Abstract from the Thirty-ninth Annual Meeting of the Scandinavian Society for Electron Microscopy

JOURNAL OF ULTRASTRUCTURE

AND MOLECULAR STRUCTURE RESEARCH 94,268-296

(1986)

Abstracts from the Thirty-ninth Annual Meeting of the Scandinavian Society for Electron Microscopy The annual meeting of the Scandinavian Society for Electron Microscopy “SCANDEM-86,” was held in Oulu, Finland, June 4-6, 1986. Given below are titles and abstracts of selected biological papers presented there. (Abstracts concerning physics and material science will appear in Ultramicroscopy.)

Immune-Electron Using the Protein

Microscopical Localization of Tubulin in Polychaete Yolk Granules A-Gold Technique. H. EMANUEISSON AND J. ALUMETS, Departments

of Zoophysiology and Pathology, University of Lund, Lund/Malmii, Sweden. In the polychaete Uphryotrocha lubronicu the full-sized (4 pm) oocyte yolk granules as well as their precursor granules, which are formed in the adhering nurse cell, contain yolk protein unusually rich in tryptophan. Accordingly, polychaete embryos, derived from females exposed to [3H]tryptophan during vitellogenesis will show a marked accumulation of label in the yolk granules. Analysis with electron microscopical autoradiography of the utilization of such [3H]tryptophan-labeled yolk granules in developing pregastrular embryos has shown that label from the disintegrating granules is preferentially associated with microtubules, suggesting identity of the labeled material with tubulin. This observation inspired us to perform an immunocytochemical investigation at the ultrastructural level of isolated polychaete oocytes and nurse cells to determine, if tubulin is present in the yolk granules. Using antibodies raised against tubulin and the protein A-gold technique we have now confirmed the presence of tubulin in the full-sized yolk granules. Moreover, we have also demonstrated that tubulin is first sequestered into the precursor granules and, following their transfer into the oocyte and incorporation into the larger granules, it is accumulated in the latter. Both the autoradiographic and the immunocytochemical investigations indicate that tubulin forms a conspicuous part of the yolk material in this species.

The Efficiency of Different Preparation Procedures for Immunocytochemistry of Hyaline Cartilage. B. ENGFELDT, Department of Pathlogy, Huddinge University Hospital, Ka-

rolinska Institute, S-l 41 86, Huddinge, Sweden. Mouse monoclonal antibodies to the chondroitin sulfates 0, 4, and 6, to keratan sulfate and to hyaluronic acid have been produced recently (B. Caterson et al., Morgantown, Va.). Using these antibodies in immunocytochemical studies on the electron microscopic level different types of hyaline cartilage in the normal and the diseased state have been examined. The postembedding procedure with a secondary antibody with a collodial gold probe was used to localize the antigenic site. Processing this tissue for immunocytochemistry involves several problems. The antigenicity may be reduced or lost as a result of the fixation. Furthermore, a considerable loss of proteoglycans occurs during the conventional procedure. To overcome these problems hyperbaric cryofixation, freeze substitution, and embedding techniques with use of hydrophilic resins have been established. We have also processed material using cationic dyes such as safranin 0 in the fixation steps. This technique has been demonstrated to prevent the loss of proteoglycans from the tissue. The differences in antigenic efficiency of the mentioned antibodies have been tested in a semiquantitative way by counting gold particles per unit area in defined areas of hyaline 268 0889-1605186 $3.00 Copyright 0 1986 by Academic Press, Inc. All rights of reproduction in my form reserved.

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cartilage treated as above. The results indicate that hyperbaric freezing or freeze substitution is superior to glutaraldehyde-safranin 0 fixation, which on its part is about three times more efficient than conventional fixation.

Differential

Shrinkage

in Tissue Processing-A

Source of Error in Morphometry.

KUOPIO,* M. VALTONEN,~ AND L. J. PELLINIEMI,t *Department ofhatomy ratory of Electron Microscopy, University of Turku, Turku, Finland.

T.

andthzbo-

Fixation and dehydration are generally known to cause shrinkage of tissue specimens prepared for light and electron microscopy. However, it is only recently that the possibility of differential shrinkage has been taken into account in morphometric studies. H. Haug et al. (1984, J. Hirnforsch. 25, 354-374) have made morphometric measurements of changes in the number of neurons in the aging human brain. They demonstrated that young brains shrink more than old ones and that these changes in tissue shrinkage have been neglected by earlier investigators. This has led to the common misinterpretation that the number of neurons in the human brain decreases considerably with age. We have observed several morphological changes that may influence the tissue shrinkage due to the processing of specimens from different stages during the postnatal development in the rat testis. We measured the effects of immersion fixation (5% glutaraldehyde in 0.16 mol/ liter s-collidin-HCl buffer, pH 7.4) and embedding in Epon on the testicular volume at the ages of 5 and 40 days. The fresh weights of three testes per age group were converted into volumes (sp gr = 1.045 kg/liter). The volume of the embedded tissue was estimated from serial sections (1 pm) using point counting methods and the formula V = t x ap x Xp, where t is the average distance from a section to the next, up area associated with one point, and 2p number of points hitting one section. The results (Table I) of our limited material suggest that young testis would shrink 4% less than the old one. However, this change is statistically not significant (Student’s t test, P < 0.5). TABLE1 TISSUE SHRINKAGEINSPECIMENPREPARATIONOFRAT TESTIS AT DIFFERENT AGES

Age (days)

5 40

Fresh volume (mean (SEW, ccl) 7.0 (0.7) 737.9 (28.6)

Volume after embedding (mean (SEW, 4)

Shrinkage (% (SEM))

5.4 (0.3) 547.2 (22.1)

21.8 (3.8) 25.7 (3.2)

The results of Haug et al. in the human brain and ours in the developing rat testis indicate that possible changes in shrinkage should always be measured if morphometric analysis of numerical densities is made on specimens at different ages. Similar changes should also be taken into account when tissue specimens from same organs are analyzed at different functional or pathological states.

Sputter-Coated

Cytoskeletons in TEM, SEM, and STEM. M. LINDRorH,*Tt P. B. BELL,*

AND B. A. FREDRIKSON,* Departments University, Linkiiping, Sweden.

of *Pathology

ZZ and TApplied Physics, Linkiiping

A method to study the complex three-dimensional organization of the cytoskeleton is to extract cells with non-ionic detergent and to examine the remaining structures as whole

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mounts. Various TEM methods have been used, for example, negative staining and lowangle rotary shadowing followed by carbon replication. However, the cytoskeletons can be sputter-coated directly after dehydration and CPD and examined in TEM, SEM, and STEM. We used malignant glioma cells as test objects, which are extracted with Triton X- 100 in combination with high-salt buffer, leaving only the intermediate filaments. They were sputter-coated with Pt, W, or Ta at thicknesses from 1 to 5 nm with a Microvac magnetron sputter-coater installed in an Edwards apparatus equipped with a quartz crystal thickness monitor. The samples were examined at 160 kV in a fully equipped JEOL 2000EX analytical instrument. By this method one avoids the somewhat cumbersome replication techniques while it is still possible to study details of the cytoskeleton at high resolution. Particularly, the inverted STEM mode proved to be very useful in showing delicate structures of the filaments. For the SEM mode it is important not to coat the samples with too much metal because this will obscure the details, but still enough to obtain a reasonable secondary electron emission.

Immunoelectron Microscopical Localization of Cathepsin H and Its Tissue Inhibitor: A Fixation Study. A. RINNE,* R. SORMUNEN,* M. JARVINEN,* H. KIRSCHKE,~’ B. WIEDERANDERS,? AND V. K. HoP~u-HA~LJ,$ *Department ofPathology, University of O&u, Oulu, Finland; tlnstitut ftir Medizinische Biochemie, Martin Luther Universitlit, Halle, Deutsche Demokratische, Republik; and $Department of Dermatology, University of Turku, Turku, Finland. We have raised specific polyclonal antibodies against rat liver cathepsin H and its tissue inhibitor. In the present communication we describe immunoelectron microscopical localization of the enzyme and its inhibitor using an indirect immunogold technique on thin sections of Lowicryl-embedded tissues. The tissues (the kidney and the squamous epithelium of the tongue) were fixed in 4% formaldehyde, or in 4% formaldehyde, 0.1% glutaraldehyde, in isotonic buffered sucrose, or in PLP fixative, for 4 hr at 4°C. Cathepsin H was localized in the lysosomes of the proximal tubule of the kidney. The best results were obtained by fixing the tissue with formaldehyde-glutaraldehyde in isotonic sucrose. Formaldehyde alone in the sucrose preserved the antigenicity very well and the structure well, while the PLP fixative destroyed both the structure and the antigenicity. Polymerization at -25°C preserved the tissue structure better than conventional polymerization at 4°C. The inhibitor (cystatin ol) was seen in the cytoplasm of the squamous cells of the tongue. The gold label was distributed diffusely, but some clusters of the label were seen on tonofilaments. The results of the fixation experiments were the same as in the case of cathepsin H.

Preparation

Microscopic Survey Sections in Cryoultramicrotomy. K.kE E. GUNNAR KOPSTAD, AND OLAV A. HAUGEN, Department of Pathology and Institute of Cancer Research, University of Trondheim, Regionsykehuset, N- 7000 Trondheim, Norway. The preparation of freeze-dried cryosections of prostatic epithelium is time consuming because in benign prostatic hyperplasia the stromal component may be the dominant feature. Time and effort may be saved by examining sections at lower magnification in the light microscope before proceeding to electron microscopy. We have developed a simple and inexpensive method to collect, stretch, and stain unfixed cryosections. A coverslip (5 mm in diameter) is used to support the frozen section. The coverslip rests TVEDT,

of Light

JOSTEIN HALGIJNSET,

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271

on a stage close to the knife stage. A small amount of ethanol is applied to the coverslip where it is allowed to cool. The stage contains a heating coil and the temperature is kept just above the melting point of ethanol. The highly viscous alcohol efficiently retains and stretches the section. By increasing the temperature, the alcohol is evaporated and the section is firmly attached to the coverslip. With fine forceps the coverslip is removed from the stage, and the section is stained with hematoxylin and erythrosin. After staining the coverslip is placed with the section downward on a glass microscope slide with a small drop of mounting medium on it. The introduction of a heat source into the cryo chamber did not affect the temperature of the air close to the specimen. Sections with 0.5 pm thickness were easily cut, and the staining was sufficient to provide the necessary morphologic information. This method should prove useful also in other instances where the feature of interest is widely scattered among other tissue components.

