DNA laddering. To investigate the signalling pathway of apoptosis, protein levels of bcl-2 and bax were determined by western blot analysis. The relationship between NF-B and apoptosis was investigated after treatment of phosphorothioate antisense oligonucleotide (AS) for the p50 subunit of NF-B to gastric AGS cells. As a result, H. pylori induced apoptosis in gastric AGS cells. H. pylori-induced apoptosis was accompanied with the decrease of bcl-2 protein and the increase in bax protein. H. pylori-induced apoptosis was prevented in the cells treated with AS for the p50 subunit of NF-B. In conclusion, oxidant-sensitive transcription factors such as NF-B might be a critical step in regulating H. pylori-induced apoptotic signalling in human gastric epithelial cells. This study was supported by a grant (HMP-00-CH-02-0002) from the Korea Ministry of Health and Welfare.

Cell Biology International, Vol. 25, No. 10, 2001

DIFFERENTIAL EFFECT OF THAPSIGARGIN ON APICAL AND BASOLATERAL ATP-RELEASABLE CALCIUM POOLS IN POLARIZED EQUINE SWEAT GLAND EPITHELIA C. H. Y. Wong and W. H. Ko Department of Physiology, The Chinese University of Hong Kong, Shatin, Hong Kong, China

In this study, a technique which allows us to monitor nucleotideevoked short-circuit current (ISC) and [Ca2+ ]i concurrently in a polarized epithelium was employed (Ko et al., 1999; Wilson et al., 1998). Previous studies have shown that the equine sweat gland epithelia will form polarized monolayer on permeable support and express multiple P2Y receptors (Ko et al., 1997; Wilson et al., 1998). Moreover, apical and basolateral ATP releases calcium from two separate stores. Only calcium released from the apical ATP-sensitive store activates a substantial increase in ISC, which is mainly due to Cl
secretion. Apically applied ATP, however, partially releases Ca2+ from the basolateral ATP-sensitive calcium pool but not vice versa (Wong and Ko, 2000). The aim of this study was to examine the effect of thapsigargin (Tg), a sacro/endoplasmic reticulum Ca2+ -ATPase (SERCA) pump inhibitor, on apical and basolateral ATP-evoked [Ca2+ ]i signalling and anion secretion in equine sweat gland epithelia. Cells were grown on Transwell-Col membranes for 4 days using standard culture techniques. Confluent monolayers were clamped in modified Ussing chamber and perfused bilaterally in normal Krebs solution containing 2.5 mM Ca2+ . Thapsigargin and/or ATP were then delivered to either apical or basolateral aspect of the epithelia under Ca2+ -free condition to characterize the nature of the internal Ca2+ stores. Experiments were carried out to test whether Tg could access all internal Ca2+ pools including the ATP-releasable Ca2+ stores. Results indicated that the thapsigargin-releasable calcium pool and the apical ATP-releasable calcium pool were partially overlapped. However, the basolateral ATP-releasable calcium pool appeared not to be affected by Tg and apical ATP. Data therefore suggests that Tg may release Ca2+ mainly from the apical ATP-sensitive Ca2+ pool. In addition, emptying the apical or basolateral ATP-releasable pools triggered a store-operated Ca2+ influx predominately through the basolateral membrane. The effect of apical and basolateral ATP on Ca2+ influx was not additive, suggesting that the two separate pools were coupled to same calcium entry channel/pathway. References K WH, L VW, W HY, W SM, 1999. The simultaneous measurement of epithelial ion transport and intracellular free Ca2+ in cultured equine sweat gland secretory epithelium. J Membr Biol 170: 205–211. K WH, W SM, W PYD, 1997. Purine and pyrimidine nucleotide receptors in the apical membranes of equine cultured epithelia. Brit J Pharmacol 121: 150–156. W SM, L VW, P JD, A EA, W G, K ZE, K WH, 1998. Nucleotide-evoked calcium signals and anion secretion in equine cultured epithelia that express apical P2Y2 receptors and pyrimidine nucleotide receptors. Br J Pharmacol 124: 832–838. W HY, K WH, 2000. Calcium-dependent and -independent mechanisms of P2Y receptor regulated anion secretion in polarized epithelia. J Korean Med Sci 15(Suppl, S63–S64.

ROLES OF CTX AND APAMIN SENSITIVE KCa CURRENTS IN ENDOTHELIAL CELL HYPERPOLARIZATION AND ENDOTHELIUM-DEPENDENT RELAXATION IN RABBIT AORTA Xingya Kuang1,2, Willmann Liang1, Ismail Laher1, Cornelis van Breemen1 and Xiaodong Wang1 1 Department of Pharmacology & Therapeutics, University of British Columbia, Vancouver, Canada; 2Department of Occupational Diseases, Yang-Pu District Central Hospital, Shanghai, China

Smooth muscle tone is modulated by a variety of vasoactive factors released by endothelial cells including nitric oxide (NO), prostagland-

1057 ins, and/or an endothelium-dependent hyperpolarization factor (EDHF). While the function of NO and prostacyclin have been known for more then 20 years, the properties and identity of EDHF remain elusive. Recent evidence suggests that charybdotoxin (CTX) and/or apamin sensitive Ca2+ activated K + channels play important roles in the response to EDHF after inhibition of NO and prostacyclin synthesis. In this study we characterized ACh-induced, non-NO, nonprostacyclin mediated relaxation in the intact rabbit aorta and investigated the participation of KCa in such responses. In addition, using the patch-clamp technique in freshly isolated rabbit aortic endothelial cells, we examined the role of KCa channels in ACh and CPA induced membrane hyperpolarization. The results shown that after inhibition of NO and prostacylin production using L-NAME (20 to 200 M) and Indomethancin (10 M), ACh (10 M) induced vasodilation was reduced to 20.50.2% of the control. Pretreatment of the aorta with 2 M bradykinin potentiated the subsequent ACh response to 400.8% of control. Addition of CTX (4 M) and apamin (30 M) abolished the remaining relaxation. In isolated endothelial cells, Ach (10 M) induced transient membrane hyperpolarization (from
359 mV to
785 mV). This transient hyperpolarization is converted to a sustained response following pretreatment with either bradykinin or the SERCA blocker cyclopiazonic acid (CPA, 15 to 30 M). Pretreatment with either CTX or apamin abolished the maintained phase of the hyperpolarization response, while the maxi K channel inhibitor Iberiotoxin or opener NS1619 had no effect on membrane potential in these cells. In conclusion, the intermediate conductance (CTX sensitive) and the small conductance (apamin-sensitive) KCa channel mediate Ach induced endothelial membrane hyper-polarization, which is responsible for non-NO, non-prostacyclin mediated vasodilatation (e.g. EDHF type response) in the rabbit aorta. This study was support by HSFC (XW, IL, CvB) and Yang-Pu District Central Hospital, Shanghai (XK).

REGULATION OF INSULIN-LIKE GROWTH FACTOR-1 AXIS BY DIETARY PHOSPHATE DEPRIVATION IN ADULT RATS Wan-Ping Lai1, Tsui-Shan Chau1, Samuel C. L. Lo1, Meng-su Yang2 and Man-Sau Wong1 1 Open Laboratory of Asymmetreic Synthesis, Department of Applied Biology and Chemical Technolgy, Hong Kong Polytechnic University, Kowloon, Hong Kong; 2Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chi Avenue, Kowloon, Hong Kong

Dietary phosphorus deprivation alters the signalling protein expression through IGF-1 axis in adult rats. Previous studies showed low phosphate condition is more responsive to IGF-1 stimulation than the normal phosphate condition. Studies were undertaken to determine whether LPD causes the alteration of the signalling proteins expression along the IGF-1 signalling pathway in the renal proximal tubules cell. Here we have examined the alteration of the protein expression of renal IGFI-R, IRS1, Shc, IR during 0, 1, 2 ,3, 5, 7 days of LPD (0.1% P) of adult rats (12–14 weeks). Serum chemistries on both phosphorus and calcium were done and detected by the kits from Sigma. The rat proximal tubule cells were harvested and were lysed by extraction buffer, supplemented with protease inhibitors. The cell lysate was resolved in the SDS-PAGE and immunodetected by the western blotting. The signal was visualizad by the Lumi-Imager. The serum phosphorus was decreased significantly in day 1 (3.20.58 mg/dL), P>0.005; day 3 (3.800.38 mg/dL), P>0.01; day 5 (3.190.75 mg/ dL), P>0.005 and day 7 (4.200.46 mg/dL), P>0.05 when compared with day 0 (6.620.53 mg/dL). There was a constant serum calcium content trend throughout 7 days of LPD, except in day 2 (11.770.19 mg/dL) when compared with that of day 0 (9.9360.38 mg/dL) (P<0.05). Results showed a fivefold (P<0.005) increase in renal IGF-1 receptor protein during 3 and 5 days of low P diet treatment compared with rats fed a normal P diet (0.65% P), and remained threefold (P<0.005) increase through 7 days. Similarily, expression pattern of renal IR was found upregulated in day

1058 3 and day 5. Both the IRS1 and Shc also showed an up-regulation through 3 and 5 days low P treatment, but their basal levels were far less than that of the IGF-1 receptor. Data were analysed by the one-way ANOVA software and the Tukey’s posttest. These findings might explain why LPD is more responsive to IGF-1 than normal phosphate condition, as more IGF1-receptor expressed responds to IGF-1. Later on, we will try to stimulate the renal cell with IGF-1 to see the phosphorylation level of the protein along IGF-1 axis.

BILE INDUCES Ca2+ -DEPENDENT CELL DAMAGE IN RAT PANCREATIC ACINAR CELLS Joo Young Kim, Jin Ah Lee and Min Goo Lee Department of Pharmacology and Brain Korea 21 Project for Medical Sciences, Yonsei University College of Medicine, Seoul 120-752, Korea

Acute pancreatitis is a disease with a relatively high morbidity and mortality. About 25% of patients need intensive care support, which becomes a significant burden for health service systems in most developed countries. Although cholelithiasis is highly associated with acute pancreatitis in clinical situations, it is not clear how gallstone causes the disease. In the present study, we found that the low concentration of bile acids, of which concentration may be present in pancreatic tissue either by simple luminal diffusion or by interstitial leakage when the lower biliary tree is obstructed, causes pancreatic cell damage by deteriorating intracellular Ca2+ -signalling. These constitute the first evidence that pancreatic acinar cells have multi-specific bile uptake transporters in both luminal and basolateral membranes, transported bile acids caused a sustained [Ca2+ ]i elevation, and they cause pancreatic cell damage by Ca2+ -dependent manner. The present findings may have important implications not only in understanding the pathophysiologic mechanism of pancreatitis, but also in finding applicable therapeutic targets for bile-associated pancreatic disorders.

CHANGES OF PLACENTAL AT1 RECEPTOR IN PATIENTS WITH PRE-ECLAMPSIA P. S. Leung1, S. Y. Lam1, T. P. Wong1, S. J. Tsai2, T. N. Leung3 and T. K. Lau3 1 Department of Physiology, Faculty of Medicine, Chinese University of Hong Kong, Hong Kong; 2Department of Physiology, National Cheng Kung University Medical College, Taiwan; 3Department of Obstetrics and Gynecology, Prince of Wales Hospital, Hong Kong

The human placenta has been believed to possess its own reninangiotensin system (RAS), which may play a role in regulating the utero-placental blood circulation. The changes of such a placental RAS during pregnancy could be important for the physiological and pathophysiological aspects of some clinical disorders such as pre-eclampsia, a pregnancy-induced hypertension. In our present investigation, the alterations of expression and localization of placental angiotensin II receptor subtypes, namely AT1 in patients with preeclampsia (elective caesarean delivery) were examined and compared with its controls (vaginal delivery and elective caesarian delivery) using semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), Western blot and immunohistochemistry respectively. RT-PCR analysis revealed an upregulation of placental mRNA for AT1 receptor subtype in patients with pre-eclampsia when compared with that in the controls. Moreover, there was a significant activation of angiotensinogen mRNA in preeclamptic placenta. Western blot analysis showed that the expression of AT1 receptor was consistently upregulated. Immunohistochemistry further demonstrated that enhanced immunoreactivity for placental AT1 receptor was predominantly localized to the thin layers of syncytiotrophoblasts and, to a less extent, the capillaries of the term placental villi. All these data indicate that changes of placental RAS components, notably AT1 receptor in the syncytiotrophoblasts, could have a pathophysiological role in patients with pre-eclampsia.

Cell Biology International, Vol. 25, No. 10, 2001

A NEW CYTOKINE: A POSSIBLE EFFECTIVE PATHWAY OF METHIONINE ENKEPHALIN Li Gang2, Huang Dong-Ai, Yang Fei-Yi, Liu Xin-Hua, Wang Dan and Li Ping-Feng2 The Department of Biochemistry and Molecular Biology, Health Science Center, Peking University, Beijing 100083, China

The aim of this study is to investigate the effects of methionine enkephalin on signal transduction of mouse myeloma NS-1 cells in this experiment. The method we used are the antigen determinate of delta opioid receptor was designed in this lab and the polypeptide fragment of antigen determinate with 12 amino acids residues was synthesized. Monoclonal antibody against this peptide fragment was prepared. Proliferations of Mouse NS-1 cells treated with methionine enkephalin of 110
6 mol.L
1 were observed. The activities of protein kinase A (PKA) and protein kinase C (PKC) were measured and thereby the mechanism of effect of methionine enkephalin was postulated. The results demonstrated that methionine enkephalin could enhance the proliferation of NS-1 cells and the effect of methionine enkephalin could be reversed by monoclonal antibody. The activities of PKA were increased in both cytosol and membrane. With reference to PKC, an opposite result was observed in cytosol and membrane. The activities of PKC in cytosol was elevated at 110
7 mol/L of methionine enkephalin and then declined gradually as the concentration was raised. An opposite result was exhibited in membrane. The effects of methionine enkephalin might be reversed by both naloxone and monoclonal antibody. In Conclusion, it indicated that the signal transduction systems via PKA and PKC were involved in the effects of methionine enkephalin by binding the traditional opioid receptors, and therefore resulted in different biological effects.

ISOLATION AND IDENTIFICATION OF TWISTED GASTRULATION PROTEIN GENES IN HUMAN TESTES AND MOUSE TESTES BY CDNA MICROARRAY Li Jianming, Zhou Zoumin and Sha Jiahao Kay Laboratory of Reproductive Medicine Nanjing Medical University, China

A number of specific genes are expressed restrictive in space-time in spermatogenesis. Spermatogenesis occurs in successive mitotic, meiotic and post-meiotic in all animals. Genes expression of development genetics in spermatogenesis has been the evolutionary conservation of molecular mechanisms and homologous molecular. Human full-length twisted gastrulation protein complementary DNA (hTSG cDNA) related to bone morphogenetic protein have been isolated by using testes cDNA library microarray with cDNA hybridization probes from total mRNA of testes of adult, six-month embryo, one-week mouse and four-weeks mouse. The hTSG cDNA encoding a novel protein that contains xTSG modif was isolated by is cDNA microarray from human testis library. This cDNA clone consists of 1934 nucleotides and encodes an open reading frame of a 223 amino acid protein that shares similarities with the cysteine-rich domains of Chordin, and is part of the BMP synexpression. hTSG was more highly expressed in adult testes compared with fetal testis, and in 4 week-old testis compared with 1 week-old testis by cDNA microarray. The mouse TSG (mTSG) full-length cDNA with mouse expression sequence tags (EST) was obtained from mouse testis by polymerase chain reaction with reverse transcription (RT-PCR). In situ hybridization showed that is lower expression in spermatogonia and higher expression in spermatocytes and spermatid, and mTSG compared with BMPs are expressed in similar pattern in male germ cells. It was suggested that TSG is associated with process of spematogenesis.

GENE CLONING, EXPRESSION AND BIOLOGICAL CHARACTERS FOR HUMAN TISSUE INHIBITOR OF MATRIX METALLOPROTEINASE 1 Keqin Li1, Fang Tian1, Fengjun Xiao1, Yun Zhao2, Hong Zhang3 and Chunhai Li1 1 Department of Tumor Oncology, Institute of Beijing Basic Medicine; 2Department of Gynaecology, Peking University

Cell Biology International, Vol. 25, No. 10, 2001

affiliated People Hospital; 3Department of Gynaecology and Obstetrics, Military General Hospital, Beijing, China

MMP and TIMP form noncovalent complex by the ratio of 1:1, the high ratio of MMP/TIMP is closely related to the occurrence of tumour metastasis. Human TIMP-1 natively is a glycosylated protein, and can The invasion and metastasis of malignancy tumour cells is one of the important cause for the cancer patient’s death, and it’s very significant to seek for the tumour metastasis related factors and according to them to design anti-cancer drugs as well as gene therapy. Tumour cell’s invasion and metastasis require proteolytic enzymes to hydrolyze the excellulary matrix, Matrix Metalloproteinases (MMPs) are the key proteinases be involved in these process. There are native inhibitors called tissue inhibitors of matrix metalloproteinases (TIMPs) in human tissues, especially binding to the type IV collagenase B (MMP9); type IV collagenases are the only type of proteinase that can hydrolyse the skeletal structure of excellulary matrix (three helix structurally type IV collagens), so they play a very important role in the tumour metastasis. To investigate the tumour related biological characters of TIMP-1 will be help to the anti-cancer invasion drug’s design and development. This paper reports the cloning, expression and purification of human TIMP-1 in yeast system, as well as the biological characters study of rTIMP-1. (1) The TIMP-1 gene’s encoding sequence was cloned by PCR, and it was been constructed as pPIC9-T1 expression vector. The vector was transformed to Pichia Pastoris yeast system, and the secreted rTIMP-1 was gotten; content of the interested protein is 40 mg/L, 60% of the total proteins is the interested protein. (2) By the Preparation High Pressure Liquid Chromatogram (FPLC) protein purification system, the crud were purified by desalting, anion/cation exchange and gel-filtration steps, the purity of the rTIMP-1 is more than 95% showed by SDS-PAGE silver stain. (3) By the method of western blot, reverse zymogram and ‘hyperiodic acid–schiff reagent’, it is proved that rTIMP-1 has the same inhibit MMP activity as the native TIMP-1, also the rTIMP-1 is glycosylated as same as the native protein. (4) By the method of in vitro invasion and chicken chorioallantoic memberane (CAM) model, it is proved that the rTIMP-1 has the activity of inhibit tumour in vitro invasion and has the ability of anti-angiogenesis. In conclusion, our recombined TIMP-1 protein is high yield, high stability and high biological activity same as the native protein. Our results of rTIMP-1 are the highest yield and activity as we known. Because of the TIMPs’ unique ability of anti tumour metastasis and anti tumour angiogenesis, this report is very significant to investigate tumour happening and processing. It is also practicable for the development of anti-tumour drugs.

STUDY OF GENE EXPRESSION LEVEL IN OVARIAN CANCER AND TISSUE DISTRIBUTION OF A NOVEL MATRIX METALLOPROTEINASE-MMP23 Ke Qin Li1, Ya Li Li2, Yun Zhao2, Hong Zhang2, Duan Qiang Pei3, Chun Hai Li1 1 Department of Tumour Molecular Biology, North Taiping Hospital for the Beijing Institute of Basic Medical Science, Beijing (100850), P. R. China; 2Department of Obstetrics and Gynecology, 301 Hospital, Beijing (100850), P. R. China; 3 Department of Pharmacology, University of Minnesota, 6-120 Jackson Hall, Minneapolis, MN 55455, U.S.A.