Local Effects of Hydrocarbon Solvents on Rat Tail Skin and Nerves. E. VERKKALA, J. NICKELS, AND T. STJERNVALL, Institute of Occupational Health, Haartmaninkatu 1, Helsinki 29, Finland. Occupational exposure to hydrocarbon solvents can cause dermatitis and neuropathy in workers. Very few experimental studies are, however, done with percutaneous exposure, although skin is a notable route of entry for these lipophilic chemicals. We exposed 12 cm* of male Wistar rat tail skin to 1 ml white spirit formula for 3 hr daily, 5 days a week up to 6 weeks. After 2 weeks macroscopic scaling and hyperkeratosis was seen in the treated area and the dermatitis worsened with exposure. Neurophysiological measurements showed polyphasia and increased duration of the motor responses in the sixth week. The exposed axons of the tail nerves showed prenodal swelling and slight widening of Ranvier nodes in groups treated with white spirit formula, containing more than 10% n-nonane. Demyelinative foci were seen in the nerves from rats treated with white spirit containing 17O/6aromatics and almost no nonane. Electron microscopy of the skin revealed morphological changes in the epidermis. The mitochondria in the keratincytes were swollen with broken inner structures. Edema vacuoles were seen in the epidermal cells and in the subepidermal space. The intercellular spaces were widened in the epidermis and especially around the hair channels. The nuclei of many epidermal cells were pyknotic. Inflammatory cells and cell debris were seen in the upper dermis. The cutaneous nerves were more frequent in the treated groups than in the controls, perhaps as an answer to the irritant effect of the solvent. In the myelinated fibers degenerative changes were seen in the treated groups. The axons contained myelin figures and the arrangement of neurofilaments was often disorganized. Many axons also appeared shrunken. Some unmyelinated fibers also appeared degenerated in the treated groups.

The Value of X-ray Microanalysis in Determining the Etiology of Rheumatoid Pneumoconiosis. SISKOANTTILA,* SEP~~SUTINEN,*PGWOPG~~G,*B~FINELL,~ANDJERROLD L. ABRAHAM,$ *Department of Pathology, University of Oulu, Oulu, and tounasrinne Hospital, Rovaniemi, Finland; and *Department of Pathology, Upstate Medical Center, Syracuse, New York. Rheumatoid pneumoconiosis (Caplan’s syndrome) was first described in coal miners suffering from rheumatoid arthritis. Rheumatoid lung lesions have also been reported in patients with silicosis and asbestosis, and in workers in various dusty occupations. One way to study the etiology of pulmonary rheumatoid nodules occurring without apparent

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pneumoconiosis is to assess the dust accumulated in them by X-ray microanalysis. A 46year-old woman with rheumatoid arthritis developed numerous round opacities at the apex of the right lung 11 years after exposure to dolomite. Resected lung showed discrete 0.8- to 2-cm nodules, with central necrosis surrounded by palisading fibroblasts and a prominent inflammatory zone. A large number of birefringent dust particles were seen in the necrotic centers and around the nodules. By electron microscopy the particles were dense, mostly elongated, and lamellar. For X-ray microanalysis, 5-pm sections were cut from paraffin blocks, mounted on carbon blanchets, and deparaffinized. Particles observed in a backscattered electron image were analyzed by the SEM/EDS and their number was estimated morphometrically: 8.4 x 1O9particles/cm3 of tissue were found. Of the particles, 4% were silica, 94O/baluminum silicates consistent with mica, 1% miscellaneous silicates and 1% metals. No dolomite was found. As a bagger in a dolomite quarry, the patient had been exposed to the material consisting up to 93O/b dolomite, the rest being other minerals as impurities. Because the Caplan’s nodules did not develop until 11 years after exposure, it is apparent that other minerals, such as mica and silica, were the promoting factor for the lung lesion.

X-ray Microanalysis of Freeze-dried Cryosections as a Method to Demonstrate Intracellular Iodine after Roentgen Contrast Media Exposure in Viiro. ASBJDRN NORDBY,* K&w E. TVEDT,*$ JOSTEIN HALGCJNSET,* GIJNNAR KOPSTAD,* KETIL THORSTENSEN,# AND OLAV A. HAUGEN,* *Department of Pathology, j-Institute of Cancer Research, and *De-

partment of Clinical Chemistry, University of Trondheim N-7000, Trondheim, Norway. Vascular roentgen contrast media (CM) are composed of highly hydrophilic molecules containing three or six atoms of iodine. Six hours after an ordinary urographic examination 99% of the intravenously administrated CM will be excreted by glomerular filtration. An evaluation of possible harmful effects on the endothelial cells implies the need of sensitive methods capable of determining whether CM cross the cell membrane. We have demonstrated that the human cell line NHIK 3025 and human umbilical endothelium grown as monolayers contained about 0.1 O/6CM after 2 hr of exposure, as measured by high performance liquid chromatography. In order to confirm a possible entry of CM into the cells, X-ray microanalysis was used to measure intracellular iodine. A complete arrest of soluble compounds was obtained by quickly freezing cell monolayers after 2 hr of exposure to CM (20 mg iodine/ml). Freeze-dried cryosections (0.25 pm thick) were analyzed with a Kevex 7000 energy-dispersive spectrometer on a JEOL 1OOCX electron microscope with scanning attachment. Inside cytoplasmic electron-dense bodies, iodine was found in concentrations significantly above the detection limit. Thus, CM cross cell membranes in vitro. Studies should be undertaken to see whether this also takes place in the human body.

Characterization of Environmental Inorganic Particles in Human Lungs in Northern Finland. 0. TAIKINA-AHO,* S. ANTTILA,~ P-L. KALLIOM~I,* T. KnRor.&t P. P;i;iKKij,t S. J. WONEN,* AND S. SuTmm,t *Institute of Electron Optics and tDepartment of Pathology, University of Oulu, SF-90570 Oulu, and Slnstitute of Occupational Health, SF-

00290 Helsinki, Finland. Many inhaled inorganic dust particles stay permanently in the lungs. Traditionally fibers have been identified and counted from digested or ashed lung samples and only recently have other inorganic particles also been studied. The aim of this preliminary study was

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273

to assess the basic mineral burden of human lungs in Northern Finland. The material consisted of unselected autopsy lungs of two male smokers and of two nonsmokers, aged 80-85 years. The lungs were fixed intrabronchially with formalin-polyethylene glycolalcohol solution and air dried. A sample of 1-2 g was taken from the apicoposterior segment of the upper lobe and another from the basal part of the lower lobe. After chemical hypochlorite digestion the filtered samples were studied using scanning electron microscopy and energy dispersive X-ray microanalysis. Mineral particles were identified by converting characteristic peak intensities of the elements detected to oxide weight fractions and identification was confirmed by calculating the mineral formula. The mean particle size was 1.3 pm and the number of particles in the digested lung tissue was 2-l 1 x lo61 cm3. The inorganic particles were silicate minerals typical of Finnish bedrock: plagioclase 16-23%, quartz l&22%, K-feldspar and clay minerals 6-37%, kaolinite 5-26%, and talc O-26%. Occasional particles of micas, sphene, antophyllite, mullite, titanium, aluminum, and molybdenum were also seen. Mineralogic decomposition of silicates was evident and 6% of the particles remained unrecognized.

Determination of Mineral Fiber Concentration in Lung Tissue. T. TUOMI* AND A. TOSSAVAINEN,?*Institute of Occupational Health, Haartmaninkatu 1, SF-00290 Helsinki, Finland; and f-International Agency for Research on Cancer, F-69372 Lyon Cedex 08,

France. A method to assess the mineral fiber burden in lung tissue with electron microscopy and X-ray microanalysis is presented. The objective of the continuing work is to establish a reproducible method of estimating the cumulated exposure to asbestos and other mineral fibers. Blocks of lung tissue were taken from autopsies or biopsies. The sampling sites were selected to represent different parts of the lung. Usually the hilar lymph node was also sampled. Tissue blocks were air dried to constant weight and low-temperature ashed for 12 hr (T < 70°C). The remaining ash was dispersed in 0.1 N HCl and sonicated for 5 min with power output less than 0.1 W/ml. The suspension was filtrated on 25 mm Nuclepore filter with pore size 0.2 pm. The piece of filter was gold coated for SEM analysis or carbon coated for STEM analysis. When STEM was used the sample was placed on a copper grid and the filter material was dissolved with chloroform. A blank sample was always used. Fiber counting by SEM was preferred because of the simplicity of the sample preparation which is more reproducible with less losses of fiber. The fiber counting was done at 5000 x magnification from a tilted sample. Fibers were counted and sized and the collected X-ray spectrum was stored. Quantitative analysis and mineral classification were performed. Fiber counts were normalized to the dry weight ofthe tissue. The counting of 400 fields of view and the collection of spectra took 2 hr per sample. The analytical sensitivity obtained was 5 x lo4 to 1 x lo5 fibers/g dry tissue and the detection limit was 2 x 1OSto 4 x 1O5fibers/g dry tissue. The present results show excessive accumulation of fibers with an average length and diameter of > 5 and 0.5 pm, respectively.

X-ray Microanalysis of Crystalloids in Prostatic Carcinoma: Light Microscopic Identification of Small Particles for Subsequent Scanning Transmission Electron Microscopy. KARE E. TVEDT, JOSTEIN HALGIJNSET, GUNNAR KOPSTAD, AND OLAV A. HAUGEN, Department of Pathology and Institute of Cancer Research, University of Trondheim, Re-

gionsykehuset,

Norway.

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Small, distinct crystalloids are sometimes present in the acinar lumina of highly differentiated prostatic carcinomas. The rare occurrence of crystalloids in hyperplastic glands has been proposed to be of diagnostic value, indicating an adjacent tumor. X-ray microanalytical information may help elucidate the nature of prostatic crystalloids. However, this presents some technical difficulties which must be overcome. First, unequivocal identification can only be made with the light microscope, and consequently the same section must first be examined by light microscopy and thereafter analyzed with the electron microscope. Furthermore, crystalloids are rare and widely separated, and in order to save time and effort the area available for investigation should be as wide as possible. We have sandwiched paraffin sections between two formvar films supported by a carbon retainer, providing a 3.5-mm2 area transparent to both light and electrons. The delicate film was handled gently during deparaffination and staining using a leaky vial, floating the various solutions slowly over the film and section. In 13 crystalloids from three patients, sulphur was the dominating element, but phosphor, calcium, zinc, and small amounts of magnesium were also present. The elemental composition was not statistically different from that of 16 corpora amylacea from two patients. It may well be that the two kinds of particles contain the same constituents. This method should also prove useful in other situations when X-ray microanalysis of small and rare particles is performed.

Ultrastructural Demonstration of Endogenous Peroxidases in Rat Testis. J. L. COURTENSAND L. PLOEN, INR4 37380 Nouzilly, France, and Swedish University of Agricultural

Sciences, S-750 07 Uppsala, Sweden. Endogenous peroxidases have been demonstrated after light glutaraldehyde (1.25%, 30 min) or PAF-glutaraldehyde (4). fixations of rat testis. Incubations were conducted either before embedding, the substrates (diaminobenzidine (DAB) and H202) being applied through a perfusion of the aorta, or on ultrathin sections. The enzymes were revealed at 20°C with low concentrations of DAB (0.12 mg/ml). The addition of H202 (1. 10-50h) was only necessary when sections were used, confirming that hydrogen peroxide is already present in the testis (F. Palombi and M. Bataglia, 1986, IV. Eur. Works. Mol. Cell. Endocrinol. Testis, Capri). In the interstitial tissue, the lymphocytes and few endothelial cells were the only structures to contain strongly positive peroxisomes, while some of the mitochondria of Leydig cells were stained as well (catalase activity?). Staining with acidicPTA according to A. Rambourg (1971, Znt. Rev. Cytol. 31, 57-114) demonstrated glycoproteins in all the structures positive for peroxidases, but mitochondria. Inside the seminiferous tubules, the enzymes could not be detected in any of the different germ cells. They were located exclusively in lysosome-like organelles of Sertoli cells after both preor postembedding techniques. The peroxisomes of Sertoli cells were grouped in certain areas displaying cyclical changes of location with the stages of spermatogenesis (C. P. Leblond and Y. Clermont, 1952, Ann. N. Y. Acad. Sci. 55, 548-573). They were concentrated in the basal cytoplasm in stages III to VII, being mixed with small lipid droplets, and were numerous around the heads of elongating spermatids in stages X to V, without any permanent association with lipid inclusions. They were rarely observed towards the adluminal side in the steps preceding the spermiation (VI to VIII). The significance of these observations remains obscure, but cyclical changes in Sertoli cell have been demonstrated (J. B. Kerr and D. M. De Kretser, 1975, J. Reprod. Fert. 43, 1-8; M. Parvinen, 1982, Endocrine Rev. 3, 404-417).

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Fine Structure of Three Different Neuronal Populations in the Feline Lateral Cervical Nucleus. I. ERIKSSON, R. FLINK, B. A. SVENSSON,AND J. WESTMAN, Department ofdnatomy, Uppsala University, Biomedical Centre, Box 571, S-751 23 Uppsala, Sweden. The neurones in the lateral cervical nucleus (LCN) have been retrogradely labelled with injections of native or conjugated horseradish peroxidase (HRI?) or choleratoxin subunit B (CTb) in 14 adult cats. HRP was visualized with tetramethylbenzidine and CTb with monoclonal antibodies. CTb labelled more cells than HRP especially from collateral projections, whereas the fine structure was better preserved in HRP positive neurones. Three different neuronal populations could be distinguished. The ascending cervicothalamic projection neurones were largest and most numerous. They have a relatively small nucleus, the highest mitochondrial volume fraction, a high somatic bouton covering ratio (40-50°h), and numerous somatic spines. The descending cervicospinal cells are smaller and very elongated in the length axis of the spinal cord. They have the largest nuclear surface membrane, few somatic spines, and a somatic bouton covering ratio of about 25%. The third and least numerous population is the supposed internuncial neurones, very small cells unlabelled after injection into all known external termination areas of the LCN. They have the highest volume fraction of nucleus, smooth and granular endoplasmic reticulum, the most folded cell nucleus, no somatic spines, and a somatic bouton covering of about 10%.

Ultrastructural Adaptations to Parasitism in Anoplodium sfichopi (Dalyellioida: Platyhelminthes). ULF JONDELIUS, Department of Zoology, University of G&eborg, Box 250 59, S-400 31 Giileborg, Sweden. The parasitic flatworm groups Trematoda, Monogenea, and Cestoda are generally believed to be descendants of free-living flatworms closely related to the group Dalyellioida. The Dalyellioida contains both free-living and endosymbiotic species. The epidermis of Anoplodium stichopi was studied by transmission and scanning electron microscopy. The species is a dalyellioid living endosymbiotically in the coelomic cavity of the sea-cucumber Stichopus tremzdus. The studies revealed a considerable enlargement of the epidermal surface. The apical portion of the epidermal cells is folded into anastomosing ridges that create the impression of a honeycomb in scanning electron micrographs. This, together with the presence of numerous coated vesicles in the epidermal folds and numerous mitochondria just below them, supports the idea that A. stichopi absorbs nutrients via the epidermis from the coelomic fluid of the host. The ciliation of the epidermal cells is sparse compared to that in free-living species. The findings are regarded as adaptations to parasitism in A. stichopi. Adaptations similar to those found in the dalyellioid A. stichopi are known from many species of the presumably phylogenetically closely related groups Trematoda and Cestoda.

Ultrastructural and Functional Pathology of the Compound Eye of the Retinal DegenerationMutant. M. J~VILEHTO,* M. WECKSTR6M,j- R. HARJULA,*AND E.KOUVALAINEN,~. *Department of Zoology and TDepartment of Physiology, University of Oulu, Oulu, Finland. A new inheritable mutation in the blowfly Calliphora erythrocephala has been discovered which shows prominent retinal degeneration. Characteristically the receptor cells in the retina appear morphologically and functionally normal at eclosion. The degeneration of the receptor cells proceeds gradually in time. The mutant has appeared in the laboratory

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stock probably through an inbred line. The behaviour of these flies shows highly reduced visual performance in escape reaction. The virility shows an apparent increase. The function of the receptor cells was tested with intracellular recordings using KCl-microelectrodes. The retinal ultrastructure was studied in compound eyes fixed at different time intervals after eclosion (1 day, 1 week, 2 weeks) using 3% GA in cacodylic buffer, + 4“C, postfixed in buffered 0~0~ for 2 hr, dehydrated, and embedded in Epon &add). The eyes examined at different ages show no superficial difference compared with nonmutant ones, but the degenerative changes appear in the fine structure in all receptor cells. During the aging of the mutant, the receptor cells increase in volume, the mitocondria are displaced towards the periphery, the cell interior becomes less well structured, the rhabdomeres shrink and exhibit vacuoles, and the microvilli become disintegrated. Osmoregulation in the tissue seems to be disturbed. The increase in cell volume and the fragmentation of desmosomes during the final stage point to a strong osmotic stress in the tissue. The receptor cell function is characterized by a rapid reduction of the receptor potential during the first week after eclosion and finally the loss of all photic responses. The spectral and polarized light sensitivity seems to stay normal as long as the response can be measured. A similar type of mutant has earlier been reported in the fruitfly Drosophila melanogaster. A degeneration of this type has also been found in vertebrates and some other invertebrates, indicating a more general, but still poorly understood, phenomenon.

Morphometric and Ultrastructural Study of the Perinatal Regression of Rat Testicular Leydig Cells. T. KUOPIO,* LEO PALJ,XRVI,~ AND L. J. PELLINIEMI,$ *Department ofdnatomy, TDepartment of Pathology, and *Laboratory of Electron Microscopy, University of Turku, Turku, Finland. The development of androgenic steroids producing testicular interstitial Leydig cells is biphasic in most mammals. The first phase of the development starts during the fetal life and hence the cells are called fetal Leydig cells. At puberty a new generation, the adult Leydig cells, arises via differentiation from mesenchymal precursors and the fetal cells apparently disappear. The final fate of the fetal cells is still controversial. Marked regressional changes and a considerable decrease in the total volume take place in the fetal Leydig cell population between the late days of pregnancy and the early days of postnatal life. In the present investigation we wanted to study whether this decrease is due to cell loss or a decrease in the size of individual cells, and to correlate this with the changes in the morphology and ultrastructure. At the age of 19.5 days of fetal life and at the ages of 1 and 5 days postnatally, rat testicular specimens for morphometric and ultrastructural examinations were fixed in 5% glutaraldehyde in 0.16 mol/liter s-collidin-HCI buffer, pH 7.4. The specimens were embedded in Epon. Semithin sections (1 I,cm) were used for the morphometric estimation of the total volume and the total number of Leydig cells. The volume density was measured with a point counting method and the numerical density of the cells was obtained using the Floderus equation. The volume of an average Leydig cell was calculated by dividing the total volume of cells with the total number of cells. The considerable decrease in the total volume of Leydig cells during the perinatal period was accompanied by a significant decrease in the volume of an average Leydig cell. The total number of cells remained unchanged (see Table I). Ultrastructural examination revealed signs of cell degeneration in some of the fetal Leydig cells. Occasional mitoses were also observed in the well-developed Leydig cells and some were still under differentiation. The main conclusion is that the perinatal regression of rat fetal Leydig cells is explained by the shrinkage of individual cells. The simultaneous constancy of the total