Matrix metalloproteinases (MMP) are involved in a range of biological processes such as signal transduction, tumour invasion, angiogenesis and the turnover of extracellular matrix (ECM). In order to find the relation between the MMPs genes expression and the ovarian cancer progress, we investigate the gene expression of a newly discovered and reproductive related gene- MMP23 in the ovarian cancer tissues. Firstly, we extracted the total RNA of ovary tissue (normal and cancer), human ovarian and endometrial cancer cell lines, as well as some organs of human embryo tissue. Reverse the mRNA to cDNA with the oligo-dT as 3 primer, PCR the c-terminal half molecule of MMP23, and investigate the MMP23 gene distribution in each tissue. Secondly, we co-amplified MMP23, and investigated the MMP23 gene distribution in each tissue, and semi-quantitatively determinated the expression levels of the MMP23 gene. Furthermore, we collected the

1059 MMP23 fragment from the gel, and inserted it into pGEM-T cloning vector and constructed a pGEM-MP23C plasmid and then sequenced this plasmid with T7 promoter. Results: (1) By the method of semi-quantitative RT-PCR, we found that the newly discovered MMP gene mainly exists in the ovary of normal women (gene expression level is 71.4338.32%), but disappeared or has low expression in the ovarian cancer tissue (28.0531.88%, P<0.001). (2) We further demonstrate that this gene is distributed in some special organs (spleen, colon and kidney) of human embryo stage. (3) The primary results of this gene suggest that it may play an important role in the functions of ovary and embryo development, this gene possibly is the first MMP gene that existed in normal tissue but disappeared or low expressed in the cancer. K: ovarian cancer; MMP23 gene; semi-quantitative PCR; tissue distribution.

STRESS: EFFECT ON LEARNING AND MEMORY Li Pingyang1, Tan Runchu1 and Feng Jian-qiang2 1 Department of Physics, Sun Yat-sen University of Medical University of Medical Science, 2Department of Physiology, Sun Yat-sen University of Medical University of Medical Science

Stress is a necessary survival mechanism, while prolonged stress conditions can have severe impairments in the physiology and psychology. Stress has been shown to have a profound effect on cognitive functioning. It has been the studying focus on the neurobiology mechanism of learning and memory. A great deal of work suggests that stress could act in many ways to affect the process that underlie learning and memory. Stress has been shown to affect synaptic plasticity, dendritic morphology, neurotoxicity and enhanced neuronal cell death. At present, the long-lasting acivity-dependent changes in synaptic efficacy in hippocampus (long-lasting potentiation LTP; long-lasting depression LTD) are the possible neurobiology mechanism of learning and memory. In animal model, stress has shown to impair hippocampal-dependent forms of learning by inhibiting LTP while enhance LTD distinctly. Particularly, glucocorticoid has been shown to have a number of effects on the hippocampus. The hippocampus is enriched with both the high-affinity (type I) mineralocorticoid and the low-affinity (type II) glucocorticoid receptors. The activation of type I receptors increases the magnitude of LTP, while The activation of type II receptors attenuates LTP and enhances the induction of LTD. Behavioral studies in rats have shown that impaired spatial learning is found with selective of type II receptors activation. In addition, glucocorticoid can affect the intrinsic properties of hippocampal neurons (such as prolong the after hyperpolarization of hippocampal neurons by increasing the level of Ca2+ influx). So hippocampal neurons are very sensitive to glucocorticoid because of enriched with type II receptors. The chronic effects of nomal level of glucocorticoid in the blood can make some hippocampal neurons repercussions, death and affect the function of hippocampus. Since the hippocampus take part in the negative feed back regulation of epinephros-hypophysis axis, the impairment of hippocampus might enhance the secretion of glucocorticoid and found vicious circle as time goes on. When the level of glucocorticoid enhanced to an extent, it might affect widespread brain cells by activating type II receptors and increasing Ca2+ influx. So the function of learning and memory might be severely impaired, even inducing Alzheimer’s disease. K: stress; learning and memory; hippocampus.

MECHANISM OF IL-1-INDUCE CALCITONIN GENE-RELATED PEPTIDE (CGRP) RELEASE IN A549 HUMAN PULMONARY EPITHELIAL CELL LINE Li WenJing, Hou LingFei, Cao Ju and Wang Xian Department of Physiology, Peking University 100083, Beijing, China

Calcitonin gene-related peptide (CGRP) is a 37-amino acid neuropeptide that plays an important biological role in cardiovascular, respiratory and digestive systems. Our previous data have shown that rat and human lymphocytes can synthesize CGRP, which provided

1060 and evidence that CGRP may act as a neuroendocrine immune modulator. It is known that CGRP is a mediator that modulates the acute inflammatory response induced by interleukin 1 and results in polymorphonuclear leukocyte migration and edema in lung, skin and colon where are abundant of epithelial cells. However, whether IL-1 can directly induce CGRP release from epithelial cell is still unknown. Therefore, we tested that effect of IL-1 on CGRP release in A549 human pulmonary epithelial cell line. We used RIA technique to detect CGRP release in supernatant and in cytosol, used RNase protection to detect CGRP transcription. The results showed that IL-1 could induce both CGRP release and synthesis in a dose- and timedependent manner. Actinomycin D and cycloheximide markedly attenuated the IL-1 induced and NF-B inhibitor, pyrrolidine dithiolidine dithiocarbamate also can attenuate IL-1 induced CGRP release and expression, where as PKA inhibitor Rp-cAMPS had no effects. These data indicate for the first time that PKC and NF-B pathway, but not PKA pathways are involved in IL-1-induced synthesis and release of CGRP in human pulmonary epithelial cells line (A549). And we present another evidence for CGRP may act as a neuroendocrine immune modulator.

EFFECT OF LOW LEVEL HELIUM-NEON LASER IRRADIATION ON CYTOSKELETON IN RAT LUNG FIBROBLASTS Li Xiao-yuan1, Liu Jian-zhong2, Li Yan2 and Tan Run-chu1 1 Department of Laser Medicine, Sun Yat-sen University of Medical Sciences, Guangzhou, and 2Department of Biology, Sun Yat-sen University of Medical Sciences, Guangzhou, P.R. China

The objective was to study the effect of low lever Helium-Neon laser on cytoskeleton in rat lung fibroblasts at different laser dosages. Primary culture and subculture of rat lung fibroblasts were established in vitro. The lung fibroblasts of third passage were randomly divided into 4 groups, 3 laser irradiation groups and 1 control group, 6 holes each group. The lung fibroblasts of 3 laser irradiation groups were irradiated by He-Ne laser with an output of 20 mW, spot size 1 cm in diameter, 2.5, 5 and 10 min (energy density of 3.8 J/cm2 7.6 J/cm2 15.2 J/cm2), respectively, per day for consecutive 5 days. The lung fibroblasts were stained using Coomassie BB staining technique. The changes of cytoskeleton in lung fibroblasts and the average number of fibroblasts each group were calculated under microscope and compared with that of control group. The results show changes of cytoskeleton were not found after He-Ne laser irradiation 2.5 minutes with a light dose of 3.795 J/cm2. The reduction of cytoskeleton and the increase of cell number were observed after 5 minutes and 10 minutes laser irradiation with a laser does of 7.59 J/cm2 and 15.18 J/cm2 respectively. In conclusion, the result show that the alteration of cytoskeleton and cell number were induced by low power He-Ne laser irradiation.

SIGNALLING OF SHEAR STRESS-INDUCED IL-8 mRNA EXPRESSION IN HUMAN VASCULAR ENDOTHELIAL CELLS Liang Feng1, Huang Ning1, Wang Boyao1, Chen Huaiqing2 and Wu Lizhi2 1 Department of Pathophysiology, Research Unit of Biomedical Engineering, West China Medical Center, Sichuan University, Obengdu 610041, China

It has been demonstrated that Monocytes contribute to the development of atherosclerotic lesions and the lesion development occurs predominantly in regions of the vasculature exposed to low wall shear stress. Although monocytes chemoattractant protein-1 (MCP-1) has a powerful effect on monocytes, it has been described that IL-8 also plays an important role in the monocyte-endothelial interactions under flow conditions. MCP-1 gene has been demonstrated to be upregulated by shear stress. This study is to examine IL-8, TLR-2 and TLR-4 mRNA expression and activation of NF-B in human vascular endothelial cells when exposed to low laminar fluid shear stress. RT-PCR detection indicated that there was a marked increase in IL-8 mRNA expression in HUVECs at 2 h of exposure to laminar flow with 4.2 dyne/cm2 shear stress. NF-B P65 immunocytofluorescent staining

Cell Biology International, Vol. 25, No. 10, 2001 of HUVECs showed that when exposed to the same flow shear stress for 0.5 or 1 h, the cell nuclei became stained, and after 1.5 or 2 h, the staining was very strong. A markedly increased P-IB of the cell lysates, which was detected by Western blot, occurred at 10 minutes of exposure and the blot density almost dropped down to the baseline after 60 minutes of the expose. The density of IB blot dropped down with increasing exposure time. Both RT-PCR detection and Northern blot analysis showed that HUVECs constitutively expressed TLR-2 and TLR-4 mRNA, when exposed to above flow shear stress for 1 hour, TLR-4 mRNA expression was markedly increased. These experiments suggested that the inflammatory Toll/ NFkB signalling pathway would probably be involved in flow shear stress-induced II 8 gene expression.

EFFECTS OF HISTAMINE AND CYCLOPIAZONIC ACID ON INTRACELLULAR CALCIUM IN CULTURED HUMAN VALVULAR MYOFIBROBLASTS Willmann Liang1, Casey van Breemen1, Paul McDonald2, Bruce McManus2 and Xiaodong Wang1 1 Department of Pharmacology and Therapeutics; 2 Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, B.C., Canada

Although valvular myofibroblasts (VMF) are the most predominant cell type in cardiac valves, little is known about how they are regulated by Ca2+ signalling. In this study, the effects of histamine and cyclopiazonic acid (CPA), an inhibitor of sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA), on intracellular Ca2+ were investigated. Myofibroblasts were isolated by enzymatic digestion from a normal female human mitral valve and grown on glass coverslips. Intracellular Ca2+ concentration ([Ca2+ ]i) was measured by fluorescence imaging microscopy of fura2-loaded cells. The result showing that in the presence of extracellular Ca2+ histamine induced a transient increase in [Ca2+ ]i followed by several oscillations in [Ca2+ ]i, which decayed within 200 s. Removal of extracellular Ca2+ did not alter this response and Mn2+ quenching experiments failed to provide evidence for agonist-induced divalent cation entry. Blockade of SERCA by CPA induced a slow transient [Ca2+ ]i increase, which was independent of extracellular Ca2+ . Addition of CPA during the measurement of Mn2+ -quenching of fura2 fluorescence failed to provide evidence for the presence of Store Operated Channels (SOC). When histamine was added to cells pretreated with CPA for 10 minutes in Ca2+ -free PSS it elicited one rapid transient [Ca2+ ]i spike with no subsequent oscillations. In conclusion, histamine causes release of Ca2+ from the ER of cultured VMF, but does not stimulate Ca2+ influx. The initial release is followed by partial SERCA-mediated reuptake of Ca2+ into the ER, which supports several diminishing [Ca2+ ]i oscillations. Depletion of the Ca2+ stores by either opening of IP3 receptors or SERCA inhibition does not cause Ca2+ entry. This study was supported by Wyeth Ayerst Research and the Canadian Institutes of Health Research.

EXPRESSION OF TRANSFORMING GROWTH FACTORSIGNAL TRANSDUCER SMAD-2 AND SMAD-4 IN RAT ENDOMETRIUM DURING PERI-IMPLANTATION Haiyan Lin1, Juan Wang2, Hongmei Wang1, Xuan Zhang1, Longjiang Shao1, Donglin Liu1, Huitu Liu2 and Cheng Zhu1 1 State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080; 2Key Laboratory for Cell Proliferation and Regulation of Ministry of Education, Institute of Cell Biology, College of Life Sciences, Beijing Normal University, Beijing 100875, China.

The invasion of the blastocyst trophoblast and the reception of the uterine endometrium are both important factors for successful embryo implantation. During this tightly regulated process a variety of growth factors secreted by the uterus and conceptus exert pivotal effects along with steroid hormones. Smad proteins are a family of intracellular signalling molecules that transmit signals elicited by members of

Cell Biology International, Vol. 25, No. 10, 2001 transforming growth factor  (TGF-) superfamily. Unlike our knowledge regarding TGF-s and their receptors participate in implantation, far less is known about how Smads are involved in this process. To decipher the mechanism of TGF- signaling in implantation, we performed in situ hybridization and immunohistochemistry to investigate whether, when and where Smads mRNAs expressed in rat endometrium during peri-implantation and whether the detected transcripts were translated into proteins. The results demonstrated that expression of Smad-2 mRNA shifted from uterine glandular epithelium and luminal epithelium on D5 and D6 to embryo and decidualizing stromal cells on D7. The protein expression pattern was similar to its mRNA expression except on D5 Smad-2 protein was mainly observed in stroma and luminal epithelium. High levels of Smad-4 transcripts were noted in glandular epithelium and luminal epithelium on D5 while this high expression level decreased on D6. Smad-4 mRNA was only detected in decidua on D7. No signal of Smad-4 protein expression was detected on D5 whereas it distributed to luminal epithelium on D6 and decidua and embryo on D7. These data suggest that Smad-2 and Smad-4 express in a temporal and spatial pattern during rat peri-implantation and may play some roles in initial decidualization thus providing molecular evidence for TGF- signal transduction in rat implantion.

ROLE OF DOPAMINERGIC NEURONS OF AMYGDALA ON PARKINSON’S DISEASE Liu Bin and Xie Jun-xia Medical College, Qingdao University, 26602, China

Parkinson’s disease (PD) represents an age-related movement disorder, which results from degeneration of the nigrostriatal dopaminergic system. Marked sex differences have been reported to occur in the incidence of PD, with male to female ratios ranging from 1.36–3.70 to 1. Gonadal steroid hormones, especially estrogen, have been shown to exert substantial modulatory effects upon nigrostriatal dopamine system, and may likely contribute to the gender differences that exist in the incidence of PD. Moreover, recent researches have reported that the amygdala undergoes severe pathological changes during the course of PD. Our former studies indicate that the maximum dopamine (DA) release from the central amygdala nucleus (CAN) evoked by electrical stimulation in the female rats significantly higher than that of male rats. In the present study, fast cyclic voltometry (FCV) was employed to monitor DA release from CAN of female rats that were ovariectomized (OVX) and/or treated with different doses of estradiol benzoate (EB). Radioimmunoassay was used to measure the serum estradiol concentration of the rats. And we used high performance liquid chromatography (HPLC) to detect the contents of DA and its metabolites in the amygdala and striatum of the rats under different levels of serum estradiol concentration. In addition, DA release was measured from CAN following electrical stimulation of ventral tegmental area (VTA) in the PD model rats, which were unilaterally lesioned with 6-hydroxydopamine (6-OHDA). The influence of EB on rotational behavior induced by apomorphine (APO) in ovariectomized PD rats was investigated. The results were as follows: DA release from CAN was significantly higher in EB-treated OVX rats compared to OVX rats, and there was a dose-dependent relationship between DA release from CAN and the serum concentration of estradiol in rats. The contents of DA and its metabolites in Amy but not Str were significantly decreased when the OVX rats were treated with high doses of EB. The turnover rate of DA in Amy of the rats was about twice of that in Str, while the content of DA in Amy was six times less than that in Str. The turnover rate of DA in Amy of the OVX rats was lower than that of normal and EB-treated OVX rats. After the administration of EB (10 g/0.1 ml oil/d) subcutaneously for 3 days in OVX PD rats, DA release from CAN of both sides of PD rats was increased significantly, particularly the unlesioned side, compared to the oil-treated group. DA release from CAN could hardly be measured in lesioned sides of 6-OHDA PD rats, while DA release from unlesioned sides in PD rats was not affected. In the OVX PD rats, rotational behavior induced by APO was attenuated after EB administered, and the difference of rotational numbers between EB-treated group and oil-treated group had statistic value. Taking the above results together, it suggests that estrogen plays an important role in the regulation of DA release from mesolimbic

1061 dopamine system. Estrogen administered properly could increase the dopamine release as well as lower symptomatic severity in PD rats. Our data provide further experimental evidence for understanding the epidemiology of the gender differences in PD, and for a promising future strategy in the therapy of PD. K: amygdala; estrogen; dopamine; Parkinson’s disease; rat. The project was supported by the Key Project for the Ninth Five-Year Plan of China (No. 96-906-05-08) and the Natural Science Foundation of Shandong Province of China (No. Y98D0149, No. L2000C01).

TWO ENDOMETRIAL MARKERS OF UTERINE RECEPTIVITY Liu Cheng-Quan, Wang Zhong-Xing and Yuan Yao Shanghai Institute of Planned Parerthood Research, Shanghai 200032, China

Establishment of uterine receptivity is still a great biological mystery that remains unsolved. The complex and species-specific interactions between embryonic and endometrial cells requires synchronous development of cellular endomertrial components and coordinated endocrine, paracrine and autocrine communications. The subject of this study is to identify two markers in the guines pig endometrium that participate in the implantation process. The first biomarker is leukaemia inhibitory factor (LIP). We have demonstrating that LIP is expressed at the time of implantation in the guinea pigs. Immunoreactivity of this cytokine was compared between the control and treated with anti-progestin (mifepristore) endometria. Immunoreactivity of LIF in the endometrium of the mifpristonetreated (0.03 mg/kg/g) guinea pigs was light in the glandular and luminal epithelium and the stroma, as compared with intense staining for the control guinea pig endometria. The second marker belongs to the uterine pinopodes. Pinopodes are ultrastructural features of receptive endometrium, and have been proposed as a reliable marker of the receptive endometrium. The pinopodes principal value resides in the temporal correlation between the time of appearance of pinopodes and the window of implantation. These characteristics are an index of good responsiveness of the epithelium to progesterone. It is likely that the endometrium will be a viable target for novel forms of contraception in the future and more remains to be discovered about the role the endometrium in facilitating implantation of the embryo.

EUKARYOTIC 3 URT AND CANCER: A NOVEL ‘TRAVERSE’ SIGNAL TRANSDUCTION PATHWAY MAY EXIST IN CELLS Liu Ding-Gan Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological Science, Chinese Academy of Sciences, Shanghai 200031

Since more than 10 years, our research team has been engaged with studies on the tumour suppressor genes and tumour suppressor regulation elements. We found an RNA element with activity of tumour suppression to malignant cell lines of different species (mouse and human), i.e. the RNA of 3 untranslated region (3 UTR) of human nuclear factor for interleukin-6 expression (NF-IL6). Up to date, 5–6 types of such 3 UTRs with tumour suppressor function have been found worldwide. They do not produce protein products; so the molecular mechanisms of their tumour suppressor activity should be explained by investigation of their own functions rather by assuming any protein factor encoded. As a part of an intact gene, one of the functions of the 3 UTR is able to accept the interaction of protein factors from outside, i.e. to bind them. Because these bindings are all reversible, we guess that some 3 UTRs may carry signals related with the functions of their genes, and they may transfer the signals to the trans protein factors which interact with them; and the signals may arrive their target gene elements through interactions of the factors with the other protein factors. We call this novel signal transduction pathway ‘traverse signal transduction pathway’. In preliminary work, we have isolated a cDNA fragment, by using a yeast three-hybrid system, and found the polypeptide expressed from it was able to specifically bind NF-IL6 3 UTR RNA. Now we are trying to isolate

1062 protein factor(s) that directly interact with NF-IL6 3 UTR RNA, and secondary protein factors interacting with it. It is apparent that, is this novel signal transduction pathway can be found, some of its protein factors may be developed into novel gene drugs for cancer gene therapy. We hope to have cooperation from Hong Kong colleagues who are interested in this subject.

SIGNAL PEPTIDASE ACTIVITY OF -LACTAMASES H. B. Liu and Y. C. Leung Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong, China

The emergence of new strains of bacteria that are resistant to currently available antibiotics is increasing the difficulty of treatment of common bacterial infections. New strategies and new drug targets are required for the development of antibacterial agents to treat diseases due to drug-resistance pathogens. Toward that goal, bacterial protein secretion pathways can provide logical targets for identification of new antibacterial drugs. It has generally been accepted that although DD-peptidases and -lactamases can recognize -lactams, as well as ester and S-ester analogues of the substrates of the former, -lactamases are unable to hydrolyze ‘true’ peptide bonds. However, about ten years ago, Black et al. proposed a striking similarity between the mechanisms of catalysis of signal peptidase and -lactamase. Instead of the classic catalytic trial of Ser, His and Asp, the proposed mechanism of catalysis involves a Ser-Lys dyad without the apparent involvement of aspartic acid. This unusual catalytic mechanism may explain why none of the commonly used inhibitors of proteolytic enzymes inhibit signal peptidase and is consistent with the generally low catalytic efficiency of the enzymes. During the purification of -lactamases, we surprisingly found that MBP--lactamase (or GST--lactamase) could cleave itself at specific site into two parts—MBP (or GST) and -lactamase with low catalytic efficiency. It seems that -lactamase might also function as a signal peptidase. Thorough study of these phenomena may lead to a deeper understanding of the functions of -lactamases and will be instrumental in the rational design of novel antibiotics.