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number of Leydig cells does not need to be in disagreement with the observed signs of degeneration, because the degenerating cells are apparently replaced by cell differentiation or mitosis. The present results suggest that a turnover mechanism controls the size of the perinatal fetal Leydig cell population. TABLE MORPHOMETRIC

(LC)

Age (days) 19.5 1 5

Total volume of LC (mm3 (SEM)) 0.13 0.08 0.05

I

MEASUREMENTS

(0.02) (0.01) (0.01)

AT DIFFERENT

OF LEYDIG AGES

CELL.S

Total number of LC (SEMI

Volume of an average LC km3 (SEW)

59 600 (3600) 65 700 (12 000) 64 600 (8300)

2223 (434) 1203 (121) 777 (75)

Structure of the Exopeptidase Tripeptidyl Peptidase II from Human Erythrocyte and Rat Liver. E. MACPHERSON,* R.-M.BA~ijw,t S.H~GL~ND,* B.TOMKINSON,~ ~ZETTERQvIs-r,t *Institute of Biochemistry, Uppsala University, Uppsala, Sweden, and TDepartment of Medical and Physiolog.cal Chemistry, Uppsala University, Uppsala, Sweden. Protein turnover in living cells involves peptidases, which includes endo- and exopeptidases of both lysosomal and nonlysosomal origin. The structure of a newly identified exopeptidase, tripeptidyl peptidase II, from human erythrocytes and rat liver is presented here. This enzyme which is classified as a serine protease has neutral pH optimum and a subunit polypeptide of M, 13 5 000. Three different structural representations, all with an average length of 60 nm were revealed. A substructure with dimensions of 3 x 10 nm is interpreted as corresponding to the subunit of h4, 135 000. The results suggest that detailed studies of the interrelations between the structure and function of this enzyme may be possible.

Is There Any Transtubular

Transport

of Lysozyme in Renal Proximal Tubules? Ss~nv Department of Cell Biology

NIELSEN, JSRN THEIL NIELSEN, AND ERIK 1~s~ CHRISTENSEN, University of Aarhus, DK-8000 Aarhus, Denmark.

It is well documented that the cationic protein lysozyme is taken up by endocytosis from the lumen of renal proximal tubules and degraded in lysosomes. However, it has been suggested that intact lysozyme might be subject to transtubular transport in the range of up to 50% of the luminal uptake. In a recent study we have demonstrated a small but significant transtubular transport of cationized ferritin, 0.5% of the amount taken up. The present study was performed to elucidate the controversy of whether or not a significant transtubular transport of lysozyme exists. For this purpose proximal tubules from a rabbit kidney were dissected and perfused in vitro for 20 min with 1251-lysozyme and i4C-inulin and then with tracer-free perfusate for an additional 40 min before fixation. The uptake of lysozyme was visualized by electron microscopic autoradiography, and the possible transfer of intact lysozyme was compared to the transfer of inulin by measuring the amount of inulin and intact lysozyme in the bath. The results showed that 2.7% of the perfused amount of lysozyme was taken up and that 2 1.3% of the amount taken up was degraded. The autoradiographic analysis showed that 26.5% of the grains were localized over endocytic vacuoles and 48.4% over the lysosomes. Calculated as the autoradiographic grain

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density (% grains over organelle/% area occupied by the organelle), only endocytic vacuoles and lysosomes showed accumulation of the tracer with values of 11.5 and 20.0, respectively. The transfer from lumen to bath of lysozyme and inulin was 1.08 + 0.46% and 1.05 +- 0.46O/b, respectively, of the perfused amount. These values are not significantly different and therefore the present study does not support the idea of a major transtubular transport of intact protein.

A Colonial

Nervous System of a Primitive Polyp Community. HELENA REIJONEN AND of Zoology, University of Oulu, Linnanmaa, Oulu, Fin-

PATTI J;~RVILEHTO, Department

land. A coral colony consists of numerous individual polyp animals, which are partly embedded in either a calcareous hard or elastic material. If one polyp is mechanically stimulated, its tentacles and entire body will contract, and the reaction will proceed throughout the whole colony of animals. Even if this phenomenon can be well observed in both soft (Octocorallia) and hard (Hexacorallia) corals, its morphological basis has remained obscure. The conduction stability and velocity may suggest a cellular pathway between individual polyps. The morphology of the tentative conduction system has been studied in four Alcyonarian species and examined by various histological methods, including nerve-specific staining, and transmission and scanning electron microscopy, the latter also combined with X-ray analysis. An extensive network of cells was found in the keratinous substance connecting individual polyps. The structure of the cells resembles that of a typical nerve cell, and their processes were found to contact the epithelial tissue of the polyps. TEM study as well as X-ray data of the nerve-cell-specific staining show nerve fibers and also cell junctions, similar to chemical synapses between the cells located outside the polyps. According to the common morphological criteria typical for nerve cells, it seems likely that a colonial nerve net mediates the information transmission throughout a single colony of these types of soft corals.

Immunoelectron Microscopic Localization of Carbonic Anhydrase III in Rat Skeletal Muscle. H. K. Vji;i~Am~, Department of Pathology, University of Oulu, SF-90220 Oulu, Finland. Carbonic anhydrase III is one member of the multigene family of carbonic anhydrase. Previous biochemical and radioimmunoassay measurements have suggested the high specificity of carbonic anhydrase III to skeletal muscle. Light microscopical immunohistochemical studies have further suggested that carbonic anhydrase III is mainly concentrated in red skeletal muscle fibers, fibers containing acid-resistant actomyosin ATPase. In this study the distribution of carbonic anhydrase III in rat soleus and vastus lateralis muscles at the electron microscope level was studied using the immunogold technique. Small pieces of muscle were fixed in a paraformaldehyde-glutaraldehyde fixative and embedded into Lowicryl K4M. Thin sections were cut on grids and incubated with monospecific polyclonal rabbit antihuman carbonic anhydrase III serum. Colloidal gold particles coated with antirabbit IgG were used to localize primary antibodies. The enzyme protein was found to be diffisively distributed in cytoplasm of skeletal muscle cells. In white muscle only trace amounts of the specific reaction were observed, whereas in red skeletal muscle a very strong specific reaction was noticed. There was no reaction in mitochondrias or in other intracellular organelles.

SCANDEM-86 Chloroplast Ultrastructure in Relation to the Chlorophyll-Protein Photosynthetic Capacity. EVA-MARI ARO, Department of Biology,

279 Composition

and the

University of Turku,

SF-20500 Turku, Finland. Ultrastructure of mesophyll chloroplasts has been studied in plants grown under different environmental conditions. The effect of the quantity and the quality of light on the thylakoid organization and on the chlorophyll-protein composition was investigated. Lowlight chloroplasts had higher ratios of appressed to nonappressed thylakoid membranes and relatively more light-harvesting Chl a/b-protein than high-light chloroplasts. This is in accordance with the well-known lateral heterogeneity in the distribution of the Chlprotein complexes in the thylakoid membranes. Responses to darkness and different artificial light qualities were more complicated, suggesting divergent distribution of the Chl-protein complexes in the thylakoid network. The relationship between the chloroplast ultrastructure and the photosynthetic capacity of the leaf discs was studied in plants growing wild in different biotopes. The higher the photosynthetic capacity of the leaf discs, the lower was the ratio of the total length of appressed to nonappressed thylakoid membranes and the smaller the grana content in the mesophyll chloroplasts. This relationship was evident during early season when the environmental conditions were most favorable. The size of grana increased in all the species as the season progressed, while no diurnal variations were observed. The content of starch grains in the chloroplasts, however, fluctuated widely both diurnally and seasonally.

Variations in Oxidase Activity in Procambium Cells of Willow Buds during Winter and Early Spring as Shown by Cytochemistry. B. BERGGREN,Department of Botany, University

of Stockholm, S- IO6 91 Stockholm, Sweden. This report is part of an ultrastructural study on morphogenesis and seasonal variations in willow buds. The specimens were sampled outdoors, fixed in cold glutaraldehyde, and cut into loo-pm sections. The sections were incubated with H202 and diaminobenzidine, alternatively tetramethyl benzidine or Hanker-Yates’ reagent, and treated with Os04 overnight. In January reaction was found at pH 7 in the cell walls, especially in the middle lamella region, at the plasmalemma, and in the endomembranes, many minute vacuoles, and mitochondrial cristae. The enzyme activity in walls and plasmalemma appears insensitive to treatment with diethylcarbamate (DIECA) and to some extent aminotriazole (AT) and omission of H202. The endomembrane reaction is not inhibited by DIECA or KCN. After treatment with AT or omission of H202 some activity is retained in the cristae. At pH 5 the cristae appear inactive. At pH 9 the activity is mostly weaker. In February no reaction product was found in the cristae. At pH 5 more precipitate appeared over walls and smaller vacuoles (or microbodies?) surrounding lipid bodies. Mature sieve cells had peroxidases at ER and P-protein. A slight reaction appeared at pH 9. During March and April enzyme activity at pHs 7 and 9 decreased in most cellular components. The stacked ER in sieve cells retained its activity. The results reveal that there is a considerable metabolic activity in seemingly dormant buds during January and February. Several peroxidases with different pH optima and inhibitor sensitivity were recognized.

Differentiation of the Protophloem in Adventitious Roots of Sufix vimindis. INGEMAR FJELL, Department of Botany, University of Stockholm, S-106 91 Stockholm, Sweden.

Most studies of sieve element differentiation are based on short series of cells of the metaphloem and secondary phloem. In this contribution is presented the morphogenesis

SCANDEM-86 280 in an uninterrupted series of as many as 32 future sieve elements in adventitious roots of Salix viminalis. The cells were numbered, starting from the apical me&em. The nucleus was lobed at an early stage and in older cells the nucleoplasm became electron lucent. In the first or second cell from the first mature sieve element the nuclear envelope broke open. From the 10th cell the plastids contained starch. ER increased in amount and began to form stacks in the 20th cell. C’allose platelets were first observed on the transverse walls in cell 18. The sieve pore sites were covered with ER cistemae. Gradually the middle lamella was dissolved and callose formed cylinders around the pores of the sieve plate. Aggregations of tubular P-protein were present from cell 15. In mature sieve elements disaggregating P-protein bodies were seen. Occasionally P-protein formed plugs in the sieve plate pores. P-protein bodies were also present in parenchyma cells adjoining mature sieve elements. The sieve elements had no ontogenetically related companion cells.

Wax Layer Erosion in Spruce Needles-An Indicator of Air-Borne Pollution. GULLVAG AND H. ~STENSEN, Department of Botany, University of Trondheim,

B. M. 7055

Dragvoll, Norway. The epicuticular wax of spruce needles has been studied with the aim of correlating structural degeneration of the wax layer with air-borne pollution. Needles (O-7 years old) with no damage visible in the stereo microscope were studied by scanning electron microscopy after gold coating. Branches with a succession of needles were sampled from areas with possibly negligible pollution and from localities with differing levels of airborne long distance and/or local pollution. The best preserved specimens from the cleanest localities were chosen as controls and a comparison is made with similar investigations. The epicuticular wax above the stomata forms a three-dimensional network of anostomosing wax rods covering the guard cells and the outer vestibule. In a natural environment the fine structure becomes more coarse after 2-3 years but remains visible after 6 years or more. Several investigations have shown damage to the wax layer of Conifer needles due to air-borne industrial pollution. Long-distance air-borne pollution may damage the wax so severely as to cause melting of the stomata1 wax fine structure shortly after the needles are formed. The gradient of damage is related to the amount of pollution and the age of the needles. As the erosion of the wax layer is a direct effect of air-borne pollution, it may possibly be used for monitoring. A possible classification of damage is suggested.

Comparisons of Leaf Surface Structures of Elm, Oak, and Maple in Urban and Rural Trees. S. HUTTUNEN AND K. RUONALA, Department of Botany, University of Oulu, SF-

90570 Oulu, Finland. Scanning electron microscopy was used to study changes of leaf surface structure in urban trees. The elm (Ulmus glabra), oak (Quercus robur), and maple (Acer platanoides) leaves were obtained from test trees growing in the center of the town of Pori and in the rural area, Yline, in southern Finland. The surface structure of elm was wavy and covered with amorphous wax. On the upper surface a lot of scutellate trichomes were observed. The upper surface of the oak leaf was flat and covered with crystalloid waxes. In maple leaves the upper surface waves were located in groups and these groups formed a netlike structure with amorphous waxes. In urban elms a less wavy structure and more scutellate trichomes were observed. Broken trichomes were typical of urban elm trees. The amount of chrystalloid wax was less in urban elm trees. In oak leaves the amount of crystalloid

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wax was less in urban trees. The wax morphology in urban oak trees was destroyed in 1 month. In the maple, urban leaves had a less wavy structure. The amount of waves decreased in August maple samples. The waves were fused into larger groups and the netlike structure was disturbed. The total amount of waxes in maple leaves was about 1%. The amount decreased from July to August.

Effects of Ozone on the Ultrastructure

of Botany, University of Stockholm,

of Leaf

Cells. MONICA

S- 106 91 Stockholm,

JOHANSSON,

Department

Sweden.

The objective of this study was to determine ultrastructural effects of ozone on spinach leaves that had been exposed in a fumigated chamber. The plants, which were about 30 days old and had 6-8 leaves, were exposed for 6 hr to ozone in different concentrations from 0.1 to 0.45 ppm. Symptoms of damage (necrotic spots) were apparent at 0.3 ppm. The first signs of injury noticed in the TEM were shrinkage of the cells and the occurrence of small osmiophilic droplets on the chloroplast envelope. At 0.45 ppm ozone induced severe injury of the organelles. The chloroplasts exhibited local swellings of the space between the two membranes of the envelope and these components contained vesiculate material. Crystalline bodies were formed in the stroma. The envelope of mitochondria occasionally formed spaces similar to those of affected chloroplasts. The cristae were sometimes changed in a characteristic way. The cytoplasm was vesiculate and fairly electron transparent. In seriously damaged cells the envelopes of chloroplasts and mitochondria were broken down. Some dead cells of high electron density were also found.

Effects of Aging

and Air Pollution

Department

HUTTUNEN,

on Norway

Spruce Needles. M. KARHU

of Botany, University of Oulu, SF-90570

AND S.

Oulu, Finland.

SEM images have been used to study the changes in spruce needle surface structure caused by ageing and air pollutant effects. In the clean rural forest near Oulu 65”N 12 to 13 needle years were observed. During the first year of growth, the fibril starlike structure of epicuticular wax was curled, apparently as a result of winter conditions. The changes observed in wax structure were most obvious in 3- or 4-year-old needles. The wax fibrils around stomata were fused together and had become more sheetlike. The amount of starlike fibril wax between stomata decreased. In sulphur-dioxide-polluted environments 9 needle years, and in nitrogen-oxide-polluted areas 6 needle years, were observed, and the typical changes were noticed in these cases. The changes in spruce needle epicuticular waxes were not so easily detectable as in Scats pine with more regular structures. In the same spruce branch very different levels of needle surface erosion could be detected. As a result of ageing and air pollutants the amount of eroded needles increased. When different types of air-polluted environments were compared, different needle surface injury types were observed. In addition, the accumulation of small particles in and around stomata was noticed.

Harmful

Effect of DTT, Mercaptoethanol,

LA-AHVENNIEMI

AND AIJA MUHONEN,

and Protease K on Pine Polysomes. S. KUPIof Botany, University of Oulu, Linnan-

Department

maa, 90570 Oulu, Finland. Polyvinylpyrrolidone (PVP), dithiothreitol (DTT), and mercaptoethanol prevent the formation of quinones in plant homogenates. Protease K detaches ribosomes from membranes and inhibits the RNase activity. In our studies on ribosomes isolated from vege-

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tative buds of the Scats pine we found that PVP ensured high ribosome yield with good translation capacity. The polysome profiles obtained after the sucrose density gradient centrifugation indicated good polymer separation. The scanning electron microscope studies revealed the presence of monosomes, polysomes of various size, and clusters of ribosomes. The frequency of large polysomes was high. The use of DTT or mercaptoethanol instead of PVP lowered the ribosome yields. The polysome profiles had unusually high dimer peaks. As compared to controls, the translation capacity of the samples was onethird or less. Protease K did not lower the yields or change the polysome profiles. However, the translation capacity dropped to less than one-tenth of that found in the controls. In both cases the scanning electron microscope studies suggested breakdown of the largest polysomes and ribosome clusters.

Effects of Metronidazole and Cefoxitine on the Morphology and Ultrastructure of ButL. LINKO-KETWNEN, M. K. VILJANEN, P. HUOVINEN, AND L. J. PELLINIEMI, Department of Medical Microbiology and Laboratory of Electron Microscopy, University of Turku, Turku, Finland. Bacteroides fragih was cultivated in Wilkinson-Chalgren anerobic broth supplemented with subinhibitory concentrations of cefoxitine (MIC 4 mg/liter) or metronidazole (MIC 0.5 mg/liter) in an anerobic glowbox at 37°C for 47 hr. Bacteria were harvested by centrifugation, washed twice with phosphate-buffered saline (PBS), and allowed to react with a monoclonal antibody that is directed against a B l-6 linked Dgalactose oligosaccharide in the lipopolysaccharide of B. fragilis, After incubation of 2 hr at 22°C the cells were again washed with PBS and protein A-gold conjugate (particle size 10 nm, Janssen Pharmaceutica) was incubated with the bacteria at 4°C overnight. The cells were washed as above and prefixed for 2 hr with 2.5% glutaraldehyde in 0.2 M s-collidine-HCI buffer, pH 7.4, which contained 700 ppm ruthenium red and 0.05% CX12, and then postfixed in 1% 0~0,. Bacteria were embedded in Epon. All specimens were examined with a JEOL JEM 100 C transmission electron microscope. In the lowest concentrations of both antimicrobials, strongly elongated bacteria were encountered and there were perforations and blebs in the bacterial cell walls. In the higher concentrations broken and totally destructed bacteria were seen, in addition to elongated ones. Further, ghosts consisting of the outer membrane of B. fragiZis were common, particularly in the higher concentrations. The target antigen of the monoclonal antibody occurred in the detached membrane pieces and on the ghosts. The two antimicrobials did not differ in their effect on the morphology and ultrastructure of B. fragilis. feroi&sfiugXs.