SYNERGISTIC ACTIVATION OF TRANSCRIPTION FACTOR Elk BY Ha-ras AND AIK Ya-Shih Tseng, Chi-Ying Huang1 and Hsiao-Sheng Liu Department of Microbiology and Immunology, College of Medicine, National Cheng Kung University; 1Division of Molecular and Genomic Medicine, National Health Research Institutes, Taiwan, R.O.C.

Overexpression of the oncogene AIK1 (Aurora 2, an aurora/Ipl1related kinase) has been reported in several human cancer cell lines and human carcinoma, including colorectal cancer. Colon cancer also showed high incidence of Ras mutations (about 50%). The relationship between AIK1 and Ras as well as their effects on tumourigenesis remains unclear. AIK1 can induce Xenopus oocyte maturation and activate MAP kinase in progesterone related signal pathway. Ras can activate multiple signal pathways including MAPK pathway. In this study, we investigate the effects of AIK1 and Ras on the activation of transcription factor Elk. Our results demonstrate that AIK1 can activate Elk in a dose-dependent fashion, which can be block by dominant RasN17, RafCB4, dominant negative MEK1/2 and MEK1/2 inhibitor PD98059. Moreover, coexpression of AIK1 and RasV12 enhances the phosphorylation on of ERK1/2 and activity of Elk. Furthermore, the RalGDS/Ral/RalBP pathway also involves in this pathway, which was demonstrated by dominant negative mutants of RalGDS pathways. Taken together, AIK1 can synergistically activate Ras downstream Elk activity through the RalGDS related pathway.

MECHANISMS OF HYPOXIC ACCLIMATIZATION ON CARDIOVASCULAR SYSTEM OF RATS Long Chao-Liang, Yin Zhao-Yun and Wang Hai Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences, Beijing 100850, China

Cell Biology International, Vol. 25, No. 10, 2001 As a stress factor, hypoxia almost can affect all functions and activities of human. Among them, the effects of hypoxia on cardiovascular system are most remarkable. The effects of hypoxia on cardiac functions have been an important question in clinical medicine, aerospace medicine and high altitude medicine. The studies about the mechanisms of hypoxic acclimatization (HA) began 1960s. Though almost 40 years have passed, the mechanisms were still not very clear. To investigate the effects of HA on functions of cardiovascular system and its mechanisms have important theoretical and practical values. The present study was designed to investigate the functional changes and its mechanisms of sarcoplasmic reticulum (SR) and mitochondria under hypoxic acclimatization condition. The high altitude HA mechanisms of cardiovascular system were studied from ion transport and energy metabolism. The results shown that: (1) After HA, the content of CPK in plasma and the content of MDA in myocardium were decreased significantly. At the same time, the myocardial total protein content and the content of NO2
(a metabolite of NO) were increased significantly. These results indicate that decreased CPK releasing from myocytes, alleviated damage of oxygen free radicals and lipid peroxidation, increased NO releasing and myocardial systolic protein expressing are the biochemical mechanisms of improved cardiac functions and alleviated myocytes injury after HA. (2) After HA, the activities of Ca2+ , Mg2+ -ATPase of myocardial SR, the phosphorylation of phospholamban, a regulative protein of SR calcium pump, and the ability of 45 Ca2+ uptake of SR were increased significantly. These results suggest that the increased function of myocardial SR calcium pump, the strengthened phosphorylation of phospholamban to alleviate the inhibition of calcium pump and the increased function of Ca2+ transport in SR were the mechanisms of HA protecting cardiac functions from injury induced by severe hypoxia. (3) After HA, the protein content and activity of Ca2+ , Mg2+ -ATPase of mitochondria were increased significantly. The activities of enzyme complex, ?? and ?? of respiratory chain were also increased significantly. These results suggest that the proliferation and improved functions of mitochondria and the strengthened energy metabolism are the mechanisms of HA improving cardiac systolic and diastolic functions. In conclusion, the improved functions of myocardial SR, strengthened calcium transport, ameliorated functions of mitochondria, strengthened energy metabolism were the main mechanisms of HA of cardiovascular system. And it was suggested that strengthened phosphorylation of phospholamban, a regulative protein of SR, was one of the biochemical mechanisms of HA for the first time. K: hypoxic acclimatization; cardiovascular system; sarcoplasmic reticulum; mitochondria; phospholamban; respiratory chain; enzyme complex; rat.

INVESTIGATION OF THE STRUCTURE-FUNCTION RELATIONSHIPS OF PORCINE PYRIDOXAL KINASE BY SITE-DIRECTED MUTAGENESIS M. K. T. Ng and Y. C. Leung Department of Applied Biology and Chemical Technology, Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong

Pyridoxal kinase (PK) catalyzes the phosphorylation of vitmain B6, generating pyridoxal-5 -phosphate (PLP), an important cofactor for many enzymatic reactions. This enzyme has been detected in virtually all mammalian species as well as in many microorganisms. Although the major pathways of vitamin B6 metabolism have been established for many years, there is very little information about PK catalytic activity. The study of structural and catalytic properties of this enzyme can enhance our understanding of the relationship of vitamin B6 metabolism to brain disorder and some doubts about the connection between vitamin B6 metabolism and several genetic diseases, such as Down’s syndrome and autoimmune polyglandular disease type I. In the absence of the information about the PK detailed threedimensional structure, site-directed mutagenesis is one of the methods to understand the structure-function relationship of this enzyme. Three conserved amino acids that identified by primary sequence alignment of known PK sequences were altered. The mutant enzymes including Y135A, Y135F, G240A, G242A, and H186A were expressed and purified to great homogeneity. The H186A mutant had 70%

Cell Biology International, Vol. 25, No. 10, 2001 residual activity, whereas the Y135F mutant exhibited less than 10% residual activity. The mutagenesis in Y135A, G240A, and G242A completely abolished enzymatic activity. The results of substrate binding analysis showed that mutation of tyrosine 135 to alanine (Y135A) or phenylalanine (Y135F), and mutation of glycine 242 to alanine (G242A) affected pyridoxal binding, while the G240A mutant did not show any interaction with both pyridoxal and ATP. These results suggest that Y135 and G242 play a significant role of pyridoxal binding, and G240 is critical for catalysis. In addition, the unfolding processes of the sited-directed mutants were monitored by circular dichroism and fluorescence spectroscopy. No dramatic change in the midpoint transition was observed, implying that the residues substituted are not essential for maintaining the protein conformation.

RECEPTORS AND SIGNALLING PATHWAYS IN ENDOTHELIN-1-INDUCED VASOCONSTRICTION IN HUMAN RADIAL ARTERY C. E. Black, N. Huang, P. C. Neligan, C. R. Forrest, J. E. Lipa and C. Y. Pang The Hospital for Sick Children Research Institute, and Departments of Surgery and Physiology, University of Toronto, Toronto, Ontario

Despite the increasing use of radial artery (RA) as a conduit coronary artery bypass graft, little is known about the mechanisms regulating vasospasm in this blood vessel. The aim of the present study was to investigate the receptor and postreceptor signal transduction pathways mediating endothelin-1 (ET-1)-induced vasospasm in human RA. Human RA specimens (0.5 cm) were obtained from the redundant portion of the vascular pedicle in the radial forearm skin free flap used in reconstructive surgery, with an approved clinical protocol. A 4 mm long RA ring was perfused in an organ bath containing aerated (95% O2, 5% CO2) Krebs bicarbonate buffer at 37C and pH 7.38. The isometric tension elicited by 50 mM KCl was used to standardize vascular response to each dose of drug added to the organ bath. It was observed that ET-1 caused a cumulative concentration-dependent (510
11–210
8 M) contraction in RA rings with high contractile potency (pD2 =8.970.051; n=5). The selective ETA receptor antagonist BQ 123 (10
5 M) blocked the ET-1-induced contractile response by >95%. However, the selective ETB receptor antagonist BQ 788 (510
6 M) did not have any significant antagonistic effect on the contractile response to ET-1 in RA rings. The L-type Ca2+ channel antagonist nifedipin (510
6 M; n=4), the PKC inhibitor chelerythrine (10
5 M; n=4) and the intracellular Ca2+ chelator BABTA-AM (10
5 M; n=4) shifted the cumulative concentration-dependent contractile response curves of ET-1 to the right. Nifedipine, chelerythrine and BAPTA-AM significantly (P<0.05) reduced the contractile potency of ET-1 by 4.4, 4.9, and 3.1 folds, respectively. Similarly, nifedipine, chelerythrine and BAPTA/AM significantly (P<0.05) reduced the maximal contractile response to ET-1 in RA rings by 25%, 26% and 15% respectively. We conclude that the contractile response to ET-1 in human RA is predominantly mediated by ETA receptors, and the postreceptor mechanism involves L-type Ca2+ channels, PKC and intracellular Ca2+ . Supported by the Canadian Institute of Health Research.

CHARACTERIZATION OF TWO DISTINCT Ca2+ SIGNALS ASSOCIATED WITH APOPTOSIS IN SINGLE LIVING HeLa CELLS Yongmei Pu, Kathy Q. Luo, Wenhua Gao and Donald C. Chang Department of Biology, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China

Using living cell imaging technique and our newly developed in vivo FRET probe of caspase-3 (CFP-DEVD-YFP), we characterized the Ca2+ signals associated with apoptosis at single cell level, and studied their functional roles in regulating the progression of apoptosis. Our results showed: (1) Two types of Ca2+ events were observed during the process of apoptosis; they included early repetitive Ca2+ spikes and later sustained Ca2+ elevation. (2) The Ca2+ spikes appeared much earlier than cyt c release and caspase-3 activation; while the sustained

1063 Ca2+ increase was associated with later cell morphological changes after the activation of caspase-3. (3) The IP3R antagonist-heparin was found to be able to abolish these cytosolic Ca2+ changes, as well as blocking cyt c release, caspse-3 activation, cell blebbing and chromatin condensation. Taken together, our findings suggest that Ca2+ signal may have multiple roles in regulating the progression of apoptosis: (1) It plays an important role in the commitment phase of apoptosis, by participating in regulating the signal upstream of cyt c release and caspase-3 activation; (2) It is required for the progression of the execution phase, by controlling the initiation of cell blebbing and chromatin condensation.

CHARACTERIZATION OF tSH3p13 GENE ENCODING A DEVELOPMENT PROTEIN EXPRESSED DURING SPERMIGENESIS Yuan Qiao, Juxiang Yang, Xinyong Tian, Shiying Miao and Linfang Wang National Laborary of Medical Molecular Biology, 4 Dong San Tiao, Beijing, China

A cDNA, designated as tSH3p13, was isolated from a rat testis cDNA library. It consisted of 1463 bp, containing an open reading frame of 936 bp, coding a protein composed of 312 amino acids and assigned the GeneBank accession number AF227439. The deduced tSH3p13 protein is related to endophilins members of the SH3p4/p8/p13 protein family isolated previously from a mouse brain cDNA expression library. Its sequence is identical to tSH3p13 except for a deletion of a 35 amino acid segment in the N-terminus. The tSH3p13 gene is expressed in the testis and brain, based on Northern blot analysis of various tissues. During postnatal development of the rat, tSH3p13 is detected in the testis on day 18 and increase gradually thereafter to attain a maximum level on day 60. Using anti-tSH3p13 anitserum raised against the recombinant specific region of tSH3p13, the antigen was immunolocalized in epermatids at stages 8–19 of spermiogenesis and found to be situated in the acrosome region by immuno-electron microscopy. A 35 kDa potential binding partner of tSH3p13 was isolated and identified in germ cells by the paplication of the surface plasmon resonance and GST ‘pull-down’ assays. This additional component is distinctly different from the previously reported binding parterns of SH3p4/p8/p13 protein. The N-terminal segment of tSH3p13 including conserved region and various region was constructed as bait protein to screen the mouse testis cDNA library. A positive clone, which was subsequently named tSH3p13NBP, was found with an open reading frame encoding a 185 amino acids peptide. Considering tSH3p13 is highly related to endophilins, which is thought to participate in the formation of trans-Golgi network, together with the results of immunohistochemical analysis, we hypothesize that the complex of tSH3p13 and 35 kDa protein as well as tSH3p13NBP may play a functional role during the transformation of sperm cells and the formation of acrosome.

INVESTIGATION OF THE POSSIBLE MECHANISMS OF BAK FOONG PILLS IN TREATING DYSMENORRHOEA D. K. Rowlands, Y. G. Cui and H. C. Chan Epithelial Cell Biology Research Centre, Department of Physiology, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong

Bak Foong Pills (BFP) have been used for many centuries in China in the treatment of dysmenorrhoea, whereas Western treatments for dysmenorrhoea rely on the use of non-steroidal anti inflammatory drugs and compounds which cause reduction in uterine tension (e.g. oxytocin and vasopressin antagonists) (Akerlund et al., 1995). This study aims to determine if BFP works in a similar manner to modern medicines or whether it has it own novel mechanisms of action. The analgesic role of BFP was investigated using the acetic acid induced writhing model. Briefly, ICR mice (40 g, male) were treated with BFP (250 mg–5 g/kg, p.o.) daily for three days to assess semichronic, and for 1 day to assess acute treatment of BFP. Water (10 ml/kg, p.o.) was administered as vehicle in control animals and indomethacin (1 mg/kg, p.o.) as a positive control. Acetic acid was injected (0.5% solution, 5 ml/kg, i.p.) one hour following the last

1064 treatment, and writhing response assessed over a 30 min period. Immediately following the test, peritoneal washes were taken for evidence of prostaglandin production. BFP was shown to have greatest analgesic action following 3 days of treatment (EC50 =900 mg/kg, n=12), with 2 g/kg exerting a maximal analgesic response of 62% inhibition when compared to control animals. Acute treatment with BFP (2 g/kg) only produced a 17% inhibition of the writhing response. Indomethacin was effective following acute and semi-chronic treatment, giving 87% and 89% inhibition of writhing respectively. Radio immunological assay (RIA) of the peritoneal washes demonstrated that BFP is unable to reduce levels of both PGE2 and 6-keto PGF1??, whereas, indomethacin is able to significantly reduce the amount of both prostaglandins. The uterine relaxant effect of BFP was investigated using an isolated organ bath preparation. Uteri were suspended in Tyrodes solution containing indomethacin (3 ?M) (37C, 5% CO2/O2) and contracted using either KCl (40 mM) or oxytocin (0.1 U/ml). Results showed that BFP caused uterine relaxation in a concentration dependant manner (EC50 =5 mg/ml, n>3) following contraction with either KCl or oxytocin. In conclusion, BFP exerts significant analgesic effect following semi-chronic treatment but not acute treatment. The analgesic effect is not due to inhibition of cyclooxygenase, since prostaglandin levels were not affected. BFP also induces relaxation of uterine tissue in vitro, following contraction with either KCl or oxytocin, indicating that BFP is unlikely to be an oxytocin antagonist. The precise mechanisms of BFP mediated analgesia and uterine relaxation is yet to be determined, but the present study has demonstrated that the anti-dysmenorrhoeal effect of BFP is not dependant on a single action but due to a combination of two or more separate mechanisms. References A M., et al., 1995. Adv Exp Biol 395: 595–600.

EFFECTS OF ETHANOL EXTRACTS FROM CHINESE HERBS FOR HYPERTROPHIC SCAR ON THE GROWTH AND PROLIFERATION OF HYPERTROPHIC SCAR SKIN FIBROBLAST IN VITRO Shen Danbei, Xia Longqing, Ju Qiang, et al. Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College, Nanjing 210042, China

Hypertrophic scar and keloid are characterized by proliferation of dermal fibroblasts, overproduction of extracellular matrix by dermal fibroblasts. Various conservation therapies have been used, but definite and effective therapy has not yet been established. The traditional Chinese medicine complexes (Heibu Salve et al.) have been certain remedies for hypertrophic scar and keloid since long time ago, but by some unknown mechanism. People do not know what is the effective ingredient of Heibu salve. That is why the traditional Chinese medicine complex for hypertrophic scar and keloid could not obtained a satisfied effective therapy. The objective of this study is to screen potent inhibitors of cellular growth and proliferation and to determine their effective inhibitive dosage from the traditional Chinese medicines complex treated for hypertrophic scar in cultured normal human dermal fibroblasts and hypertrophic scar skin fibroblasts. The methods that we used: (1) To prepare the ethanol extracts from Chinese nut-Gall (Rhus chinensis Mill), Safflower (Carthamus tinctorius Linn.), Centipede (Scolopendra subspinipes mutilans L Koch). (2) Hypertrophic scar sample was obtained from a woman after surgical excision. Normal skin sample was obtained from a young man after circumcision. Primary cultures of dermal fibroblasts were established as common tissue culture technique. Fibroblasts at passages 5–8 were used in this study. (3) With MTT Colorimetric assay and cell growth curves, to compare the influence among ethanol extracts from Chinese medicine herbs for hypertrophic scar on fibroblasts growth activity and growth cycle. The results shown that: (1) Chinese nut-Gall can obviously inhibits the proliferation of human fibroblasts derived from hypertrophic scar and normal human skin, and has a more effective inhibition on hypertrophic scar fibroblasts than on normal human skin fibroblasts in a dose-dependent manner (0 g/ml–400 g/ml) during

Cell Biology International, Vol. 25, No. 10, 2001 72 h of treatment. But Safflower and Centipede cannot (500 g/ml– 2 mg/ml). (2) The reversibility of inhibition do not appeared to correlate with changes in fibroblast morphology with Chinese nut-Gall at 400 g/ml and below, but dose with colchicine at <50 g/ml. The IC50 of colchicine is 50 /ml. The IC50 of Chinese nut-Gall is 100 g/ ml. (3) Chinese nut-Gall at 50 g/ml can delay and shorten the Log phase and extend the population doubling time (PDT) in cultured normal human and hypertrophic scar skin fibroblasts in an manner of time-dependent. (4) The cell growth curve of hypertrophic scar fibroblasts differs from that of normal human skin fibroblasts. Chinese nut-Gall, Safflower, and Centipede are the main ingredients of traditional Chinese Medicines complex for hypertrophic scar and keloid. But only Chinese nut-Gall exhibits anti-proliferative activity on hypertrophic scar fibroblasts, and has less cytotoxicity than colchicine on cell size and morphology. In conclusion, Chinese nut-Gall can inhibits the proliferation of human fibroblasts derived from hypertrophic scar and normal human skin. The dose- and time-dependent inhibition suggests that Chinese nut-Gall could be investigated as a novel potential therapeutic agent in treating abnormal dermal scarring.