Ultrastructure

of Cell Envelope of Gram-Negative Bacteria with “Extra” Proteins. K. Departments of Electron Microscopy and General Microbiology, University of Helsinki, Helsinki, Finland. In many gram-negative bacteria there are “extra” proteins in their cell envelope. Some of those proteins are fimbrillines or flagellines which are known to be important in the adhesion and the motion of the bacteria. There are also in some bacterial species certain proteins, such as paracrystalline surface (S layer) proteins and tack-like protein structures. The function of these proteins is so far unknown. On the surface of the plasmid-containing virulent strain of Yersinia enterocolitica oultivated at 37°C there is tacklike or fibrillar material, which has an important role in the autoaggregation of the cells. The role of this protein (MW 47 kDa1) in the adhesion of the bacterial cells to the host cells is still unclear. LOUNATMAA,

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283

The S layers are important for the virulence of some patogens, whereas the S-layerless strains are no more virulent. Structurally those layers are composed by periodically arranged protein (or glycoprotein) particles. In Bucteroides buccue strains two different hexagonal lattices were found, one with a unit cell spacing of 2 1.5 nm and another with a spacing of 7.7 nm. In these B. buccue strains a crystalline outer membrane protein with hexagonal arrangement was also found in the concave face of the outer membrane. Ultrastructure

of an Unusual Plant Cell: The Spurfina

Salt Gland. PETER OLESEN* AND

*A/S De Danske Sukketiabrikker, Biokemisk Laboratorium, Langebrogade 1, DK-1001 Kgbenhavn K, and tlnstitut for Sporeplanter, Kgbenhavns Universitet, 0ster Farimagsgade 20, DK-1353 Kgbenhavn K, Denmark. Rather few plant species are adapted to growth and reproduction in saline environments. A typical feature of such species is the occurrence on their leaves and stems of epidermal glands involved in the secretion of excess minerals and ions accumulating in the tissues. Among the different kinds of salt glands those of halophytic grasses are quite exceptional, being two-celled and displaying many structural features of animal salt glands and transporting epithelial cells. In principle, the large basal (collecting) cell of a Spartina gland is a transfer cell, i.e., a strategically positioned cell that has evolved a significantly enlarged transporting apparatus consisting of wall protuberances into the cytoplasm bordered by highly increased areas of plasma membrane. In Spurtina, however, this specialization has by far surpassed any other known transfer cell type in plants in that the whole cytoplasm is partitioned by double membranes which are continuations of the plasma membranes bordering the wall protuberances. Also the protuberances are not of the usual unorganized type but appear as hollow tubes showing some degree of organization in their walls. The large number of mitochondria are lined up in the cytoplasmic fingers between the partitioning membranes which frequently are underlined by microtubules. The large basal cell communicates with the small excreting top cell through numerous plasmodesmata. By comparison, the large basal cell of salt glands in Spartina and other halophytic grasses is structurally almost identical to chloride cells of aquatic insects, nasal salt glands of desert reptiles and marine birds, and other cell types involved in transepithelial transport of salt and water. Taken together, the ultrastructural specializations of highly evolved salt-transporting cells in a number of animal glands and salt glands of halophytic grasses provide an extremely clear-cut example of convergent evolution. LISBETH

HAUKROGH,?

Freeze-Induced Plasma Membrane Changes in Plant Cells. K. PIHAKASKI, Department of Biology, University of Turku, SF-20500, Turku, Finland. Many recent studies have suggested that alterations in the plasma membrane during freezing are the result of the freeze-induced dehydration rather than of the low temperature per se. Leakiness after damaging freezing treatment has been thought to be due to the intramembranous particle-free areas visible in the freeze-fracture faces of the plasma membrane. Changes in the lipid phase have long been thought to play an important role in freezing injuries in the membranes. A causal relationship has been suggested between the dehydration-induced lamellar-to-nonbilayer phase (hexagonal,,) transitions and the loss of osmotic responsiveness. Cryoinjury of the protoplasts isolated from nonacclimated rye leaves (Secule cereale L. cv Puma and Voima) causes osmotic unresponsiveness during thawing of the suspending medium (isotonic sorbitol) after damaging freezing. The injury is associated with changes in the ultrastructure of the plasma membrane: the appearance

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284

of the smooth-surfaced lamellae subtending the plasma membrane (at -4°C and lower), lateral phase separation, and phase transitions from lamellar to nonbilayer phase (at - 6°C and lower). The addition of DMSO (2-100/o) to the suspending medium decreases the injury of the cells significantly. It also defers the occurrence of lipid phase transitions.

Ultrastructural

Changes in Chloroplasts

of Diupensiu lupponicu L. in the Annual Cycle.

K. PIHAKASKI, Department of Biology, University of Turku, SF-20500 Turku, Finland. Ultrastructural seasonal changes in the chloroplasts in palisade parenchyma cells of a subarctic evergreen Diapensia lapponica L. were analyzed by morphometry from the conventionally prepared thin sections. Pronounced seasonal changes were found in the chloroplasts. In summer, at a time of nonrhythmic light, the chloroplasts were large in size and contained much starch. The grana were small and threadlike, resembling those of sun leaves. The volume density of grana thylakoids was of about the same magnitude as in autumn. Stroma thylakoids were poorly developed. In autumn the number of partitions per granum was more than double that found in summer resembling shade leaves. The stroma thylakoids were well developed. The chloroplasts continued to be oblongate in shape from summer through autumn and early winter, corresponding to the photosynthetically active period. In midwinter and early spring the chloroplasts were rounded, corresponding to the inactive time of the annual cycle. Small starch grains were occasionally discernible still in November. Spring chloroplasts were characterized by low volume density for the grana but relatively high volume density for the stroma thylakoids.

Plastid Structure KOSKI, M. F~RDIG,

and in Anther-Derived Haploid Plants. M. RAUDASof Botany, University of Helsinki, UnionFinland; and *Department of Plant Breeding, University of

in Microspores

AND P. RY~~PPY,* Department

inkatu 44, 00170 Helsinki, Helsinki, Viikki, 00710 Helsinki, Finland. In Triticale haploid plants are obtained via culture of microspores (pollen grains). Anthers with microspores at the stage before haploid mitosis are chosen. A serious disadvantage of the method is the high proportion of albino plants without photosynthetic capacity arising from the plated anthers. The aim of the present study is to elucidate the background of this phenomenon by ultrastructural means. The fine structure of microspores at the time of plating and that of green and albino plants derived from the microspores were examined. Throughout the study special attention was paid to the fine structure of plastids. Samples were lixed from three different platings of anthers. The fine structure of the microspores was similar in the three samples. The microspores were mainly uninucleate with a large central vacuole. In the thin layer of cytoplasm close to the wall of the microspore proplastids were recognized by their electron-dense contents with a few lamellar structures. In both the proplastids and promitochondria light areas were distinguished with fine strands, which were interpreted as the DNA of the organelles. Some proplastids and promitochondria contained small vacuoles, which suggested that autolysis of the cell organelles may take place in the microspores. At the time of sampling, however, the metabolic activity in the microspores is probably very low, since before plating the anthers, with their microspores, are subjected to cold treatment for several days. This may have caused the frequent occurrence of microfilament bundles in the nuclei and cytoplasm of the microspores. The fine structure was examined in several green and albino plants grown from the microspores and in a mosaic plant with green and white sectors in the leaf. In the green plants all the cells contained chloroplasts with normal thylakoids.

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The division figures and areas with DNA in the chloroplasts suggested that they were increasing in number. In the albino plants the plastids were poorly developed, lacking thylakoids, but containing plastoglobuli. In light green plants, the plastids had structures which resembled the prolamellar bodies typical of etioplasts. In the mosaic plant the cells contained normal chloroplasts or plastids comparable to those of albino plants. The variation in the plastid structure was attributed to changes in the system regulating chloroplast development. Pollen Tube Cytoskeleton: Immunocytochemistry and Ultrastructure, M. RAUDASKOSKI, H. ASTR~M, AND M. FLRDIG, Department of Botany, University of Helsinki, 00170 Helsinki, Finland. Labeling of pollen tubes of Nicotiana tabacum with tubulin antibodies showed longitudinally oriented tubulin-positive fibers, especially in front of and behind the generative and vegetative nuclei, which are located close to each other in the cytoplasm-containing part of the pollen tube. The generative nucleus was visualized very effectively due to bands with bright fluorescence surrounding the spindle-shaped nucleus. NBD-phallacidin, a special probe for tiamentous actin, and anti-actin antibodies revealed another set of longitudinally oriented cytoplasmic fibers in the pollen tubes. The structure of the cytoskeleton was also examined in pollen tubes grown on solidified germination medium containing a microtubule-stabilizing agent, tax01 (1 pg ml-l), dissolved in DMSO (dimethylsulfoxide). Since DMSO is known to interfere with cytoskeletal elements, pollen tubes were also grown on medium containing only DMSO ( 1%). Both drugs clearly affected the growth of pollen tubes. Taxol decreased and DMSO increased the tube length. Both drugs improved the visualization of the cytoskeleton. Exposure to tax01 frequently led to a change in the orientation of tubulin-positive fibers from longitudinal to transverse and to a decrease in the fluorescence of the generative nucleus. Exposure to DMSO increased the number of pollen tubes with generative cell division. Ultrastructural examination likewise indicated improved preservation of microtubules when pollen tubes were grown with taxol or DMSO. Two types of microtubules were detected, close to the plasma membrane occurred single cortical microtubules, which probably corresponded to the longitudinal fibers revealed with tubulin antibodies. In the narrow belt of cytoplasm of the generative cell occurred bands of parallel longitudinal microtubules, which probably caused the strong fluorescence of the generative cell after treatment with tubulin antibodies. In pollen tubes grown with DMSO, the depolymerization of microtubular belts and the repolymerization of tubulin to spindle microtubles could be followed during the division of the generative cell into two sperm cells. No microfilaments were detected in electron microscopic examination of pollen tubes, in spite of the abundance of microfilaments revealed by fluorescence microscopic techniques. This suggested that conventional methods of preparing specimens for electron microscopic studies give poor preservation of microfilaments in pollen tubes. In fungal hyphae fixation by freeze substitution has been shown to give good preservation of microfilaments. The application of freeze substitution in the study of pollen tube cytoskeleton is under research. Observations

on the Ultrastructure

of Winter

AND S. HUTTIJNEN,

Department

Hardened

Pine and Spruce Needles. J.

Linnanmaa 90570 Oulu, Finland. The aim of the study was to examine the ultrastructure of Scats pine (Pinus sylvestris L.) and Norway spruce (Picea a&es) needles during the coldest winter conditions with REINIKAINEN

of Botany, University of Oh,

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material collected in January 1986 in Ylikiiminki, Northern Finland. The general appearance of the mesophyll cells was compatible with earlier studies in the literature. The chloroplasts were agglomerated on cell margins and around the nucleus, no starch was found, and the cytoplasm was strongly netlike. Lipid accumulations were abundant as small droplets in the cytoplasm, and tannin appeared as small granules in the central vacuole. The curling of the outer membrane of the chloroplasts was typical for the examined cells. The chloroplast stroma often had membraneous formations including granular material. The examined spruces differed from each other mostly in the amount of grana thylakoids. Both pines also showed a decrease of granal membranes. The amount of plastoglobuli was in inverse proportion to the amount of grana thylakoids in the chloroplasts. Usually the plastoglobuli were dark, but some lightening could also be observed, especially in pine cells. The cytoplasm exhibited many vacuoles including granular material. Individual differences and other unusual phenomena noted could be the consequences of the severe winter of 1984-1985, when the needles were young.

Seasonal Changes in the Ultrastructure of Ray Parenchyma Cells of Scats Pine. PEKKA Institute of Botany, University of Helsinki, Unioninkatu 44, SF-001 70 Helsinki, Finland. The ultrastructural changes of ray parenchyma cells of Scats pine (Pinus sylvestris L.) were studied in response to heartwood formation and seasonal changes. Samples were collected from breast height of a field grown tree (D 1.3 = 30 cm). The sampling dates were June 7, August 14, October 16, 1985, January 6, and March 4, 1986. Samples of cambial zone, outer sapwood, inner sapwood, and sapwood-heartwood transition zone were taken from increment borings and fixed in 3% glutaraldehyde in 0.1 M phosphate buffer, pH 7.0, and postfixed in 0~0,. The number of lipid droplets increases from the cambial zone to the inner sapwood, but the number of other cell organelles and vacuolar size decreases. The parenchyma cells start to lose their cellular contents in the sapwoodheartwood transition zone. The parenchyma cells also reacted to environmental changes. The results show differences in the structure and starch content of plastids, the structure of plasmalemma, and chromatin condensation. SARANPA&

Changes in the Ultrastructure of Cambial Cells during the Development of Winter Hardiness in Young Shoots of Willow (S&ix dusycludos) Wim. L. SENmRBY-FoRssE*,t AND H. A. VON FIRcKs,j’ *Department of Botany, University of Stockholm, S-106 91 Stockholm, Sweden: and TDepartment of Ecology and Environmental Research, Swedish University of Agricultural Sciences, S-750 07 Uppsala, Sweden. An investigation was made to elucidate the influence of different nutrient conditions on the initiation of winter hardiness in willow plants. One group of plants was given optimum nutrient conditions and another group was grown under nutrient stress. Frost hardiness of the plants was estimated by standardized freezing tests. Cell divisions in the cambium in September produced 8-l 2 highly vacuolated cells in a radial row. Rough ER and dictyosomes with vesicles indicated metabolic activity. The xylem derivatives contained protoplasm and signs of secondary wall formation were noticed. In plants grown under optimum conditions plenty of starch appeared in chloroplasts of ray parenchyma cells. By the end of September all shoots had completed their length growth and survived -4°C without damage. During October the cambial zone contained only 3-5 cell rows indicating cessation of mitosis. In plants grown under optimum conditions highly vacu-

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olated cells still occurred. Spherosomes were observed in the cytoplasm and the amount of starch in plastids increased. Pinocytosis vesicles were abundant in phloem and ray parenchyma cells. In stressed plants the vacuole system was transformed into an assembly of small vacuoles, frequently containing whorls of membraneous material. The hardiness in all shoots was further increased so that in January they could survive -80°C. This was accompanied by an augmentation of dormant features in the ultrastructure of cambial cells.

Transmission Electron Microscopy of Bacterial Attack of Wood Cell Walls. A. P. SINGH,* T. NILSSON, AND J. A. BurcHna,t Department of Forest Products, Swedish University of Agriculture Sciences, Box 7008, S-750 07 Uppsala, Sweden. Pinus radiata horticultural posts were first examined by light microscopy to locate areas of bacterial attack. Selected areas containing bacterial attack were then processed for transmission electron microscopy. Wood sections were fixed in glutaraldehyde, postfixed in osmium tetroxide, dehydrated in acetone, and embedded in low viscosity Spurr’s medium. Ultrathin sections obtained on an LKB ultramicrotome with a diamond knife were stained in 1% aqueous potassium permanganate and examined in a Phillips 300 transmission electron microscope. Several patterns of bacterial attack of wood cell walls, including tunneling, cavitation, and erosion were observed. This presentation will be concerned with the description of the erosion type of attack. Ray cells were attacked first and bacteria gained entry into tracheids via ray cells at ray crossings. The erosion of the tracheid cell wall by bacteria progressed from lumen outwards and had a distinct conical form. Initially, bacteria degraded the S3 wall at the point of entry into the wall but the attack soon spread into the S2 wall in preference to the S, wall. Conical erosion troughs were often seen to be tiled with a granular residual wall material and small cavities containing bacteria. Eroded wall areas eventually became empty of all contents. Bacterial attack in some cases spread to the S, wall but the middle lamella remained largely unaltered. * Visiting scientist from Forest Research Institute, New Zealand. t Forest Research Institute, Private Bag, Rotorua, New Zealand.

Morphogenesis

of Tapetal Cells in pinus @vest&

BJ~RN WALLES

AND JOHN R. ROWLEY,

Department of Botany, University of Stockholm, S-106 91 Stockholm, Sweden. The tapetum of microsporangia functions is nourishing the microspore mother cells and the microspores during their extensive growth. Since no physical contact exists between those cells and the tapetal cells, the nutrients have to be secreted into the locular fluid. Using annual fixations of Pinus sylvestris microsporangia from over 9 years we have documented changes in tapetal structures from before the start of meiosis in sporogenous cells until the first microspore mitosis. We discovered that the tapetal cells repeatedly go through cycles of high activity reflected in a characteristic ultrastructural appearance and intervening periods of reversion to a dedifferentiated, i.e., a meristematic, condition. About 1 day before the start of meiosis the cell wall of the tapetal cells is lysed. The cytoplasm looks hypersecretory, e.g., rER is dilated, dictyosomes produce many vesicles, and numerous autophagic bodies appear. Subsequently the cells dedifferentiate with a reduction of ribosomes and rER and the appearance of amoeboid plastids and mitochondria. The cells are also interconnected by plasmodesma-like processes, may have

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plasmatubules, and undergo mitosis (but not cytokinesis). During microspore mother cell meiosis five cycles of high activity can be identified in the tapetum, followed by two further cycles during the free microspore period. Typically, activation of the cells includes a radial extension into the sporangial locus. The number of tapetal cells appears constant although the loculi grow in size from about 150 pm in diameter to 500 x 1500 pm. Injuries in Mesophyll Ultrastructure of Needles of Scats Pine (pinus @vest&) by Fluoride. A. WULFF AND L. KXRENLAMPI, Ecological Laboratory, Department vironmental Hygiene, University of Kuopio, P.O. Box 6, 70210 Kuopio, Finland.

caused

of En-

The 4-year-old Scats pine saplings growing in containers in open field were exposed to fluoride by spraying hydrogen fluoride solutions on the plants 5 days a week. The concentrations used were 2, 15, and 70 mgF/liter. The control saplings were sprayed by distilled water. The samples for EM examination were taken after exposure of 10 weeks duration (9/9/1985). The samples were fixed in 2% glutaraldehyde in 0.075 Mphosphate buffer and postfixed in 1% 0~0,. Thin sections were stained with uranyl acetate and lead citrate. Injuries were quite local and usually situated immediately beneath the hypodermis or near the stoma. There were no differences in injuries between the lst- and 2nd-year needles. The ‘IO-mgF-/liter treatment caused slight, medium, and severe swelling of chloroplast thylakoids. The stroma of chloroplasts was granulated and the chloroplast envelope occassionally disappeared. The granulation of mitochondrial matrix and the swelling of mitochondrial cristae could also be seen. The treatment with 15 mgF-/liter caused slight or medium swelling of the thylakoids. Both the 15- and 70-mgF-/liter treatments increased the amount of large lipid accumulations. In the mesophyll cells immediately beneath the hypodermis the amount of thylakoids was decreased and the number and size of plastoglobuli was increased after the 15- and 70-mgI-/liter treatments. In these cells the plasmalemma was frequently severely curled. The ultrastructure of mesophyll cells exposed to the 2-mgF-/liter treatment was normal. Scanning Electron Microscopy and Transmission Electron Microscopy of the Esophageal Mucosa of the Rabbit Treated with cis-Diamminedichloride Alone and in Combination wiht Ionizing Radiation. M. ALBERTSSON, C. H. H~ANSSON, AND C. MERCKE, Department of Oncology, University Hospital, S-221 85 Lund, Sweden.

cis-Diamminedichloride (cis-platin) has for more than 20 years been used in the therapeutic arsenal of oncology. Most of our knowledge of its biological action is based on clinical investigations and there is a need for an examination of its influence at the cellular and subcellular levels. Five milligrams of c&P was given to ten rabbits. Ultrastructural examinations were performed on the upper and lower part of the esophagus each day after the ip injection for 10 days. Another 50 rabbits obtained 5 mg c&P and were irradiated in an area just below the larynx. They were given 2 Gy at each irradiation and were maximally treated to 20 Gy. Examinations were made starting on the first day after the final treatment and then on each day during 10 consecutive days. Five animals were used as controls. c&P was shown to have an extremely deleterious effect on the epithelial layer of the esophageal mucosa with cell loss and structural disarrangement of the microridges and whorls on the surface. This was an early observation and the early phenomenon lasted for all 10 days of examination. The changes were even more exaggerated when irradiation was added. Repopulation of new cells from the matrix was noticed at about 5 days after the administration of c&P alone and at the 7th or 8th day within the irradiated area in those animals also treated with cis-platin.