ZONA PELLUCIDA/Ca2+ INDUCES RAPID ACTIVATION OF PHOSPHOLIPASE A2 AND RENDERS GUINEA PIG SPERMATOZOA CAPABLE OF UNDERGOING ACROSOMAL EXOCYTOSIS Q. X. Shi1, Y. Y. Yuan1, E. R. S. Roldan2, L. Z. Mao1, S. Q. Yu1, W. Y. Chan1 and X. Fang1 1 Zhejiang Academy of Medical Sciences, Hangzhou, Zhejiang, 310013, China; 2Museo Nacional de Ciencias, Naturales (CSIC), C/Jose Gutierrez Abascal, 28006-Madrid, Spain

The acrosome reaction (AR) is an exocytosis event induced physiologically by the zone pellucida (ZP) and progesterone (P4). It is now clear that PLA2 plays an essential role in the release of fatty acid and lysophospholipids in sperm membrane fusion during acrosomal exocytosis (Roldan and Fragio, 1993). PLA2 is activated in human (Baldi et al, 1993) and boar sperm (Roldan and Vazquez, 1996) in response to P4, but no studies have yet reported ZP-induced activation of PLA2 (Roldan, 1999). Our objectives were investigate whether PLA2 is involved in the AR induced by ZP and if Ca2+ is required for activation of PLA2, as well as its signal transduction pathway during the AR in guineas pig spermatozoa. Spermatozoa were preincubated in MCM-Low Ca2+ medium (containing 23 M Ca2+ , MCM-L Ca2+ ) with 4 mg BSA/ml and 20 mM Hepes for 1 h in a capped vase in a shaking water bath and then labelled with 2  Ci/ml [methyl-14C] choline chloride or 0.5  Ci/ml [1-14C] arachidonic acid (1-14C AA) for 6 h, respectively. Spermatozoa were washed through a three gradient percoll (30–55–85%) and resuspended in MCM-LCa2+ with or without 2 mM Ca2+ . Spermatozao were exposed to several reagents alone or in different combinations for 15 min before lipid extraction and separation using onedimensional TLC, and radioactivity in spots determined by scintillation counting. The AR was assessed by phase-contrast microcopy. Mobility of spermatozoa was 85–90% after all additions. The main results were as follows: (a) Spermatozoa exposed to 2 mM Ca2+ resulted in an increase in release of arachidonic acid (AA) and the generation of lysophosphatidylcholine (Lyso PC) and the AR. The combination of both Ca2+ and soluble ZP caused a further increase in the release of AA and the generation of Lyso PC, whereas accompanied by a decrease in Phophatidylcholine (PC). These effects as mentioned above were prevented if spermatozoa were challenged with 160 M aristolochic acid (ATA, a PLA2 inhibitor). (b) To understand the possible role of G-protein transducing pathways during the acrosomal exocytosis stimulated by ZP, we used 1–2 g/ml pertussis toxin (PTX, a G-protein inhibitor) to treat with capacitated spermatozoa. The release of AA and the generation of Lyso PC as well as the occurrence of AR induced by ZP were inhibited. (c) Activation of PLA2 is required for extracellular Ca2+ influx. In the presence of 23 M Ca2+ in MCM, spermatozal acrosome exocytosis did not happen, whereas the release of AA and the generation of Lyso PC were maintained at a low level. When spermatozoa were treated with 2 mM EGTA or 250 M La3+ , an essential role of PLA2 in both exocytosis and AA release was prevented. (d) When spermatozoa were treated

Cell Biology International, Vol. 25, No. 10, 2001 with DAG kinase inhibitor R59022, the release of AA, the generation of Lyso PC and the increase in exocytosis induced by ZP/Ca2+ were significantly increased. Taken together, these results indicate that PLA2 is involved in exocytosis in guinea pig spermatozoa and its activation is required for low Ca2+ and regulated by signal transduction pathways via DAG. Supported by the National Natural Science Foundation of China (No.39870364) and the National 973 Programmes (199905992)

INVOLVEMENT OF Ca2+ -ACTIVATED K+ CHANNELS IN RECEPTOR-REGULATED SPERM MOTILITY S. C. So, C. X. Zhou and H. C. Chan Epithelial Cell Biology Research Center, Department of Physiology, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong

Previous voltage-clamp studies have demonstrated the modulation of sperm Ca2+ -activated K + (KCa) channels expressed in Xenopus oocytes by angiotensin II (Ang II) and extracellular ATP via AT1 receptor and P2U receptor, respectively. In the present study, we investigated the involvement of KCa channels in receptor-regulated sperm motility of the rat using a computer-aided sperm analysis system, HTM-IVOS, in conjunction with Ca2+ -moblizing agents, receptor agonists/antagonists and KCa channels blockers. The percentage of motile sperm was increased by ionomycin (0.5 M), which could be inhibited by K + channel blockers, tetraethylammonium (TEA 1 M ) or charybdotoxin (ChTX, 300 nM) indicating the presence of KCa channels. Ang II, at low concentration (10 nM) was found to increase motility; however, at higher concentration (1 M) percentage of motility was suppressed. Both stimulatory and inhibitory effects of Ang II could be reversed by losartan, a specific antagonist of AT1 receptors, but not the AT2 antagonist PD123177, indicating that the involvement of AT1 but not AT2 receptor in mediating both effects. ChTX also abolished both stimulatory and inhibitory effects of Ang II, suggesting the involvement of KCa channels. The percentage of motility was also enhanced by extracellular ATP, a factor involved in sperm activation. The ATPenhanced sperm motility was mimicked by UTP, and inhibited by ChTX and reactive blue, an antagonist of P2 receptor, indicating the involvement of both P2U and KCa channels. RT-PCR study was also conducted to confirm the expression of KCa channels, AT1 receptors and P2U receptor, but not AT2 receptor, in rat caudal epididymal sperm. The present findings suggest an important role of KCa channels in the regulation of sperm motility by AT1 and P2U receptors.

MECHANISM OF IL-6 SIGNAL TRANSDUCTION IN HUMAN MYELOMA CELLS Song Lun, Li Yan and Shen Bei-fen Institute of Basic Medical Sciences, Academy of Military Medical Sciences , Beijing, 100850, P. R. China

Interleukin-6 (IL-6) is the major growth factor for the survival and proliferation of multiple myeloma (MM) cells. In this study, IL-6 signal transduction pathways and regulation mechanism were investigated in three different kinds of human myeloma cell lines which can match the different living states of myeloma cells in vivo during MM pathogenesis. According to the growth stimulation effect of IL-6 on myeloma cells, MM cell lines can be divided into three kinds: (I), IL-6dependent MM cell line; (II), IL-6- independent MM cell line, but exogenous IL-6 can promote the proliferation of the cells; (III), IL-6-independent MM cell line, and exogenous IL-6 cannot exert any growth or growth inhibition effect on the cells. From MTT results, the four MM cell lines used in this research displayed different proliferation effect with the stimulation of IL-6, so they belonged to I (XG-7), II (KM-3, U266) and III (Sko-007) MM cell lines, respectively. Up till now, two IL-6 signal transduction pathways have been revealed, one is JAK/STAT pathway, and another is Ras/MAPK/NFIL-6 pathway. To investigate the IL-6 signal transduction mechanism in the four MM cell lines above, electrophoresis mobility shift assay (EMSA) and immunoprecipitation (IP) were firstly used to detect the activation states of the transcription factors-STAT3. NF-IL-6 and the protein kinases-JAK1, MAPK in the two IL-6 signal transduction

1065 pathways, respectively. And we observed that Ras/MAPK/NF-IL-6 pathway can be activated by IL-6 in all of the four MM cell lines, but the activation of JAK/STAT pathway by IL-6 can only be detected in Sko-007 and U266 cells. These results gave us a concept that Ras/ MAPK/NF- I L-6 is a general IL-6 signal transduction pathway in MM cells; but JAK/STAT pathway preferred to be activated in the myeloma calls that is related to the late MM developmental phase. The second part of this study is to disclose the regulation mechanism between two IL-6 signal transduction pathways in Sko-007 and U266 cells with the specific protein tyrosine kinase inhibitor- Genistein, specific inhibitor for Ras pathway PD98059, antagonist for Ras pathway-PMA and the anti sense nucleic acids for STAT3, ERK and NF-IL-6.According to the results, in Sko-007 cells some tyrosine phosphorylation process that related to JAK/STAT pathway can participate in the activation of Ras pathway, at the same time, Ras pathway can also give a positive regulation on the activation of JAK/STAT pathway through the direct binding between the protein kinase ERK and the transcription factor STAT3. In contrast to Sko-007 cells, we found there was an antagonism affect between the two IL-6 signal transduction pathways activation in U266 cells. In the third part of this research, we focused on the relationship between two IL-6 signal transduction pathways activation and its biological function in the four MM cell lines with the same specific protein kinase inhibitors and sense and antisense nucleic acids as in the second part. The results showed that IL-6 can keep the survival and normal cell cycle proceeding in XG-7 cells by the activation of Ras pathway; and the promotion effect of IL-6 on the proliferation of KM-3 and U266 cells were also mediated by Ras pathway’s activation. In addition, a primary research to look for the unknown biological functions on Sko-007 cells corresponding to the two IL-6 signalling pathways activation was also developed. And we found the induced expression of a new IL-6 target gene-tyrosine phosphatase CD45, is related to JAK/STAT pathway’s activation in this target cell line. K: IL-6; multiple myeloma; signal transduction; regulation; biological. function.

ROLE OF CALCIUM IN THE EFFECT OF ANGIOTENSIN II INDUCED ATRIAL NATRIURETIC PEPTIDE RELEASE IN SUPERFUSED RAT ATRIAL TISSUE Hayet Soualmia, Franc¸ oise Masson, Christiane Barthelemy, Joelle Eurin and Alain Carayon Faculte´ Medecine Pitie´ -Salpeˆ trie`re, France and Faculte´ Sciences Gabe`s, Tunisie

This study presents an investigation of the mechanism of angiotensin II (Ang II) induced atrial natriuretic peptide (ANP) release in sliced right atria of rats. Using a superfusion method, ANP outflow was significantly increased in presence of Ang II (10–7 M). To understand the mechanism of Ang II on ANP release, the role of calcium (Ca+ +) in Ang II signal transduction was examined with Ca+ + antagonists. The use of TMB-8, a blocker of intracellular calcium mobilization, showed a completely inhibition of Ang II stimulated ANP release. To more evaluate the role of intracellular calcium, the effect of calmodulin inhibitor was tested by W-7. Perfusion of W-7 at 10–5 M prevented ANP increased by Ang II. Further, to understand the mechanism of Ang II, we used Bapta-AM, a chelator of intracellular calcium. We observed that this Ca+ + antagonist suppressed the effect of Ang II on Table Drugs (M)

ANP (pg/min/mg tissue) Control Ang II (10
7 M) 16.11.0** 10.60.5

Bapta-AM (10
5) TMB-8 (10
7) W-7 (10
5) A23187 (510
7)

9.90.4 9.00.9 9.91.2 13.30.5

10.10.5§ 10.40.6§ 9.90.7§ 20.31.5§

N>6, **P<0.01 versus control, §P<0.001 versus Ang II.

1066 ANP release. In contrast, A23187, a calcium ionophore potentiated the Ang II effect on ANP release. In conclusion, all these results confirmed the important role of calcium in the effect of Ang II on ANP release in right atrial tissue.

EFFECTS OF HISTAMINE AND CYCLOPIAZONIC ACID ON INTRACELLULAR CALCIUM IN CULTURED HUMAN VALVULAR MYOFIBROBLASTS Willmann Liang1, Casey van Breemen1, Paul McDonald2, Bruce McManus2 and Xiaodong Wang1 1 Department of Pharmacology and Therapeutics; 2 Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, B.C., Canada

Although valvular myofibroblasts (VMF) are the most predominant cell type in cardiac valves, little is known about how they are regulated by Ca2+ signalling. In this study, the effects of histamine and cyclopiazonic acid (CPA), an inhibitor of sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA), on intracellular Ca2+ were investigated. Myofibroblasts were isolated by enzymatic digestion from a normal female human mitral valve and grown on glass coverslips. Intracellular Ca2+ concentration ([Ca2+ ]i) was measured by fluorescence imaging microscopy of fura2-loaded cells. The result showing that in the presence of extracellular Ca2+ histamine induced a transient increase in [Ca2+ ]i followed by several oscillations in [Ca2+ ]i, which decayed within 200 s. Removal of extracellular Ca2+ did not alter this response and Mn2+ quenching experiments failed to provide evidence for agonist-induced divalent cation entry. Blockade of SERCA by CPA induced a slow transient [Ca2+ ]i increase, which was independent of extracellular Ca2+ . Addition of CPA during the measurement of Mn2+ -quenching of fura2 fluorescence failed to provide evidence for the presence of Store Operated Channels (SOC). When histamine was added to cells pretreated with CPA for 10 minutes in Ca2+ -free PSS it elicited one rapid transient [Ca2+ ]i spike with no subsequent oscillations. In conclusion, histamine causes release of Ca2+ from the ER of cultured VMF, but does not stimulate Ca2+ influx. The initial release is followed by partial SERCA-mediated reuptake of Ca2+ into the ER, which supports several diminishing [Ca2+ ]i oscillations. Depletion of the Ca2+ stores by either opening of IP3 receptors or SERCA inhibition does not cause Ca2+ entry. This study was supported by Wyeth Ayerst Research and the Canadian Institutes of Health Research.

EFFECT OF Na + DIET ON ENDOMETRIAL EPITHELIAL Na + CHANNEL (ENaC) SUBUNITS AND CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR (CFTR) EXPRESSION DURING THE ESTRUS CYCLE IN MICE L. L. Tsang, L. N. Chan, D. K. Rowlands, Y. W. Chung, Y. M. Chan and H. C. Chan Epithelial Cell Biology Research Center, Department of Physiology, Faculty of Medicine, The Chinese University of Hong Kong

Previous studies have demonstrated cyclic changes in ENaC and CFTR expression during the estrus cycle. The present study investigated the regulation of uterine ENaC subunits and CFTR expression by aldosterone in vivo using a low Na + diet mouse model. Estrus cycle of 10–12 weeks old ICR mice was synchronized by injection of estrogen (5 g/100 g) two days before experiment. The mice were fed with a low sodium diet for at least two weeks, during which circulating aldosterone is expected to be elevated. The mRNA level of uterine CFTR and ENaC subunits for all stages of the estrus cycle was examined by competitive reverse transcription-polymerase chain reaction (RT-PCR). The results showed a cyclic expression pattern of uterine ENaC throughout the estrus cycle with maximal expression level at the metestrus for  subunit and at diestrus for
and  subunits. On the other hand, the expression pattern of CFTR reached maximal expression level at proestrus and estrus, and decreased at metestrus and diestrus. When fed with a low Na + diet, which was expected to elevate the circulating aldosterone, a steroid hormone which controls

Cell Biology International, Vol. 25, No. 10, 2001 the rate of transepithelial Na + absorption in a variety of epithelia, the cyclic characteristics of expression pattern for all ENaC subunits remained unchanged but for CFTR was changed, it shows lower mRNA expression in all stage of estrus cycle. And the maximal level of ENaC expression was considerably elevated, 121.9%, 35% and 283.3% for
,  and  subunit, respectively. The results indicate that when undergoing a low Na + diet, uterine ENaC expression was maximally elevated, presumably in response to elevated circulating aldosterone. These results suggest that in addition to the regulation by ovarian hormones, uterine ENaC and CFTR expression level also responds to changes in circulating aldosterone, contributing to the overall body electrolyte and fluid homeostasis. Our data also indicated a predominant role of ENaC subunit in regulating ENaC activity and thus the rate of Na + absorption across mouse endometrium.

NEW GENERATION OF CARBAMOYLASE WITH DESIRABLE FEATURES FOR ANTIBIOTICS PRODUCTION Jin Caike, Shen Dong, Wang Jun and Tsang Wai Kei Paul Department of Biochemistry, The Chinese University of Hong Kong, Shatin, Hong Kong SAR

Carbamoylase is one of the most crucial enzymes for large-scale production of D-amino acids used in antibiotics industry. Applying in vitro recombination between homologous genes and high throughput screening scheme developed in-house, we have isolated a number of recombinants that carry enzymatic activities 20 to 60 times higher than the existing commercial products and more thermostable. The same approach should facilitate the breeding of new generation of protein drugs in general.

NEUROKININ RECEPTOR EXCITATION AND SIGNALLING IN CULTURED HUMAN ESOPHAGEAL SMOOTH MUSCLE CELLS Jian Wang, P. S. Krysiak, L. G. Laurier, S. M. Sims and H. G. Preiksaitis Departments of Medicine and Physiology, The University of Western Ontario and The Lawson Health Research Institute, St. Joseph’s Health Care London, London, Ontario, Canada, N6A 4V2

We recently demonstrated that tachykinins play an important role in non-cholinergic neuronal activation of human esophageal smooth muscle (Gastroenterology 120: 39, 2001). Three subtypes of neurokinin (NK) receptors (NK1, NK2 and NK3) are expressed in mammalian systems. All three NK receptors are thought to share a common signalling pathway involving phospholipase C, IP3 and release of intracellular Ca2+ , however the distribution of the NK receptors varies between tissues. We examined NK receptor expression and function in human esophageal smooth muscle cells in primary culture. We previously demonstrated that this preparation contains >96% smooth muscle and displays features similar to the tissue of origin (AJP 279: G1059, 2000). Receptor expression was examined by RT-PCR, immunocytochemistry, and immunoblotting using subtype selective antibodies. Functional response was studied by Ca2+ fluorescence using fura-2. Messenger RNA encoding all 3 subtypes was detected by RT-PCR and confirmed by sequencing. Immunoblotting revealed single bands corresponding to the predicted molecular mass for NK1 and NK2. The result with anti-NK3 was variable. Free intracellular calcium ([Ca2+ ]i), measured by fura-2 fluorescence was 13020 nM at baseline. Stimulation with 1 mM b-Ala8-NKA (Neurokinin A analogue, 20 s) produced a transient increase of [Ca2+ ]i to 31524 nM. This transient persisted with removal of extracellular Ca2+ but also was partially blocked by 1 mM nifedipine (425%) indicating both release of Ca2+ from intracellular stores and influx contribute to the response. The selective NK2 antagonist (SR48968, 2 nM) blocked 903% of the response with partial recovery on washout, whereas 2 nM SR140333 (NK1 antagonist) and 2 nM SR142801 (NK3 antagonist) had more modest effects (163% and 171%, respectively). In conclusion, human esophageal smooth muscle cells express multiple NK receptor types. Excitation is mediated mainly by the NK2 receptor with a rise in [Ca2+ ]i due to both influx of Ca2+ and release from intracellular stores.

Cell Biology International, Vol. 25, No. 10, 2001

MECHANISMS UNDERLYING HCO3
TRANSPORT ACROSS APICAL MEMBRANE OF MOUSE ENDOMETRIAL EPITHELIUM X. F. Wang and H. C. Chan Epithelial Cell Biology Research Center, Department of Physiology, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong

The mechanisms of HCO3
transport across apical membrane of endometrial epithelial cells is responsible for high HCO3
concentration in the uterine fluid which provides an optimal environment for sperm capacitation and embryo development. Using intracellular pH (pHi) measurement, we investigated the apical cellular mechanisms involved in mediating HCO3
secretion across cultured mouse endometrial epithelium. pHi was measured using the PTI Ratio-Master fluorescence system. Polarized monolayer of endometrium cells was loaded with BCECF-AM, a pH-sensitive fluorescent dye, and then mounted in a miniature Ussing chamber perfused with different media. The HCO3
transport mechanisms across apical membrane was explored by monitoring the recovery of pHi from an alkaline load. Cells were initially perfused in a physiological medium buffered with 25 mM HCO3
and CO2. Removal of HCO3
/CO2 from the bathing medium (replaced by HEPES) resulted in an elevation (alkalinization) of pHi subsequent to immediate exit of CO2 from the cells, and the cells were allowed to recover from the alkalinization. In the present study, the HCO3
efflux rate from alkalinization was 8.480.9810
2 pH/ min in apical medium containing Cl
. In contrast, when Cl
was replaced with gluconate anion, the recovery was greatly alternated with a HCO3
efflux rate of 3.610.6110
2 pH/min, indicating the presence of a Cl
-dependent HCO3
extrusion, likely to be Cl
/HCO3
exchanger (AE, anion exchanger). In the presence of HCO3
, removal of apical Cl
also induced pHi increase (alkalinization), and then reintroduced Cl
resulted in pHi recovery, further confirming AE activity. The subsequent Cl
-dependent HCO3
efflux was completely blocked by apical addition of AE blocker DIDS. To investigate the involvement of CFTR, forskolin, an adenylate cyclase activator, and DPC, a Cl
channel blocker, were used. The HCO3
efflux rate was significantly increased when forskolin was added, while the forskolin-induced increase was suppressed by addition of DPC, indicating HCO3
transport may be mediated directly through CFTR channel. Using antisense transfection technique to inhibit CFTR expression resulted in inhibition of the forskolin-stimulated and AEmediated HCO3
efflux. In Conclusion, Cl
/HCO3
exchanger is critical mechanism in mediating HCO3
transport across apical membrane in endometrial epithelium under unstimulated condition. HCO3
transport was increased by activation of CFTR. On the other hand, CFTR is also likely to be an important regulator in mediating HCO3
secretion.

COLLAGEN GEL-INDUCED APOPTOSIS IN LLC-PK1 CELLS IS ASSOCIATED WITH DEGRADATION OF CALRETICULIN Yao-Hsien Wang, Yang-Kao Wang and Ming-Jer Tang Department of Physiology, National Cheng-Kung University Medical College, Tainan, Taiwan, 70101

Previous studies in our laboratory demonstrated that epithelial cells developed apoptosis upon contact with type I collagen gel. Morphologically, LLC-PK1 cells grown on collagen gel exhibited a more contracted cell shape and pulled collagen fibres before they underwent apoptosis. In the present study, confocal spectral microscopy was employed to delineate the changes of actin filament, focal adhesion kinase (FAK) and endoplasmic reticulum (ER) in response to collagen gel. Collagen gel resulted in disruption of cortical actin as well as mis-localization of FAK in LLC-PK1 cells. To understand the biochemical changes leading to collagen gel-induced cell death, we assessed changes of FAK, ER proteins and caspases. Collagen gel induced cleavage of FAK in various cell types, but did not necessarily induce apoptosis of them. In contrast, collagen gel induced time dependent cleavage of calreticulin starting from 1 h, which could be the initial trigger of collagen gel-induced apoptosis. In addition, collagen gel also triggered activation of caspase-3 within 18 h when apoptosis was actively present, but DEVD-fmk, an inhibitor for

1067 caspase-3, could not block collagen gel-induced apoptosis. Interestingly, calpain inhibitors or proteinase inhibitor cocktail alone could prevent calreticulin degradation and inhibit caspase-3 activation, but did not completely block collagen gel-induced apoptosis. Nevertheless, the combination of calpain inhibitor and protein inhibitor cocktail completely prevented collagen gel-induced apoptosis. These results indicated that activation of proteases plays important roles in collagen gel-induced apoptosis.

POSSIBILITY OF TREATING AGED DEMENTIA WITH LOW INTENSITY LASER Wang Yi-zhen, Tan Run-chu and Li Ping-yang Department of Physics, Sun Yat-sen University of Medical Sciences, China

Aged dementia (AD) is a recessive disease of central nerve system. The incidence of the disease in people more than 65 years old is approximately 10–15%. The pathogenies of AD are still being probed. The AD patients whose expirations are about 5–10 years suffer a main sympton of decline in memory and intelligence and consequently lose the ability of taking care of their own lives. It is found through body dissection that patients lose in basal forebrain large cholinergic nerve cell that emit fibres to hippocampus and provide a path closely related to learning and memory. Thus an assumption to protect and restore the injured septal-hippocampus cholinergic path is brought forward to conduct the treatment of AD. According to this hypothesis there are three therapies: substitution of nerve transmitter, transplant of brain tissue and nutrition of nerve. None of the therapies has achieved ideal effect while the new gene therapy is still in exploration. Considering the mechanism of AD, several transmitter systems like monoaminergic system and neuropeptidenergic system are injured besides intracephalic cholinergic system. These neurons also play important roles on learning and memory. Therefore trying to improve the life and function of the injured neurons of these transmiter systems will probably bring breakthrough to the treatment of AD. Low intensity laser radiation can diminish inflammation, ease pain, enhance immunity, improve microcirculation and advance renovation and regeneration of tissues. Further research demonstrates that it can enhance activities of some enzymes, stimulate phagocytosis of phagocytos, accelerate composition of gammaglobin and ATP, impact oxidation and deoxidation of cell respiration chain and improve the growth activity of cells. It is noticeable that a lot of experiments proved that proper dose of laser radiation can exert active influence on tissue growth and development and renovation and regeneration of injured tissues like skin, bone, blood vessel, nerve, enteric adrenergic structure and cholinergic structure. This clews that laser may have good effects on growth, life and restoration of various injured neurons and resume functions of injured nerve transmiter systems. So laser provide a possible way to cure AD. Memory is very closely related with electromagnetic phenomenon. Hence some kinds of electromagnetic field and electromagnetic wave could influence memory. It was reported that protein compounds could be decomposed in a very low intensity of electric field. This discovery provides a new concept to explain physiologic activities like nerve memory and transfer. Laser, a special kind of intense electromagnetic wave, has possibility to impact memory and correlative diseases.

DIFFERENTIATION AND TANSCRIPTION IN MEGAKARYOLOID CHRG-288 AND MEG-1 CELLS TREATED WITH PANAXDIOL OF GINSENG Wu Chaoqun1, Gao Ruilan2, Li Yao1, Jin Jinmai2 and Xu Weihong2 1 State Key Laboratory of Geneticengineering, School of life science, Institute of Genetics, Fudan University, Shanghai, and 2Afflicted Hospital of Zhejiang College of Traditional Chinese Medicine, Hangzhou 310006, China

The objective of this study is to explore the effects of Ginseng Panaxdiol (PDS) on megakaryoloid differentiation in CHRG-288 and Meg-1 cells, and the corresponding gene expression profiles. PDS was extracted from total panax of Ginseng fourthly. CHRP-288 and Meg-01 cells were incubated with PDS (5 g/ml) for 2 weeks. (1) Collected cells were labelled respectively with antibodies recognizing

1068 platelet glucoprotiens including CD36 (GPIV or GPIIIb), CD41 (GPIIb/IIIa), CD42b (Ib), CD61 (GPIIIa) and TSP, followed by Flow Cytometer (FACS) analysis; (2) mRNA extracted from treated cells as well as untreated cells were used as temples to synthesize probes which subjected to hybridization with cDNA chips representing 4000 of human genes. Laser scanning data was analyzed and the results showed that: (1) FACS date suggested that in the two types of cells, cd41 + , CD42b + , CD61 + and TSP + cells were significantly increased in number through PDS treatment for one month, indicating the effects of PDS in cell differentiation; (2) Microarray screening confirmed that 25 genes in CHRG-288 and 37 genes in Meg-01 cells were transcriptionally amplified, and that PDS could up-regulate the expression of cell differentiation related genes in both cell lines, such as G088 gene (Access L13463, G0/G1 switch regulator, human helix-loop-helix basic phosphoprotein) and C5-4 gene (Access M26683, human interferon gamma treatment inducible mRNA); (3) Other genes up-regulated by PDS in both two cells were mainly involved in G-protein coupled receptor, tumour suppressor p53 associated proteins, zine-finger proteins, etc. In conclusion, PDS effectively induced differentiation of megakaryoloid CHRG-288 and Meg-01 cells responses to PDS.

HUMAN SKIN CELL CULTIVATION AND ITS APPLICATION IN EPITHELIAL CELL BIOLOGY RESEARCH Xia Long Qin Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College, Nanjing 210042, China

The epidermis is a multilayered squamous epithelium and covers human skin surface. The human skin is the biggest organ of the body with approximately 5% of human body weight being skin weight. It consists of epidermis, dermis and subcutancous tissue, and has numerous blood vessels, nerves, lymphatics, hair follicles, sebaceous glands and sweat glands. Skin has many functions such as protection, secretion, excretion and absorption etc. Human skin plays an important role in keeping the stability of inside and outside environments of the organisms body. Skin cell culture in vitro is one of the most promising tools for evaluation of skin pharmacology, toxicology, physiology and pathology. We have established a series of the human skin cell cultivations such as cultivation system of keratinocytes and sebocytesm melanocytes and fibroblasts, skin organtype and organ culture. We established first the methods of isolation of human sebaceous gland and cultivation of sebocytes in 1988 in the world. We used those systems to study pathogenesis, biologic activity of related factors, expression and regulation of related genes, and screen of drugs that treat psoriasis, vitiligo, hair disorders, acne, skin tumours and skin aging. Particularly we have also studied on the effects of Chinese traditional medicines in treatment of these diseases to accelerate modernization of Chinese traditional medicines.

PHOTOACOUSTIC ABSORPTION CHARACTERISTICS OF SUNE-1, CNE-2, AND CNE-1 HUMAN NASOPHARYNGEAL CARCINOMA CELL LINES MEASURED USING PHOTOACOUSTIC TECHNIQUES Xia Yun-fei1, Zheng yun-tin2 and Tang zhi-lie2 1 Cancer Center, Sun Yat-sen University of Medical Sciences, 2 Department of Computer science, South China Normal University

To investigate photoacoustic biological characteristics of human nasopharyngeal carcinoma cell-lines. Methods: Standardized absorption photoacoustic spectroscopy and optical absorption coefficient of SUNE-1, CNE-2, and CNE-1 were measured respectively using double beam standardized photoacoustic technique. The results are shown (1) Between wave length 4000? and 6000?, optical absorption coefficient of these three cell lines is: SUNE1>CNE-2>CNE1; (2) Under the point of wave length 4200?, related photoacoustic signal mean values for SUNE-1, CNE-2, and CNE-1 are 1.36, 1.20, and 1.10 respectively, difference among them is significance statistically.

Cell Biology International, Vol. 25, No. 10, 2001 In conclusion, human nasopharyngeal carcinoma cell lines SUNE-1, CNE-2, and CNE-1 have different photoacoustic biological characteristics; this study establishes a foundation for further researchs on optical biological behaviors of nasopharyngeal carcinoma.

REGULATION OF OVINE PITUITARY ADENYLATE CYCLASE ACTIVATING POLYPEPTIDE (oPACAP) ON GROWTH HORMONE (GH) SECRETION AND SIGNAL TRANSDUCTION PATHWAYS IN PACAP STIMULATED GH SECRETION IN COMMON CARP, CYPRINUS CARPIO Dong Xiao1, Anderson O. L. Wong2 and Hao-Ran Lin1 1 School of Life Sciences, Zhongshan University, Guangzhou 510275, P. R. China; 2Department of Zoology, University of Hong Kong, Hong Kong

Regulation of ovine pituitary adenylate cyclase activating polypeptide (oPACAP) on growth hormone (GH) secretion and signal transduction pathways in PAAP-stimulated GH release in common carp were examined in vitro. The direct regulatory actions of oPACAP on GH secretion at the pituitary level were demonstrated in vitro using perifused common carp pituitary cells. OPACAP-38 (0.01 nM to 1 mM) and oPACAP-27 (0.01 nM and 1 mM) were all effective in stimulating GH release in dose-dependent manner; human vasoactive intestinal polypeptide (hVIP) (0.01 nM to 1 mM), a peptide structurally related to PACAP, was no effective in this respect. In addition, the G11 response to oPACAP-38 (100 nM) was suppressed by simultaneous treatment with norepinephrine (NE, 0.5 M) or serotonin (5-HT, 0.5 M). To further investigate the post-receptor signalling mechanism of PACAP action in the common carp, the possible involvement of the camp- and Ca2+ -dependent pathways in PACAP-stimulated GH release were examined. Ten minutes pulses of different dosages of adenylate cyclase activator Forskolin (0.1, 0.3, 1 and 3 M), phosphodiesterase inhibitor IBMX (0.1, 1, 10 and 100 M), cAMP membrane permeable analog OPT-cAMP (0.1, 0.3, 1 and 3 mM), and a calcium ionophore A23187 (0.3, 1, 3 and 10 M) all stimulated a rapid and dose-dependent increase in GH release from the perifused common carp pituitary cells, respectively. Parallel with its GH-releasing effect, oPACAP-38 (0.01 nM to 1 mM) and oPACAP-27 (0.01 nM to 1 mM) were all effective in stimulating cAMP synthesis from common carp pituitary cells in a dose-dependent manner under state incubation conditions. Furthermore, the GH response to oPACAP-38 (100 nM) was attenuated by the adenylate cyclase inhibitor MDL12330A (30 M), protein kinase A (PKA) inhibitor H89 (20 M) or was significantly suppressed or even totally abolished by removed extracellular Ca2+ with EGTA and voltage-sensitive Ca2+ -channel (VSCC) blocker nifedipine (10 M). In addition, oPACAP-38 (5 M), oPACAP-27 (0.5 M) and sGnRH (50 M) were also effective in elevating [Ca2+ ]i in common carp pituitary cells, these stimulatory actions could be abolished by removing [Ca2+ ]e using EGTA (4 mM) and recovered by adding excess CaCl (2 mM). [Ca2+ ]i increase induced by oPACAP-38 (0.5 M) of oPACAP-27 (0.5 M) was blocked by nifedipine (50 M), but oPACAP-38 (5 M) was not effective in elevating [Ca2+ ]i after common carp pituitary cells were challenged with nifedipine (50 M). These results, taken as a whole, strongly suggest that PACAP is a part of the neuroendocrine system regulating GH release in the common carp. The GH-releasing action of PACAP is probably mediated through the adenylate cyclase/ cAMP/ PKA cascade and Ca2+ entry through VSCC in common carp.

EFFECT OF INTRANIGRAL INJECTION OF IRON ON DOPAMINE RELEASE AND CONTENTS IN CPU OF RATS Jiang Hong and Xie Junxia Medical College, Qingdao University, 26602, China

Parkinson’s disease (PD) is a neurodegenerative syndrome. It always affects old people. Since many methods, such as cell transplantation and gene therapy, could not make any sense on cure for Parkinson’s disease, many new ways have been made from etiology to pathology, and from every side. Elevated iron concentrations in the substantia

Cell Biology International, Vol. 25, No. 10, 2001 nigra (SN) pars compacta have been implicated in the development of Parkinson’s disease. The ability of iron to generate free radicals by Fenton reaction, and its high affinity to melanin, which can enhance free radical formation, implicated its possible role in the etiology of Parkinson’s disease. Using fast cyclic voltammetry (FCV), atomic absorption spectrometer and high performance lipid chromotophotography (HPLC), we studied the relationship of iron contents and dopamine (DA) in FeCl3-lesioned parkinsonian rats. DA release in caudate putamen (CPu) evoked by stimulating medial forebrain bundle (MFB) was monitored by FCV. DA contents were measured by HPLC. We also studied the neuroprotective effects of iron chelator desferrioxamine mesylate. In FeCl3 lesioned rats, DA-related behavioral responses were significantly impaired. Apomorphine administration could induce contralateral rotation in 40 g FeCl3 treated rats. DA release in lesioned sides of CPu reduced, and the reduced extent was related to the dose of FeCl3. DA release was reduced by 12.2%, 23% and 84.3% at 10 g, 20 g and 40 g FeCl3, respectively. At the same time, DA contents in the lesioned sides of CPu also reduced, and the extent depended on the dose of FeCl3. DA contents were reduced by 72.7%, 85.7% and 99.5% at 10 g, 20 g and 40 g FeCl3, respectively. The contents of DOPAC and HVA reduced as well. Since iron can damage DA neurons, so we study the neuroprotective effect of iron chelator desferrioxamine mesylate. The results are promoting. When pretreated with desferrioxamine mesylate before 40 g FeCl3 injection, the release and contents of DA did not change in the lesioned sides. Taking these together, it suggests that DA neurons are susceptive to ionic iron, and iron can lead to degeneration of DA neurons, which can be seen by reduction in both DA release and contents. Iron is a strong cellular toxin. Its abnormal presence can lead cells to death. The role of iron in the degeneration of DA neurons should not be neglected. Iron chelator desferrioaxamine mesylate can reverse the neurodegenerative effects of Fe3+. More concerns should be put on the therapeutic effect of different iron chelators. Iron chelators that could go through the brain-blood barrier will sooner or later throw light on the therapy steps of PD. The project was supported by the ‘95’ National Plan of Tackling Key Problems in Science and Technology (96-906-05-08) and Youngth Foundation of Natural Foundation of Shandong Province(Q99C16).

INDUCTION OF ADIPOSE TISSUE ATROPHY BY PEROXISOME PROLIFERATORS IS BOTH PPARALPHA DEPENDENT AND INDEPENDENT IN MICE Y. Xie1, Q. Yang1, B. D. Nelson1, C. G. O } stenson2, S. E. H. 3 1 Alexson and J. W. DePierre 1 Unit for Biochemical Toxicology, Department of Biochemistry, Wallenberg Laboratory, Stockholm University, Sweden; 2Department of Molecular of Medical & Rolf Luft Center for Diabetes Research, Karolinska Institute, Sweden; 3 Department of Medical Laboratory Science and Technology, Division of Clinical Chemistry, Karolinska Institute, Huddinge University Hospital, Sweden

In the present study, we have demonstrated that potent PPs induce adipose tissue weight loss both in widetype and PPARalpha
/
mice with much stronger effect on widetype mice. In widetype mice this effect was dose- and time-dependent, furthermore, recovery is achieved within 10 days after withdrawal of PFOA although the persistence of peroxisome proliferation in liver. These indicate the effect of PPs on adipose tissue is both PPARalpha dependent and independent. Analysis of DNA content indicated that the decrease in adipose tissue weight was due to a loss of fat from adipocytes, rather than to a loss of whole cells. Enzyme assays indicated that the total activity of lipoprotein lipase (LPL) in adipose tissue is decreased, while hormone-sensitive lipase (HSL) activity is increased upon PFOA treatment. Thus, the dramatic loss of adipose tissue fat content caused by PFOA results, at least in part, from the changed fatty acid up-taking and releasing in adipose tissue. The analysis of serum lipid content showed that serum cholesterol level was closely related to the changed adipose tissue weight, in

1069 contrast, serum TG content more closely related to the peroxisome proliferation in liver suggesting serum cholesterol changes might be, in some way, a strong regulator of adipose tissue weight. In addition, the increased serum TNFalpha level and the impaired insulin secretion during glucose tolerance on treated mice lead to the hypothesis that insulin sensitivity on adipose tissue could be reduced upon PPs treatment, which is presently being examined in our Laboratory. An understanding of the exact mechanism(s) involved could, of course, have profound implications with respect to cardiovascular disease and diabetes drug design.

EXPRESSION OF PCNA, P53, P21WAF-1, RB IN FETAL ESOPHAGEAL EPITHELIUM AND THEIR RELATIONSHIP WITH CELL PROLIFERATION Xing Ying, Ning Yu and Yao yun-wei Department of Physiology, Zhengzhou University, Zhengzhou 450052, China

Cell in the fetal esophageal epithelium is one kind of cell with high proliferative activity and its cell cycle is regulated normally. Tumour suppressor genes (TSG) can suppress the proliferation of malignant cell through cell cycle. Cells in fetal esophageal epithelia proliferate actively, it is not known wether tumour suppressor genes can be involved in the proliferative activity of fetal esophageal epithelial cells through cell cycle regulation. The main modulation point is in G1 phase of cell cycle to determine whether the cell cycle progresses or is checked. The tumour suppressor genes p53, p21WAF-1, Rb plays an important role in G1 phase.To explore the expression of TSG in epithelial cells of fetal esophagus, not only can contribute to primarily understand wether tumour suppressor genes influence on the proliferation or differentiation of fetal esophageal epithelial cells,but also to provide data for the study of the proliferation character of fetal esophageal epithelial cells. Most reports about fetal esophagus etiologically probe into the mechanism that how certain chemical factors (nitrosamine) or biological factors (alternariol) or other can induce the malignant transformation of fetal esophageal epithelia, there are few reports on the proliferation characters in fetal esophageal epithelial cells by cell cycle regulation or cytokinetics. Immunohistochemical avdin-biotin peroxidase complex (ABC) technique was used to detect the expression of tumour suppressor gene p53, p21WAF-1, Rb and index of cell proliferation kinetics, PCNA in fetal esophagus. The relationship between the results and the proliferative cytokinetics are analyzed. The results of different types of esophageal epithelias were used to study the proliferative character of fetal esophageal epithelial cells and the effect of tumour suppressor genes on the proliferation of fetal epithelial cells. The results shown that: (1) PCNA immunostain-positive cells were located mostly in the epithelial basal cell layer and sporadically observed in submucosa layer or muscle layer. Positive IR was localized in the nuclei in dark brown colour. The average of positive IR cells per mm2 is 506239. There were significant differences (P<0.05, t-test) between the fetal PCNA positive IR cells with the adult normal basal cell proliferation and malignant epithelia. (2) Positive-IR cells of p53 were located in the nuclei in dark brown. p53-positive- IR rate was 10%. There were significant difference (P<0.05, Chi-square test, Fisher’s exact test) when fetal positive rate were compared with normal, basal cell proliferation, dysplasia, and cancerous epithelia. (3) No p21WAF-1 positive IR cell was observed in either cell nuclei or cytoplasm. (4) No Rb positive IR cell was observed in either cell nucle or cytoplasm. In conclusion, (1) The positive IR of PCNA reflect the active proliferative status of fetal esophageal epithelia. (2) The expression of p53 was only in 3 fetal esophageal epithelia, suggest that p53 seldom participates in the active proliferation or differentiation process of fetal esophageal epithelia. (3) Neither p21 nor Rb expression could be detected in fetal esophageal epithelia, suggest that they seldom participate in the active proliferation and differentiation process of fetal esophageal epithelia. (4) The different expression of p53 and Rb between fetal and other types of adult epithelia suggest that fetal esophageal epithelia may process speical proliferative activity. K: fetal esophagus; cell proliferation; tumour suppressor gene; immunohistochemistry; proliferation-cell-nuclear-antigen.

1070

MODULATION OF ENDOTHELIAL CELL FUNCTION BY INTRAVENOUS IMMUNOGLOBULINS— POSSIBLE MECHANISM OF ACTION IN VASCULAR DISEASES Chen Xu1, Bruno Poirier, Jean-Paul Duong Van Huyen, Newton Lucchiari, Odile Michel, Jacques Chevalier and Srinivas Kaveri 1 Department of Histology & Embryology, Shanghai Second Medical University, Shanghai, China; The Unite de Recherche Immunopathologie Humaine, INSERM U430, Paris, France

Intravenous Immunoglobulin (IVIg) is increasingly used in the treatment of autoimmune and inflammatory diseases, including vasculitides and Kawasaki disease. However, the outcome of IVIg interaction with endothelial cells of the vascular bed is not clear as yet. We have investigated the effect of IVIg on the in vitro activation of human endothelial cells, as assessed by cell proliferation and reverse transcription-polymerase chain reaction-detected expression of mRNA coding for adhesion molecules (intercellular adhesion molecule-1 and vascular cellular adhesion molecule-1), chemokines (monocyte chemoattractant protein-1, macrophage colony-stimulating factor, and granulocyte-macrophage colony-stimulating factor) and proinflammatory cytokines (tumour necrosis factor-
, interleukin-1, and interleukin-6). IVIg inhibited proliferation of endothelial cells in a time-dependent manner. This effect was dependent on both Fc and F(ab )2 fragments of the immunoglobulin molecule and was fully reversible. Tumour necrosis factor-
and interleukin-1 also inhibited thymidine incorporation, but to a lesser degree. IVIg had no effect on basal levels of mRNA coding for the adhesion molecules chemokines and proinflammatory cytokines. IVIg fully down-regulated the expression induced by tumour necrosis factor-
or interleukin-1 of mRNA coding for these molecules. Thus blockade of cellular proliferation and of cytokine-induced expression of adhesion molecules, chemokines, and cytokines may explain the therapeutic effect of IVIg in vascular and inflammatory disorders.

NEURON APOPTOSIS AND EXPRESSION OF bcl-2, bax GENE AFTER SLEEP DEPRIVATION IN RAT HIPPOCAMPUS Cheng Jiangtao and Zhang Qian Department of Physiology, Zhengzhou University Medical School, Zheng Zhou 450052,China

Sleep is one kind of physiological processes under the meticulous adjustment of central nerve system (CNS), but its physiological functions are not clearly known. A lot of studies on sleep functions in human or animals by sleep deprivation found that sleep deprivation could induce body temperature fallen, feeble, brain damage, even death. Histological studies discovered that sleep deprivation could cause brain cells chromatolysis, protoplasmic vacuolization, nuclear breakage. There are two kinds of neuron death as other cells: necrosis and apoptosis. Many studies showed that neuron apoptosis not only exist extensively in the processes of nerve system’s development and damage repair, but also in many nervous system diseases. Whether sleep deprivation could induce neuron death, apoptosis or necrosis has not been shown so far .In the present work, we observed the neuron apoptosis in rat hippocampus by TUNEL staining and the expression of bcl-2 and bax genes by in situ hybridization technique after sleep deprivation. 32 health adult SD rats with weighted 200 g20 g of both sexes were divided into four groups randomly. Each group had eight rats. They are: (1) total sleep deprivation group (total sleep deprivation, TSD); (2) rapid eye movement sleep deprivation group (or paradoxical sleep deprivation, PSD), small pedestal diameter 7 cm, (3) rapid eye movement sleep deprivation control group, small pedestal diameter 13 cm; (4) normal control group. The group (1) and (2) were named as experimental group, group (3) and (4) as the control group. The TSD rats were deprived of sleep on the rotational disc (2 cycles/min, referred to Rechtschaffen’s TSD method), and PSD model derived of Cohen’s small-pedestal sleep deprivation method. Each rat was deprived of sleep for 72 hours beginning at 8 am of experimental day. The normal

Cell Biology International, Vol. 25, No. 10, 2001 control rats stayed within their home cage throughout the experiment keeping their normal sleep-awake cycle. Animals were perfused with paraformaldehyte following normal serum after test, then removed the brain and fixed 24 hours with paraformaldehyde, paraffin sections were prepared at 6 m, TUNEL staining for counting apoptosis ratio and in situ hybridization for calculating integral scores of bcl-2 and bax mRNA positive signals, and the data were analized with statistical treatment. The results showed as follow: 1.TUNEL staining: Apoptosis positive cell nuclei appeared blueish purple granules. The apoptosis percentage of hippocampal CA1, CA2, CA3, CA4 areas of TSD rats was: 7.861.67, 1.560.41, 8.361.87 and 1.510.39, respectively. The apoptosis percentage of CA1, CA2, CA3, CA4 areas in PSD rats was: 6.602.24, 1.690.45, 6.811.32 and 1.740.98, respectively. More apoptosis positive cells could be found in hippocampal CA1 and CA3 areas in the experimental groups, and mainly located in hippocampal pyramidal layer, There were more positive cells in initial part of CA1 and middle part of CA3. No positive cells can be seen in each hippocampal area of the control groups. The neuron apoptosis between the experimental and control groups had significant difference. The cell apoptosis ratio between hippocampal CA1, CA3 and CA2, CA4 areas had significant difference in the experimental group too (P<0.05). There was no obvious difference between the corresponding areas in hippocampus of the experimental groups (P>0.05).

CONSTRUCTION OF EXPRESSION VECTOR p64C-ANG-AAD FOR DUNALIELLA SALINA CHLOROPLAST Pan Weidong and Xue Lexun Biotechnology Laboratory, Zhengzhou University Medical College, 40 Daxue Road, Zhengzhou, Henan 450052, China

Angiostatin is an inhibitor of angiogenesis, which is a 38 kDa fragment degraded from plasminogen consisting of kringle 1 to 4, and is believed to inhibit tumour growth by blocking blood vessel formation. It has been considered as a new effective approach to treat tumours. Recombinant human angiostatin (hANG) has been produced in E. coli expression system but has too high costs and low activity of the products because prokaryotic cells cannot perform the glycosylation of the recombinant proteins and the proteins cannot be correctly folded into the functional conformation. To obtain hANG with higher biological activity and to reduce production costs, we have been studying the expression of hANG in the chloroplast of a unicellular eukaryotic alga Dunaliella salina and constructed an expression vector p64C-ANG-AAD for the Dunaliella salina chloroplast. The sequence of kringle 1 to 4 of human plasminogen was cloned from liver cell cDNA by RT-PCR and added with initiation codon ‘ATG’ and termination codon ‘TAA’ to form a completely expressible hANG sequence. This sequence was subcloned into the plasmid pUC-atpA-AAD that has strong promotor atpA and terminator rbcL in the chloroplast. The hANG sequence was located between atpA and rbcL and was able to be expressed in the chloroplast controlled by the promotor atpA and terminator rbcL. And then this hANG expression box was ligated with atpA-aadArbcL box that can express the resistance to spectinomycin. The plasmid p64C had been cloned several chloroplast genes clpP-trnlpetB and the noncoding regions of chlL gene: the promoter sequence chlL5 and the terminator sequence chlL3 . The hANG box and aadA box were inserted between the sequences of chlL5 and chlL3 , constructing the expression vector p64C-ANG-AAD for the Dunaliella salina chloroplast, in which the transcription of hANG sequence could be enhanced under the control of double promotors chlL and atpA. This expression vector p64C-ANG-AAD can be transferred and site-directed integrated into the chloroplast genome, replacing the chlL gene by homologous recombination of noncoding sequences of chlL5 and chlL3 , forming transgenic Dunaliella salina which can express recombinant human angiostatin efficiently. Transgenic Dunaliella salina can be selected by their resistance to spectinomycin. Because of the deletion of chlL gene caused by this homologous recombination, transgenic Dunaliella salina can also be easier selected under the dark culture for it cannot synthesize chlorophyll in the dark as wild-type does.

Cell Biology International, Vol. 25, No. 10, 2001

CONSTRUCTION OF SHUTTLE VECTOR FOR KRINGLE 5 IN EUKARYOTIC PLANT CELLS Zheng Heming and Xue Lexun Biotechnology Laboratory, Zhengzhou University Medical College, 40 Daxue Road, Zhengzhou, Henan 450052, China

Angiostatin (ANG) consisting of kringle 1 to 4 of plasminogen has been documented to be a potent inhibitor of angiogenesis in vivo and in vitro. The kringle 5 (k5) domain of plasminogen, which shares high sequence homology with angiostatin, has been shown to inhibit the growth and migration of the endothelial cells. At present, E. coli expression system has been used to produce recombinant k5, which is quite complex and very high cost. In this study, cDNA of plasminogen and target gene kringle 5 were obtained by use of RT-PCR and PCR, respectively. Then the shuttle expression vectors, pB1k5 and pC33k5, were constructed by use of sub-clone technique, which contain plant promoter CaMV35S and reporter gene GUS. Here, we report for the first time that pB1k5 and pC33k5 express not only in prokaryotic cells, but also in plant eukaryotic cells. Dunaliella salina is a unicellular green alga and able to adapt some of the harshest environments known on earth. In previous studies, we have developed an expression system for angiostatin in the chloroplast of Dunaliella salina. Attempts to express kringle 5 in transgenic Dunaliella salina are in progress.

RESISTANCE OF DUNALIELLA SALINA TO THE HERBICIDE PHOSPHINOTHRICIN Zhang Guixing, Zheng Heming, Xu Peirong and Xue Lexun Biotechnology Laboratory, Zhengzhou University Medical College, 40 Daxue Road, Zhengzhou, Henan 450052, China

Dunaliella salina is a wall-less unicellular green alga. As one of the most halotolerant eukaryotic organisms ever known in the world, Dunaliella salina can proliferate in media of 0.5–5.0 mmol/L NaCl. As a green alga, the photo-autographic Dunaliella salina only needs for its propagation a relative simple media composed of inorganic compounds. Under optimal conditions, Dunaliella salina can rapidly double its population in a shorter time than higher plants or animals. All these indicate that Dunaliella salina is an ideal organism for genetic engineering and eukaryotic gene expression research. Since Dunaliella salina is edible, it may be utilized to act as a safe bioreactor host to produce many invaluable products with biological activity. But currently, it is badly in need to study some of its fundamental biological characteristics such as resistance to antibiotics or other chemical reagents, so that we can select the transgenic algae from wild untransformed ones in the future molecular biology research,. Wild Dunaliella salina cells in fine proliferating state were spread on a solid plate containing 0.8% agar. After single colonies appeared, one single colony was selected and transferred to liquid medium. Using this method, a single colony of Dunaliella salina cells was isolated. This single colony of Dunaliella salina was utilized to study its sensitivity to the herbicide phosphinothricin. Three hundred l of single colony Dunaliella salina (5.0105cells/mL) in log phase was added in 5 ml of liquid medium in a series of tubes containing phosphinothricin from 0.048 to 6.0 g/mL. A tube of Dunaliella salina in liquid medium without any phosphinothricin was used as the control. The Dunaliella salina cells were grown under an illumination strength of 4500 lux at daytime with a culture pattern of 12 hour light:12 hour dark cycle. Cell density was determinated by measuring the light absorbance at 682 nm. The results revealed that wild Dunaliella salina was not of resistance to phosphinothricin. At dose of 3.0 g/mL, phosphinothricin completely suppressed the growth and proliferation of Dunaliella salina cells in liquid medium, which provides a potential method for selecting transformed Dunaliella salina through the phosphinothricin-bar system. A visible light full wavelength screening showed that Dunaliella salina cells have a maximum absorption at 682 nm, indicating that Dunaliella salina cells predominantly contain chlorophylla, with little or no chlorophyllb.

1071

MECHANISM OF ILL-1 AND IL-1ra SIGNAL CONDUCTION IN THE LUNG INJURY PROCESS AFTER INTESTINAL ISCHEMIA AND REPERFUSION Yan Guangtao, Hao Xiuhau, Xue Hui, Wan Luhuan, Li Yingli, She Liping and Zhang Kai Research Laboratory of Biochemistry, Basic Medicine Institute, General hospital of PLA, Bejing 10085, China

As one pair of cytokines conducting antagonistic roles, ILL-1 and IL-1ra have been proved to play important role in the process of pathology in trauma, infection and shock, in which the mechanism involving in the endotoximia, activation of phosphlipaseA2, the release of oxygen radicals and the activation of neutrophils. Phospholipase A2 activation introduced activating and gathering of neutrophils in lung have been considered to be the key step of the above mechanism. We had discovered that phospholipaseA2 inhibitors have some protection role to the lung injury after intestinal ischemia/ reperfusion (I-R) and related research indicated that cytokine of IL-1 family play important role in lung injury after intestinal I-R. This experiment plans to use I-R animal model, by giving some kinds of drugs including inhibitors of phospholipaseA2, blocker of oxygen radicals and platelet activating factor receptor antagonist, to observe the changes of mRNA expression and protein level of ILL-1 and IL-1ra in blood, lung tissue and lung lavage and further discuss the relation of this change with the development of lung injury after intestinal I-R and the activation of phospholipaseA2 as well as signal conduction in cell. The results demonstrated that ILL-1 and IL-1ra increased in blood after 6 hours of intestinal I-R injury. The level of ILL-1 in injury group was significantly higher than that of the control group (0.870.22 vs 1.410.38 ng/ml, P<0.01), but without differences between the self-control group before and after injury (1.230.61 vs 1.410.83 ng/ml, P<0.05). There was no difference between injury and control group of the IL-1ra level in blood (5.932.03 vs 6.041.17 ng/ml, P>0.05), there was significant difference between self-control before injury and after injury (4.031.01 vs 4.662.59 ng/ml, P<0.001), and IL-1ra without significant changes (3.880.35 vs 3.840.47 ng/ml, P>0.05). Also ILL-1 mRNA and protein level in lung tissue was distinctly increased, and IL-1ra was not obviously increased. After the treatment of no-specific inhibitors of phospholipaseA2 chloroquine, terflazine, platelet activating factor receptor blocker SR27417A, cyclic oxygen enzyme inhibitor indomethacin and antioxidant guaiac acid, there were some changes of ILL-1 and IL-1ra in serum, lung lavage and tissue, which was mainly the decreases of ILL-1 mRNA protein and the increases of IL-1ra as well as the significant reduction of inflammatory cell gathering and infiltrating in local lung tissue. All the results demonstrated that IL-1 and IL-1ra may play important signal conduction role in lung injury after intestinal ischemia and reperfussion, in which the mechanism may involve the release of prostaglandins, platelet activating factor and oxygen radicals.

EFFECT OF cGMP ON AGONIST-INDUCED [Ca2+ ]i OSCILLATIONS IN HUMAN BLADDER EPITHELIAL CELLS H. Y. Kwan1, Y. Huang1, S. K. Kong2 and X. Yao1 1 Department of Physiology and 2Department of Biochemistry, Chinese University of Hong Kong, Hong Kong, China

Oscillatory changes in intracellular Ca2+ concentration, or [Ca2+ ]i oscillations, occur in a variety of nonexcitable cell types. [Ca2+ ]i oscillations can be triggered by a great variety of stimuli including neurotransmitters, hormones, and growth factors. Of the natural stimuli, many are calcium-mobilizing agents that bind to cell surface receptors and then activate phospholipase C. Cytosolic calcium oscillations may permit cells to respond to information provided by increases in [Ca2+ ]i while avoiding prolonged exposure to constantly elevated intracellular Ca2+ concentrations. In this study, we demonstrated that agonists could induce Ca2+ oscillations in human bladder epithelial cells. Application of 10 M acetylcholine or 200 nM bradykinin triggered an initial Ca2+ transient that was followed by periodic [Ca2+ ]i oscillations. The oscillations did not depend on extracellular

1072 Ca2+ . 8-Br-cGMP abolished acetylcholine- or bradykinin-induced oscillations. Elevation of cellular cGMP by dipyridamole, an inhibitor of cGMP-specific phosphodiesterase, also terminated the [Ca2+ ]i oscillations. The inhibitory effect of cGMP could be reversed by KT5823, a highly specific inhibitor of protein kinase G, suggesting that the action of cGMP was mediated by protein kinase G. Comparison of the effect of cGMP with that of Xestospongin C (XeC), an inhibitor of IP3 receptor, revealed similarities between the action of cGMP and XeC. Therefore, it is likely that cGMP and PKG may target on signal transduction step(s) leading to the IP3 receptor-mediated Ca2+ release.

STRETCH-SENSITIVE SWITCHING AMONG DIFFERENT CHANNEL SUBLEVELS OF AN ENDOTHELIAL CATION CHANNEL Xiaoqiang Yao, Hiu-Yee Kwan and Yu Huang Department of Physiology, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, China

Mechanosensitive Ca2+ -permeable channels have previously been reported in vascular endothelial cells. These channels are suggested to be mechanotransducers, which transform the mechanical signal of blood flow into the chemical signal of intracellular Ca2+ level. Blood flow may open these mechanosensitive channels, resulting in a subsequent rise in [Ca2+ ]i. The elevation of [Ca2+ ]i stimulates the production and release of nitric oxide, PGI2 and endothelial-derived hyperpolarizing factor (EDHF). Nitric oxide, PGI2, and EDHF are important endothelial factors that can modulate vascular tone and blood pressure. We report the recording of a mechanosensitive Ca2+ -permeable cation channel in isolated rat aortic endothelial cells. A low level of channel activity could be observed after seal formation. The channel displayed some inward rectification and had a conductance for inward current of 32 pS in Ca2+ -free pipette and bath solutions. Negative suction of
10 to
20 mmHg increased the probability of the channel being open. When the negative pressure in the pipette was raised to
35 to
45 mmHg, the channel underwent an abrupt transition to a large conductance substate that was interrupted occasionally by two other low conductance levels. Under this condition, the overwhelming majority of openings and closings were between a main level of 83 pS and the closed level. Compared to the 32 pS substate, the 83 pS large conductance substate had shorter mean open and closed times. The two channel substates had similar ionic selectivity and both were sensitive to the inhibition of cGMP and protein kinase G. This is the first demonstration showing that mechanostress can change the single channel conductance level of an ion channel in eukaryotic cells.

SUBCELLULAR LOCALIZATION AND POST-TRANSLATIONAL MODIFICATION OF A NOVEL PDZ-DOMAIN PROTEIN, PIN-1/PAPIN Man-Lung Yeung, Melissa K. Thomas, Joel F. Habener1 and Kwok-Ming Yao2 1 Laboratory of Molecular Endocrinology, Massachusetts General Hospital, Harvard Medical School, and Howard Hughes Medical Institute, Boston, MA 02114, U.S.A.; 2 Department of Biochemistry, Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China

PIN-1 (PDZ-domain protein from INS-1 cells) was first isolated as a specific interactor of the bHLH transcription factor, E12 (Thomas et al., 0000). Independently, full-length PIN-1 (named PAPIN) was reported as a multi-PDZ domain protein that interacts with NPRAP/ -catenin and p0071 (Deguchi et al., 0000). To determine the subcellular localization of PIN-1, I epitope-tag the C-terminus of the full length rat protein with V5 for expression analysis in COS-7 cells. PIN-1 could be detected in the nucleus by immunocytochemical analysis and by Western blot analysis following subcellular fractionation, consistent with its interaction with E12. However, PIN-1 expression was also detected in the ER. In database searches, PIN-1 shows the strongest match to pro-IL16 (33.3% identity and 52.1% similarity over a stretch of 235 amino acids). Pro-IL16 is proteolytically cleaved at the carboxyl terminus to generate the secreted cytokine IL-16 (Zhang et al., 0000). The extensive homology suggests that PIN-1 may be subjected to similar post-translational modification. The

Cell Biology International, Vol. 25, No. 10, 2001 detection of V5-tagged peptides (38 kDa) by Western blot analysis and Mass spectrometry in conditional medium recovered from PIN-1transfected cells support the cleavage and subsequent secretion of the carboxyl terminus of PIN-1. Generation of secreted PIN-1 (sPIN-1) could be inhibited by treatment with the general caspase inhibitor B-D-FMK. Ongoing studies include immunoprecipitation sPIN-1 for sequence analysis and determination of the cleavage site within PIN-1 by mutagenesis. This study is supported by the RGC grant 7234/99M awarded to KMY. References T MK, Y KM, T MS, W GG, H JF, 1999. Mol Cell Biol 19: 8492–8504. D M, I T, H Y, N W, H K, Y I, K H, T Y, 2000. J Biol Chem 275: 29875–29880. Z Y, C DM, W MH, C WW, Y J, A DW, K H, 1998. J Biol Chem 273: 1144–1149.

A DEVELOPMENT-REGULATED NOVEL GENE, NYD-SP8, FOUND IN HUMAN TESTIS Lan-Lan Yin1, Jian-Ming Li1, Zuo-Ming Zhou1, Hsiao Chang Chan2 and Jia-Hao Sha1 1 Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing 210029, P. R. China; 2Epithelial Cell Biology Research Center, Department of Physiology, The Chinese University of Hong Kong, Shatin, NT Hong Kong

Development of the spermatogenetic cell lineage occurs throughout most of the pre- and post-natal lifetime of male mammals, which involves progression through a series of well-characterized stages and cell types in the testis. To a certain extent, spermatogenesis is controlled or regulated by programmed genes that are differentially expressed at different stages of development in the testis. Using a human testis cDNA microarray constructed in our laboratory, we compared the genes expression in testes of human fetus and adult in a genome-wide fashion. Here we report a novel gene, NYD-SP8, which was differentially expressed at different stages, with an expression level higher in the adult testis than that in the immature testis. Tissue distribution studies using RT-PCR showed that the gene was expressed specifically in the testis but not other tissues examined except a weak signal found in the pancreas. The gene appeared to be localized in the sertoli cells according to in situ hybridization, and mapped by polymerase chain reaction on a panel of somatic cell hybrids to chromosome 19. It is composed of 6 exons extending over 3679 nucleotides. The complete cDNA contains an open reading frame of 750 nucleotides, which appears to encode a novel protein including a signal peptide and a transmembrane domain. This gene is likely to be involved in testis development or spermatogenesis. Its possible involvement in the regulation of testicular functions is currently under investigation.

RESISTANCE OF RECOMBINANT ALPHA-VIRUS CONTAINING DENGUS 2PrM GENE TO VIRUS INFECTION Yu Man, Qin Ede, Zhao Wei, Hy Zhijun, Yuan Xitong, Geng Liqing, Wang Pengcheng, Chen Shuipingm, Fan Baochang, Duan Hongyuan and Yang Peiying Department of Virology, Institute of Microbology and Epidemiology, Beijing, 10007, China

The PrM gene of dengue virus codes for virion precursor membrane protein (PrM), whose homology is high in the four stereotypes of dengue virus. The PrM protein is important to the assembly maturation of virion. The aim of our study was to insert the PrM gene of the Chinese dengue 2 virus into the alphavirus vector (pSFV), construct the recombinant alphavirus containing sense- or antisense PrM gene and observe the recombinant virus-mediated resistance to dengus viruses. The construction of recombinant aplhavirus will be helpful to the investigation of a novel way of prevention and treatment to dengue diseases.

Cell Biology International, Vol. 25, No. 10, 2001 The amplified PrM gene of dengue 2 virus was cloned into the downstream SP6 promoter of pSFV vector and the recombinant plasmid (pSPrM2) DNA which contained sense- or antisense-PrM gene was selected. pS PrM2 DNA and helper DNA linearized by the enzyme SpeI digestion were both transcribed in vitro into recombinant RNAs which contained the capping analog on the 5 end and cotransfected into BHK cells by electroporation. Then the transfected host cells were challenged with dengue-2 virus and the resistant efficiency of recombinant virus RNAs containing sense- or antisense-PrM gene to virus infection were observed, respectively. The recombinant plasmids (pSFV-PrM) containing sense- or antisense-PrM gene were selected with determination of the nucleotide sequence. The recombinant virus particles were obtained with recombinant RNA and helper RNA cotransfected into BHK cells. Host cells transfected with antisense-PrM RNA derived complete resistance to dengue-2 virus replication and the efficiency was higher than that of recombinant virus RNA containing sense-PrM gene.

ODQ, A SOLUBLE GUANYLYL CYCLASE INHIBITOR, INCREASES THE NEUROTOXICITY OF NITRIC OXIDE (NO) DONOR AND, BY ITSELF, CAUSES THE ONSET OF APOPTOTIC DNA FRAGMENTATION IN THE NG108-15 NEURONAL CELL LINE Jessie P. S. Yuen and Ronald R. Fiscus Department of Physiology, Faculty of Medicine, Epithelial Cell Biology Research Center, and the Center for Gerontology & Geriatrics, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong

Our previous studies have shown that neural cell lines, such as PC12 and C6 cells, respond to NO or atrial natriuretic peptide (ANP) with increases in production of cyclic GMP (cGMP), resulting in increased levels of cGMP in both the intracellular and extracellular spaces (Fiscus et al., 1987). The cGMP elevations resulted from NO- and ANP-induced activation of soluble guanylyl cyclase (sGC) and particulate guanylyl cyclase (pGC), respectively. However, at the time, the biological function of this signalling pathway in neural cells was unknown. Later, in collaboration with Mark Mattson’s laboratory at the University of Kentucky, we found that elevation of cGMP levels in hippocampal neurons increases their survival, specifically blocking cell death induced by stress, such as glutamate toxicity (Barger et al., 1995). Recently, we have shown that cGMP inhibits the onset of apoptosis and prolongs the survival of stressed PC12 cells, a cell culture model of catecholaminergic neurons (Fiscus et al., 2001). Specifically, elevating cGMP levels with either ANP or a related peptide, brain natriuretic peptide (BNP), inhibited the apoptotic DNA fragmentation induced by serum deprivation in the PC12 cells. We determined if cGMP has similar protective effects in another neuronal cell line, the NG108-15 (cholinergic neuronal) cells, and if lower cGMP levels (e.g. basal cGMP levels) are needed to protect against onset of apoptosis. ODQ, a potent and selective blocker of sGC, was used to determine if inhibiting cGMP synthesis increased the susceptibility to the pro-apoptotic actions of the NO donor S-nitroso-Nacetylpenicillamine (SNAP). NG108-15 cells were plated at 5106 cells/dish in 100 mm culture dishes. After one day, the cells were exposed for 24 h to SNAP, at various concentrations up to 1 mM, either with or without co-incubation with ODQ (40 M). DNA was extracted from the cells and apoptotic DNA fragmentation (DNA laddering) was analyzed on 2% agarose gels. SNAP, at 0.5 and 1 mM, caused a high level of apoptotic DNA fragmentation. Inhibition of sGC with ODQ (40 M) exaggerated this toxic action of SNAP. Interestingly, ODQ (40 M), by itself, caused noticeable apoptotic DNA fragmentation, suggesting that even basal levels of cGMP (i.e. in the absence of added NO) may be important for protecting neural cells against onset of apoptosis. Similar results were obtained in four other experiments with NG108-15 cells. These data suggest that low basal levels of cGMP (0.1–1 pmol/106 cells) may protect cells against apoptosis and that elevated levels of cGMP in the presence of added NO may serve as an important counter-balance to the toxic actions of NO. Supported by a Direct Grant for Research

1073 References F, R, W, M, 1987. J Neurochem 48: 522–528. B, F, R, H, M, 1995. J Neurochem 64: 2087–2096. F, T, C C, 2001. NeuroReport (Neurochemistry) 12: 185–189.

RESISTANCE OF M-CSF-INDUCED BONE MARROW-DERIVED MACROPHAGES TO APOPTOSIS IS ASSOCIATED WITH UP-REGULATION OF XIAP Jiyan Zhang1, Yan Li1, Hong Lin2, Ben Chen2 and Beifen Shen1 1 Department of Molecular Immunology, Institute of Basic Medical Sciences, Academy of Military Medical Sciences, P. R. China; 2Department of Hematology and Oncology, Wayne State University School of Medicine, Detroit, U.S.A.

It is known that immature hematopoietic cell such as CD34+ cells are fragile and strictly cytokine-dependent. They become more resistant to apoptosis when induced to differentiate into monocytic lineage. This finding suggests that an anti-apoptotic mechanism occurs during the differentiation process. Other types of blood cells such as granulocytes are more sensitive to apoptosis, indicating that lineage-specific regulation of the anti-apoptosis mechanism. In this study we investigate the underlying mechanism with the use of M-CSF-induced bone marrowderived macrophage and G-CSF-induced bone marrow derived granulocytes. Immunoblot analyses showed that bone marrow precursors express appreciable levels of caspase-3 and caspase-9 but no or very low levels of the apoptosis regulators X-linked inhibitor of apoptosis protein (XIAP), Bcl-2 and Bax. The differentiation of immature precursor cells into mature macrophage is associated with a steady and gradual increase in the levels of XIAP, Bcl-2 and Bax. However, the levels of caspase-3 and caspase-9 stayed essentially unchanged. Unlike immature precursor cells, mature macrophages were resistant to apoptosis induced by M-CSF depletion, which include the degradation of caspase3, caspase9, XIAP, Bcl-2 and Bax proteins in the process. The levels of the apoptosis-related proteins stated above remained unchanged during granulocyte differentiation. And mature granulocytes were still sensitive to apoptosis induced by cytokine depletion. These data suggest that the up-regulation of anti-apoptotic proteins may confer resistance on mature macrophages. Treatment of mature macrophages with XIAP anti-sense oligonucleotides (oligos), but not sense oligos, partly abolished their resistance to apoptosis. With a specific nuclear factor B (NF-B) inhibitor, we demonstrated that NF-B activity is responsible for the up-regulation of XIAP in M-CSF-induced macrophages. In addition, treatment of starved mature macrophages with M-CSF induced a rapid phosphorylation of Akt kinase. Our results showed that XIAP is one of the anti-apoptotic regulators that confer resistance on mature macrophages induced by M-CSF. K: caspase; differentiation; macrophage; granulocyte; M-CSF, G-CSF; NF-B.

EFFECT OF LIGUSTRAZINE ON INSULIN SECRETION IN THE NEWBORN RAT PANCREATIC ISLETS Xing Ying and Zhang Weihong Department of Physiology, Zhengzhou University Medical School, *Henan College of Traditional Chinese Medicine, Zhengzhou, 450052, China

The increase in cytosolic Ca2+ of the pancreatic B-cell plays a key role in the initiation of insulin secretion from the pancreatic B-cell under physiological condition. It is well known that insulin secretion is primarily dependent on the influx of the extra-cellular Ca2+ , which is mediated by the indirect activation of voltage-dependent calcium channels. It also has been reported that ligustrazine could act as an activator of calcium channel, and it has been used to prevent and treat 2-diabetes mellitus and its chronic complications. However the direct effect of ligustrazine on insulin secretion in pancreatic islets has not been reported. Our study investigated the effect of ligustrazine on insulin secretion in pancreatic islets of the rats and its mechanism

1074 in vitro. The pancreatic islets were removed and isolated from 3 to 5-day-old Sprague-Dawley rats. The isolated islets were free-floated in culture medium RPMI-1640 containing 10% (V/V) calf serum and were distributed randomly in 96 well -plastic plates according to the experimental design. The reagents were added after islets were precultured for 48 h. The insulin in medium was measured by radioimmunoassay. The results showed that after being treated with ligustrazine (0.5, 1.0, 2.0, 4.0 mmol/L) for 24 hours, the basal insulin release (BIR) (298.942.4, 426.053.9, 432.452.1, 416.2 51.2 IU/ml) was dose-dependently increased, which showed significant difference (P<0.05) compared with control group (294.0 18.0 IU/ml), except 0.5 mmol/L group. But 1 h glucose (20 mmol/L) stimulation insulin release (GSIR) had no difference. The enhancement of insulin secretion by ligustrazine was also time-dependent. After the co-culture of islets with 1.0 mmol/L ligustrazine for 12 h, 24 h and 36 h respectively, the BIR was 285.275.3, 42653.9, 488.757.9 IU/ ml respectively. In comparison with that of the control groups (19749.7, 29418, 384.733.5 IU/ml), the difference was significant (n=6, P<0.05). After being treated with Verapamil (25, 50, 100 ng/ml), a calcium channel blocker, the BIR (215.459.8, 198.731.6, 178.825.0 IU/ml) was dose-dependently inhibited significantly (n=6, P<0.05) compared with normal control group (305.739.7 IU/ml). When the islets co-cultured with Ligustrazine and Verapamil for 24 hours, the increase in BIR induced by ligustrazine (2 mmol/L) was inhibited by Verapamil (50, 100 ng/ml), which showed significant difference (n=6, P<0.05). These results indicated that ligustrazine may be an activator of calcium channel, and it may enhance insulin release through acting on calcium channel and increasing Ca2+ influx. K: pancreatic islets; insulin; calcium channel; ligustrazine; Verapamil.

MOLECULAR MECHANISM FOR CHRONIC MYELOGENOUS LEUKEMIA Robert C. H. Zhao1,2, Dong Li1, Dong Xu1, Junmin Song1, Erjin Fan1 and Catherine M. Verfaillie2 1 Sino-America Collaborative Lab, National Lab of Experimental Hematology, Institute of Hematology, PUMC & CAMA, Tianjin, P. R. China; 2Stem Cell Institute and Cancer Center, Department of Medicine, University of Minnesota, Minneapolis 55455, U.S.A.

Chronic myelogenous leukemia (CML) is a malignant disease of the human hematopoietic stem cell characterized by the bcr/abl fusion gene. The deregulated tyrosine kinase (TK) activity of bcr/abl-derived oncoprotein p210BCR/ABL is essential and sufficient for malignant transformation. At the cellular level, CML progenitors show decreased adhesion, enhanced migration, uncontrolled proliferation and delayed apoptosis. In order to understand the molecular basis in pathogenesis of CML caused by p210BCR/ABL, we developed a primary human CD34+ CML model by transducing normal CD34+ cells with b3a2 bcr/abl cDNA. We demonstrated that this recreates most pathological features in primary CML cells. Subsequently a series of studies were performed with this model. A novel and CML-related molecule as apoptosis-Inhibitor was cloned by subtractive hybridization from the bcr/abl containing CD34+ cells[1] (Genebank ID: AF332505). We also approved that up-regulation and relocation in cytoplasm of p27Kip contribute to adhesion defection and aberrant proliferation of CML cells due to the lack of p27Kip-mediated inhibition of cyclin-dependent kinase 2(Cdk2) activity (Zhao et al., 2001). Cyclin D2, a member of human G1 cyclin subfamilies, plays a pivotal role in outgrowth of progenitor cells in CML. In our study, the correlation between CML transformation and the expression of cyclin D2 was investigated. An elevated level of cyclin D2 was observed in CD34+ cells from CML patients, p210BCR/ABL transduced cells and K562 cells in comparison with normal CD34+ cells. Furthermore, an observation about the cell cycle regulation of cyclin D2 was made upon synchronized K562 cells by serum-starvation. The mRNA levels of cyclin D2 were demonstrated to oscillate periodically throughout the cell cycle. Cyclin D2 was synthesized in early G1 phase and declined during the rest of the cell cycle. For further study of the direct correlation between the expression of cyclin D2 and ABL tyrosine kinase activity in p210BCR/ABL containing cells, a second cell model

Cell Biology International, Vol. 25, No. 10, 2001 was constructed by transducing K562 cells with a retroviral vector to express intracellular single-chain antibody (intrabody, ib) directed against ABL tyrosine kinase domain. Cytoplasm expression of the intrabody markedly inhibits c-ABL and p210BCR/ABL tyrosine kinase activity followed by a down-regulation of the cyclinD2 mRNA expression. Our data suggests that cyclin D2 is a possible down-stream signal molecular of ABL tyrosine kinase signal pathway, and inhibition of the ABL tyrosine kinase activity by the intrabody could suppress the expression of this molecular and promotes apoptosis in p210BCR/ABL positive cells. The two cell models may therefore prove to be useful for the study of molecular mechanisms associated with the presence of p210BCR/ABL in CML (Jiang et al., 2000). References A novel and CML-related molecule as apoptosis-Inhibitor?Genebank ID: AF332505. Z RCH, J Y, V CM, 2001. Introduction of BCR/ ABL in normal CD34+ cells recreates the phenotype characteristic of CML-novel human CML model. Blood 97: 2406–2412. J Y, Z R, V CM, 2000. Abnormal integrinmediated regulation of CML CD34+ cell proliferation: BCR/ABL up-regulates the cdki p27, which is relocated to the Cell cytoplasm and incapable of regulation cdk2 activity. PNAS 97: 10538–10543.

A STUDY OF DNA POLYMERASE- MUTATION IN HUMAN ESOPHAGEAL CANCER Zhao Guoqiang, Dong Ziming and Zhao Qin Zhengzhou University, Medical School, Zhengzhou, 450052, PR China

DNA polymerase is an important repair enzyme involved in some steps of DNA repair and synthesis in mammalian cells. A high frequency mutation has been reported in cancers. The purpose of this study was to investigate whether DNA polymerase gene mutations occur in esophageal cancer. DNA polymerase genes in cancer and corresponding normal tissue with esophageal cancer were examined by reverse transcription and polymerase chain reaction (RT-PCR), singlestrand conformation polymorphism (SSCP) and sequence analysis. The results shown that in the operated sample group, obvious mutation was detected by SSCP in 7 of 26 esophageal cancer tissues (53.8%, 14/26) and 1 of 25 cancer corresponding normal tissue. In the early esophageal cancer sample group, DNA polymerase mutation was also detected in 6 of 14 (42.8% 6/14). In additionally, the mutation was also detected in a case of which is belong to squamocellular proliferation. The mutation hat spot appeared in 599nt T to A, 796nt G to T and 797nt T to G. Therefore, we can concluded that: (1) It is first report about POLB gene mutation in esophageal cancer. (2) Most of the mutations have lead to transform of amino acid and protein second structure. (3) DNA POLB gene mutation may be an important role in the development of esophageal cancer. It may be associated with POLB gene mutation as result of the accumulation of mutation in oncogen and tumour suppressor genes. (4) The pathogenesis of esophageal cancer associated closely with both environmental and genetic factors. (5) The pathogenetic course of esophageal cancer is a multistage progress. K: DNA polymerase; esophageal cancer; gene mutation; sequence analysis.

ESTABLISHMENT OF CELL CULTURE METHODS FOR ENDOMETRIAL AND ENDOMETRIOTIC BIOPSIES FROM ENDOMETRIOSIS PATIENTS FOR THE STUDY OF THE PHYSIOPATHOLOGICAL PARAMETERS OF ENDOMETRIOSIS Y. Zhao1, H. Zhang, Y. L. Li, F. Tian2, K. Q. Li2, F. J. Xiao2 and L. H. Wei1 Department of Gynecology/Obstetric, PLA General Hospital; 1Department of Gynecology, People’s Hospital, Beijing University; 2Department of Molecular Cancer Biology, Institute of Beijing Basic Medicine, P. R. China

Endometriosis is a common gynecological disorder. Affecting at least 10% of reproductive-aged women. It is characterized by the growth of

Cell Biology International, Vol. 25, No. 10, 2001 endometrial tissue outside the uterine cavity. Most familiar symptoms are pain before and during periods (usually worse than ‘normal’ menstrual cramps), pain during or after sexual activity, infertility, and heavy or irregular bleeding. These affect the life quality of patients seriously. It seems clear from clinical observations that endometriosis is an invasive (metastasizing) disease although its etiology and pathogenesis is largely unknown. In order to study the molecular and cellular basis of invasiveness in endometriosis, thirty-six samples of eutopic endometrial tissues and nineteen samples of ectopic endometrium were separated by filter and centrifugation method. The cells were detected though microscopy and immunohistochemical experiments. The results showed that both filter and centrifugation cell culture is powerful methods to obtain highly purified epithelial and stromal cells cultured from normal endometrial and endometriotic tissues. Then we examined the various cells for invasiveness in the collagen assay and found the invasive phenotype of endometrial and endometriotic cell is different. But the survival periods of epithelial cells were shorter than stromal cells, especially the endometriotic epithelial cells. So we conceive to establish cell lines that are either immortalized or have at least a prolonged life span in vitro. For this, we isolate cells from endometriotic biopsies that are then transfected with an SV40T antigen-encoding plasmid. SV40T antigenpositive cells are selected by G418 and overgrowth and judged by corresponding cell markers. In summary, the cellular model will be useful for us to investigate the factors that might influence/promote the degree of invasiveness of endometriotic cells and contribute to its pathogenesis.

Go
1 MEDIATES MAP KINASE ACTIVATION AND SERVES AS A TARGET FOR THE FEEDBACK REGULATION BY THE ACTIVATED MAP KINASE IN NEURONAL GPCR SIGNALLING Zhe Zhang, Yan-Xing Ni, Ya-Lan Wu, Lan Ma1 and Gang Pei Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai 200031; 1National laboratory of Medical Neurobiology, medical school, Fudan University, Shanghai 200032, People’s Republic of China

A variety of PTX-sensitive G-protein-coupled (GPCR) couple to the mitogen-activated protein kinase (MAP kinase/MAPK, also called ERK), however the signalling pathways and its physiological function are not fully understood. In the current study, using neuroblastoma X glioma NG108-15 hybrid cells, we found that activation of MAPK by stimulation of delta opioid receptor (DOR) was PTX-sensitive but independent on G subunits, and that over expression of a dominant negative form of Go
1 but not Gi
2 completely blocked the MAPK activation, indicating coupling of Go
1 to MAPK. The specific inhibitors of MPAK, PD98059 or U0126, significantly reversed the desensitization of DOR/Go
1 signalling. Interestingly, stimulation of muscarinic receptor but not nerve growth factor receptor, though both pathways activate MAPK, could crossly induce the desensitization of DOR. Further evidence from immunofluorescence confocal microscopy showed that the activation MAPK co-localized with Goa on the cytoplasm membrane in an agonist-dependent manner, which could be blocked by PD98059. Moreover, when S314 of Go
1, a potential MAPK phosphorylation site located in its GPCR coupling domain, was mutated, either uncoupling of DOR/Go
1 or desensitization of DOR/Go
1 signalling was greatly reduced. In vitro experiment further revealed that Go
1 was phosphorylated by MAPK at S3147. Thus, our data strongly suggested a novel function Go
1 as a MAK activator and a target for MAPK in the feedback regulation of neuronal GPCR signalling. K: desensitization; G protein-coupled receptors; heterotrimeric G protein Go
; MAP kinase; NG108-15 cells.

INOSITOL LIPIDS MESSENGER SYSTEM MEDIATES SIGNALLING OF SMOOTH MUSCLE CONTRACTION Zheng Guang Hua Guangdong College of Pharmacy, Guangzhou 510224, P.R.China

1075 Inositol triphosphate (IP3) and diacylglycerol (DG), the two products of phosphatidylinositol hydrolysis form two bifurcating signal pathways to regulate various important cellular functions. IP3-inducedcalcium release (IICR) through IP3 receptor/channel (tetramericcalcium channels) involves characteristics of (1) quantal nature; (2) positive and negative feedback between [Ca2+ ]i and IICR; (3) dependence on the Ca2+ content in the stores; and (4) functional coupling between Ca2+ release and influx. There are two kinds of hypotheses on the contraction mechanism of smooth muscle cell (SMC) that Ca2+ and calmodulin (CaM) participate together: one being the hypothesis of myosin phosphorylation regulating; the other the double-homeostasis (Flip-Flop) hypothesis. As the receptor enzyme and regulating intracellular cycle nucleotide and Ca2+ metabolism. Interaction between IP3 and DG is synergism and antagonism in the regulation of SMC contraction. Through release of [Ca2+ ]i, IP3 coordinates DG to activate PKC reaction not needing CaM activation. On the contrary, PKC activation inhibits irritation of an agonist to inositol lipids hydrolysis. Inducing decrease of IP3 in quantity. Phorbol ester and ion carrier (A2318 or ionomycin), to activate PKC and to increase [Ca2+ ]i respectively, inhibit the amplitude and duration of SMC contraction. These demonstrate that IP3 and DG have the action to inhibit slow wave and influx of Ca2+ in the regulation of SMC contraction, making the cellular response just right. The role and possible mechanism of IP3 receptor and ryanodine receptor in spatiotemporal Ca2+ signalling are also discensed in this article.

EVIDENCE OF THE INVOLVEMENT OF Ca2+ -ACTIVATED K + CHANNELS IN THE RAT SPERM ACROSOME REACTION C. X. Zhou and C. H. Chan Epithelial Cell Biology Research Center, Department of Physiology, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong

The sperm acrosome reaction (AR) is a crucial step for mammalian fertilization. This work describes experiment to test the effect of charybdotoxin (CTX), tetraethylammonium (TEA), angiotensin II(AngII) on the rat sperm AR in vitro. The chlortetracycline (CTC) fluorescence assay was used to study the status of the extent of induced acrosome reactions in cauda epididymical spermatozoa from rat. The results of this study show that angiotensin II (10 nM) induced stimulation of acrosome reaction, there was significant difference of the percentage of AR pattern between control and Ang II treatment. The stimulation of Ang II to the rat sperm AR was inhibited when spermatozoa were preincubated with K + channel blockers, TEA (1 uM) or CTX (300 nM). This study demonstrated the ability of angiotensin II to stimulate the acrosome reaction in capacitated rat spermatozoa and give evidence in support of a role of Ca2+ -activated K + channels in the rat sperm acrosome reaction. K: acrosome reaction; Ca2+ -activated K + channel; angiotensin II.

TRANSFECTION OF THE Hab18G/CD147 INCREASES MIGRATION AND INVASION OF 7721 HUMAN HEPATOMA CELLS Qing Zhou, Jianli Jiang, Zhinan Chen and Hsiao Chang Chan Epithelial Cell Biology Research Center, Department of Physiology, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, NT, Hong Kong

Hepatoma-associated antigen HAb18G (homologous to CD147) is a potential adhesion molecule that has been implicated in the metastasis of hepatoma cells. In order to investigate further its effect on migration and invasion of cancer cell, HAb18G cDNA was transfected to human 7721 hepatoma cells to obtain a cell line (T7721) stably expressing HAb18G, as demonstrated by Northern Blot and immunohistochemistry. Both invasion and migration assays were conducted in 24-well transwells (purchased from Costar) fitted with Millipore membrances (6.5 mm filter, 8 m pore size). In the invasive assay, membranes were coated with growth factor-reduced Matrigel (20 g/120 l/filter, Becton Dickinson). For both assays, 2.5105 7721 or T7721 cells

1076 suspending in 100 l 0.5% BSA-RPMI were plated in upper wells of transwell chambers containing 200 l the medium. Bottom wells contained 600 ml 5%FBS-RPMI. Chambers were gently shaken, followed by 24 or 72 hours incubation (37C, 5% CO2). After incubation cells were fixed and stained using 2% crystal Violet (purchased from Sigma). Then cells from upper surface of Millipore membranes were completely removed with gentle swabbing leaving the remaining migrant cells attached to the lower surface. Cellular invasion and migration indices (cells of invasion or migration % of total cells) were determined by Alpha Imager 200 (Alpha Innotech Corporation). Results show that HAb18G-expressing cell line T7721 was significantly greater in the invasive potential as measured at 36-hour and 72-hour after incubation than that observed in cell line 7721 (14.770.75% vs. 2.130.83%, n=3, P< 0.001; 49.132.58% vs. 35.204.17%, n=3, P<0.05). The ability of the two hepatoma cell types to migrate from upper compartment to lower compartment was also examined. T7721 cells (containing HAb18G) exhibited a greater migrating per cent (34.031.88%, n=3, P<0.01) as compared to that of 7721 cells (15.231.53%, n=3) after 24-hour incubation. We concluded that HAb18G is a key surface molecule of cancer cells in migration and invasiveness.

CHARACTERIZATION AND REGULATION OF CO2-ACTIVATED HCO3
secretion in T84 human intestinal epithelia Wen-Liang Zhou and Jeffrey B. Matthews Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, U.S.A.

Epithelial secretion of Cl
and HCO3
occurs by distinct yet overlapping membrane transport and regulatory signalling pathways. The human T84 intestinal epithelial cell line is widely utilized as a model of electrogenic Cl
secretion. We wondered whether T84 cells may also possess the capacity for electrogenic secretion of HCO3
, but its characteristics and underlying mechanism are poorly understood. Methods: Confluent T84 monolayers grown on permeable supports were bathed, except where noted, in Cl
-free (gluconate) buffer containing 25 mM NaHCO3. pH was maintained at 7.4 through all subsequent manipulations. To measure electrogenic ion transport, short-circuit current (Isc) was determined by voltage-clamp. Results: Introduction of 5% CO2 induced a large sustained Isc within 3–5 min (peak Isc =120.374.98 A/cm2, n=12). This Isc was comparable in magnitude to the maximal cAMP-stimulated Isc in Cl
containing solutions (94.55.8 A/cm2, n=8). The CO2-stimulated Isc occurred whether or not pH was maintained at constant pH 7.4 by NaOH titration. No Isc was observed in HCO3
free solutions, and CO2 only minimally affected Isc when 110 mM Cl
was present instead of gluconate (peak Isc =6.331.8 A/cm2, n=4). CO2-induced Isc was insensitive to bumetanide but could be completely blocked by apical DIDS (100 musignM) and by buffering intracellular Ca2+ with BAPTA-AM (29.52.6% control, n=4), consistent with apical anion exit via the Ca2+ -activated Cl
channel (CaCC). The CO2-stimulated Isc was blocked by basolateral DIDS (1 mM), ouabain, and acetazolamide and thus represents electrogenic HCO3
secretion. CO2stimulated Isc was blocked by the conventional (c) and novel (n) PKC isoform inhibitor Go¨ 6850, but not the selective cPKC inhibitor Go¨ 6976 or the PKC inhibitor rottlerin. CO2 was found to translocate PKC from cytosolic to membrane fraction, suggesting a role for this nPKC. In the absence of CO2, the cAMP agonist forskolin (10 M) induced a small but sustained Isc (peak=221.8 A/cm2 (n=8), but the Isc was not blocked by apical DIDS, suggesting involvement of a different apical anion exit pathway (e.g., CFTR). Apical addition of the mixed Ca2+ and cAMP dependent agonist ATP (100 M, n=4) induced a biphasic Isc response with an initial rapid transient rise of 41.32.9 A/cm2 that was sensitive to apical DIDS and another slower sustained rise (161.1 A/cm2) that was not DIDS-sensitive. Conclusion: In addition to Cl
secretion, T84 cells express machinery for electrogenic HCO3
secretion, a process that appears to be markedly suppressed in the presence of Cl
. This process is activated by CO2 (independent of pH effects), requires carbonic anhydrase, and utilizes transport pathways that differ markedly from those used for Cl
secretion, including a basolateral NBC as well as an apical

Cell Biology International, Vol. 25, No. 10, 2001 DIDS-sensitive anion exit step. The mechanism underlying CO2activated HCO3
secretion may involves nPKC and altered intracellular Ca2+ (possibly CaCC). HCO3
secretion in T84 cells can be activated independently by Ca2+ - and cAMP cascades that utilize different apical anion exit pathways.

SIGNAL TRANSDUCTION PATHWAY OF THE APOPTOTIC PROCESS IN RMA CELLS INDUCED BY DRUGS Zhu Hongli, Wang Yuezeng, Yu Li, Li Bin, Yao Shanqian and Lou Fangding Department of NanLou Hematology, PLA, Hospital, 28 Fuxing Road, Beijing 10085, China

The objective of the study is to explore the effect of Fas, FasL and Bc-2 on the process of apoptosis induced by drugs through detecting the expression of Fas, FasL and Bel-2 on RMA cells, Dexamethasone (DEX), etoposide (VP16), arsenie trioxide (As2O3) and all-transretinoid-acid (ATRA) were added to the murine T lymopoma cell line RMA as well as to RMA cells preincubated with interleukin-2 (IL-2), interleukin-6 (IL-6) or granulocyte-macrophage colony-stimulating factor (GM-CSF) respectively. The effect on apoptosis was observed and the expression of Fas and FasL mRNA as well as the expression of Fas and Bel-2 antigen was measured. DEX and VP16 could promote apoptosis of RMA cells while upregulating the expression of Fas and FasL without affecting the expressing of Bcl-2. ATRA may downregulate the expression of Bcl-2 without any change of Fas and FasL, no apoptosis of RMA cells induced by ATRA was observed. Although As2O3 may induce apoptosis of RMA cells, it did not affect the expression of Fas, FasL and Bc-2, which suggested that different drugs induce apoptosis on the same kind of cells by different signal transduction system and apoptosis induced by Fas system needed the coexistence of Fas and FasL. Although IL-2, IL-6 and GM-CSF upregulated the expression of Fas protein when added to RMA separately, none induced apoptosis. Apoptosis could be induced by combination of IL-2 and IL-6 clong with the upregulation of Fas and FasL. The cytokines facilitated the apoptotic action of chemotherapeutic drugs, the drug concentration of inducing apoptosis decreased and the time period of starting apoptosis shortened. Apoptosis could be observed without the expression of FasL when anti-Fas-antibody was added into RMA cells. The results demonstrated that there was synergistic effect of chemical drugs and some cytokines for induction of apoptosis. Fas-FasL system participated in the apoptotic action induced by DEX and VP16. Different drugs may induce apoptosis by different pathways of signal transduction. K: Fas and FasL, BCl-2; apoptosis; chemotherapeutic drugs; cytokine; RMA cell line.

CLONING AND CHARACTERIZATION OF A NOVEL GENE, UBIQUITIN-LIKE FUSION PROTEIN, EXPRESSED IN HUMAN ADULT TESTIS Zhu Hu1, Zhou ZuoMin1, Sha JiaHao1 and H. C. Chan2 1 Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing, 210029, People’s Republic of China; 2Epithelial Cell Biology Research Center, Department of Physiology, Faculty of Medicine, Chinese University of Hong Kong, Shatin, Hong Kong

With the aim of cloning genes involved in human testis development, we have constructed the adult human testis microarray and compared the gene expression in embryo and adult testis in a genome-wide scale. A novel gene was identified, human ubiquitin-like fusion protein (hUFP, GenBank accession number: AF311324), from human testis cDNA library. It was 3256 bases in length, and encoded a protein of 727 amino acids. There are a N-terminal ubiquitin domain (76 amino acids) and a C-terminal AN1-like zinc finger (42 amino acids) in the deduced protein of hUFP. The deduced amino acid sequence of hUFP shows 46% homology to Xenopus laevis ubiquitin-like fusion protein, especially N-terminal ubiquitin domain (82%) and C-terminal AN1like Zinc finger (83%). Xenopus laevis ubiquitin-like fusion protein locates in animal hemisphere of xenopus laevis egg, it may be inherited unequally by individual blastmeres during embryonic cell divisions and

Cell Biology International, Vol. 25, No. 10, 2001 may influence the development of blastmeres. Comparing with the genomic sequence of HTGS, we found that it is located in human chromosome 10 and has 10 exons spanning about 56,753 bp of huamn genomic DNA. The results of standord TNG radiation hybrid panel showed that it is linked with the marker SHGC-132871 and located in 10q11.2. RT-PCR results showed that hUFP mRNA is highly expressed in human adult testis. Functional studies on this gene are currently being undertaken.

CELLULAR SIGNALLING MECHANISMS UNDERLYING PHARMACOLOGICAL ACTION OF BAI FENG WAN ON GASTROINTESTINAL SECRETION J. X. Zhu, Dilys Chan, L. L. Tsang, L. N. Chan, Q. Zhou, C. X. Zhou and H. C. Chan Epithelial Cell Biology Research Center, Department of Physiology, The Chinese University of Hong Kong, Shatin, Hong Kong

Bai Feng Wan (also known as Bak Foong Pills, BFP) is a well-known traditional Chinese Medicine, which has long been used for treating gynecological disorders and improvement of overall body functions including gastrointestinal (GI) function. However the cellular signalling mechanism underlying BFP action, especially on the GI tract has not been elucidated. In the present study, human colonic epithelia cell line T84 was used as a model to investigate the effect of BFP extract on ion transport in conjunction with the short-circuit current (Isc) technique. The results showed that apical addition of BFP ethanol extract produced a concentration-dependent (10–1000 g/ml, ED50 =120 g/ ml) increase in Isc. The maximal response was observed at 500 g/ml with an increase in Isc of 24.44.5 A/cm2. The BFP-induced Isc could be inhibited by pretreatment of the cells with adenylate cyclase inhibitor, MDL-12330A (10 M). The BFP-induced Isc was not observed when extracellular Cl
was replaced. The BFP-induced Isc was partially inhibited by Cl
channel blocker, DIDS (100 M), but completely blocked by DPC (2 mM). These results demonstrated that BFP ethanol extract exerted a stimulatory effect on gastrointestinal Cl
secretion by activating adenylate cyclase and apical cAMP-

1077 dependent Cl
channels. This effect of BFP may be explored to improve GI disorders such as constipation.

EFFECT OF BAK FOONG PILLS ON EXOCRINE PANCREATIC SECRETION IN RATS J. X. Zhu, Y. W. Chung, Q. Zhou, M. K. Yu and H. C. Chan Epithelial Cell Biology Research Center, Department of Physiology, The Chinese University of Hong Kong, Shatin, Hong Kong

Bak Foong Pills (BFP, also known as Bai Feng Wan) has long been used for treating gynecological disorders and improvement of overall body functions including gastrointestinal (GI) function. Using the short-circuit current technique, we have demonstrated that BFP ethanol extract exerted a stimulatory effect on gastrointestinal Cl
secretion by activating adenylate cyclase and apical cAMP-dependent Cl
channels in T84 human colonic cells. We further examined the effect of BFP ethanol extract on exocrine secretion of the pancreas in rats. Experiments were performed on anesthetized rats prepared with bilepancreatic fistula. A polyethylene catheter (PE-10) was inserted into the common bile-pancreatic duct at the ampulla to collect bilepancreas juice. A second catheter (PE-50) was placed in the duodenum, slightly above the sphincter of Oddi, for duodenal infusion of BFP ethanol extract and other chemicals. Duodenal return of bile-pancreas juice was performed every 15 minutes. The results showed that duodenal infusion of BFP ethanol extract significantly increased the pH value of bile-pancreas juice from 7.78 to 7.94 (P<0.01), as compared to the basal control values. Similar increase in pH was also observed with forskolin, an activator of adenylate cyclase (P<0.01). It is likely that BFP may exert its effect by increasing release of secretin, which in turns stimulates cAMP-dependent exocrine pancreatic secretion, most likely bicarbonate secretion. Together with its observed effect on colonic secretion, the effect of BFP on exocrine pancreatic secretion contributes to its pharmacological actions on GI tract. Supported by Innovation & Technology Fund of the Innovation & Technology Commission of Hong Kong SAR.