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Early Effects of Cadmium on Endothelial Cells in Rat Testes. HANS EKWALL AND LEIF PLATEN,Department of Anatomy and Histology, Faculty of VeterinaryMedicine, Swedish Universityof Agricultural Sciences,S-750 07 Uppsala,Sweden. It has long been known that parenteral administration of cadmium chloride in doses of about 0.03 mmole/kg BW to male rats results in necrosis of the testes. Moreover, it is generally accepted that cadmium primarily affects testicular blood circulation. Only a few studies have been published on the early effects of cadmium, and hence we have investigated the ultrastructure of testicular endothelial cells 15 min to 24 hr after an intraperitoneal injection of cadmium chloride (0.03 mmole/kg BW) to mature male rats. Some of the testes were fixed by vascular perfusion through the testicular artery according to Forssmann (1977), the rest by immersion in 3% glutaraldehyde in 0.067 M cacodylate buffer, pH 7.2. All material was then processed for transmission electron microscopy. Lesions were already observed in the endothelial cells of arterioles, capillaries, and postcapillary venules 15 min after a Cd injection. The lesions were mainly seen as discontinuities in the cytoplasm of the endothelial cells. Within 30 min after Cd administration accumulations of platelets were seen in the lumina, and somewhat later the endothelial lining became discontinuous. Interstitial edema appeared and the vessels became filled with red blood cells. After 6 hr, perfursion was no longer possible since practically all blood vessels were obstructed. It appears that during the initial damage factors are released that induce the formation of thrombosis. The later changes are probably secondary to the disrupted blood circulation. More studies will be needed in order to further characterize the initial damage. Since the blood vessels of most other organs are unaffected, the results suggest that the testicular blood vessels differ physiologically from the vessels of most other organs. Fiber and Quartz Contents of Powdered Rocks and Phagocytosis of Particles by Human Neutrophils in Vitro. T. GUSTAFSSON, M. HEDENBORG, T. SALMI, AND M. KL~CKARS, Institute of OccupationalHealth, Haartmaninkatu 1, SF-00290 Helsinki, Finland. Powdered rocks containing minerals such as richterite, serpentine, wollastonite, tremolite, etc. were studied by electron microscopy (fiber content) and X-ray dilfractometry (quartz content). Phagocytosis of the powder particles was studied with human neutrophils in vitro using luminol enhanced chemiluminescence. The aim of the study was to compare fiber and quartz contents of the powders with the phagocytic response on neutrophils. Weighted amounts of powdered rocks were suspended in water. After ultrasonic treatment of the stock suspension (10 mg/lOO ml) and filtration of l-10 ml on membrane filters (Nuclepore, 0.2 pm) the samples were gold coated in an ion sputtering device (JFC- 1100). Fiber contents were determined from secondary electron images by counting of 50 fields of view at magnification x 3000. The fiber length and diameter varied from 5 to > 100 wrn and from 0.03 to 3 pm, respectively. In fiber counting the aspect ratio 3: 1 was required. Sensitivity was one fiber in 50 fields of view. Relative fiber contents varied from 10 to lo6 (fiber/mg). Purified neutrophils (90-9596) were obtained from heparinized human peripheral blood by HypaqueFicolI gradient centrifugation followed by NH&l lysis of erythrocytes. Phagocytosis of powder particles was studied using a Luminometer LKBWallac 125 1 and a phagocytosis program 125 1- 124 (LKB-Wallac) connected to an Apple 2e computer. One hundred-microliter cell suspensions (4 x lo6 cells/ml) in Dulbecco’s phosphate-buffered saline, 700 ~1 of 1O-4 MLuminol, and 25-l 00 ~1 of powder suspension (stock concentration 1 mg/ml) were incubated at 37°C and the chemiluminescent output was followed for 60 min. High luminol enhanced chemiluminescence output, caused by

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290

activated cellular O2 metabolism during particle and cell interaction in the early stages of the phagocytosis, was observed with high quartz contents (20-70%). Differences between powders (quartz content < 1%) were also detected. Highly fibrous sepiolite did not cause significant chemiluminescent output whereas chrysotile caused a highly increased output.

Electron

Microscopy

of the Effects of Toxic Substances on the Epidermis. L&SE ofOccupational Health, Helsinki, and Department of Dermatology, University Central Hospital, Helsinki, Finland. The effects of toxic substances on the fine structure of the skin are poorly understood. In this study we investigated the reactions ofrodent and human skin to methylmethacrylate (MMA), terphenyls, dithranol (DIT), and methylglyoxalbis (quanylhydrazone) (MGBG) by means of conventional electron microscopy. Both MMA and terphenyls caused spongiosis of the epidermis, but had no specific effects. DIT occlusion for 24 hr caused moderate to massive amounts of lipid droplets to appear in keratinocytes. Odd circular and branched Birbeck granules were found in Langerhans’ cells (LCs). Mitochondria of LCs proved more susceptible than mitochondria of keratinocytes to DIT occlusion for 3 hr. Considerable swelling of mitochondria of basal keratinocytes was detected after treatment with MGBG, the outer and inner mitochondrial membranes were distended, and the cristae were distorted. As the mitochondria of the LCs were unaffected, MGBG was probably taken up primarily by proliferative cells. The present study showed the fine structural dissimilarity caused by treating skin with different topical substances: DIT had a specific effect on the mitochondria of the LCs, and MGBG on the keratinocytes, whereas MMA and terphenyls had a classic irritant action on the epidermal cells. More basic research is needed in this field. KANERVA, ELVI VERKKALA, AND JORMA LAUHARANTA, Institute

Mammary Tumor Xenografts in Immunocompetent Mice: Ultrastructural Expression of Transplantation and Drug Treatment. A. P. KYLL~NEN, F. STENB~CK, V-M. WASENIUS, AND L. KANGAS, Department of Pathology, University of Oulu, Department of Oncology and Radiotherapy, University of Helsinki and Farmos, Turku, Finland. Mammary tumor sensitivity to hormonal and drug treatment varies depending upon tumor type and degree of differentiation, receptor status as well as for reasons not presently known. We have analyzed the effects of hormonal agents, medroxyprogesterone and tamoxifen, as well as cytostatic agents, cyclophosphamide and methotrexate, on human tumors and experimentally induced neoplasms transplanted to the subrenal capsule of rats and mice by scanning and transmission electron microscopy and immunohistochemistry and compared the results to gross tumor measurements and response to clinical treatment. The results showed a high transplantation success rate, more than 90%. Transplanted tumors exhibited glandular structures with surface microvilli, cytoplasmic CEA droplets, and basement membrane formation. Hormonal treatment resulted in mitochondrial alterations, smooth endoplasmic reticulum proliferation, and surface bleb formation. Cytostatic drug treatment caused, in less sensitive tumor cells, ballooning and shrinkage, cytoplasmic homogenization and nuclear irregularities. Moderate sensitivity reflected in nuclear disorganization, indistinct cell borders, and partial cytolysis. Successful drug treatment resulted in complete disappearance of the neoplasm with only a fibrotic acellular scar remaining. The results show the applicability of this model for evaluation of patient therapy as well as for development of new drugs. The significance of morphological alterations associated with specific forms of therapy needs further study.

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The Effect of Taxol on Cultured Prostatic P. H;~RK~NEN,

Department

ofAnatomy

Cancer Cells. K.-M. LAME,

and Pathology,

M. R~YTT& AND University of Turku, 20520 Turku,

Finland. Taxol is an experimental antitumor drug derived from a plant Taxus brevifolia. Contrary to the conventional antitumor drugs, such as colchicine, tax01 prevents the breakdown of preexisting microtubules (MT) and makes them resistant to the effect of cold and calcium. Taxol prevents the mitotic activity of HeLa cells and fibroblasts. Additionally it has been shown to cause numerous microtubules related structures both in vivo and in vitro (cf. RBytta and Raine, 1985). The effect of tax01 on LNCaP cell line, established from a metastasis of human prostatic adenocarcinoma, was studied. Several different concentrations of taxol were used from 5 M down to 0.0 1 n&K Taxol was solubilized in 0.1% DMSO and for control purposes cultured cells were treated with 0. lo/b DMSO. The cultured cells were monitored daily by phase-contrast light microscopy up to 15 days. Samples for electron microscope were fixed in 3% glutaraldehyde, dehydrated, embedded in Epon, and scanned with JEOL 100 C electron microscope. The first taxol-induced changes observed by light microscopy consisted of rounding of the contours of the tumor cells with the disappearance of the cytoplasmic extensions. Thereafter some of the tumor cells detached from the bottom of the tissue culture wells. Later on many of the remaining tumor cells became abnormally large with multiple nuclei. The appearance of these changes seemed to be dose related. Concentration of 5 M tax01 induced a marked clearance in the number of tumor cells after 2 days and after 9 days almost all the cells had died. With lower concentrations of tax01 these changes appeared at a slower rate down to 10 nM concentration, while concentrations equal to or less than 1 nM did not show any obvious alterations. By electron microscope the first changes were noted already after 1 day with an increased number of cytoplasmic microtubules, disruption of Golgi complexes, and appearance of numerous mitotic figures with a high amount of haphazardly arranged MT between chromosomes. These mitotic cells corresponded to those numerous round cells seen by light microscopy. The mitochondria were well preserved. Later on these changes increased in quantity and also multinuclear tumor cells with a high number of cytyoplasmic MT, occasionally arranged close to the smooth ER, were noted. Degenerative cells increased in number of the later phase and showed numerous membrane-bound accumulations, swollen mitochondria, and only a few Golgi complexes. The DMSO-treated tumor cells did not show changes identical to those observed in taxol-treated cells. The present study shows that tax01 has a specific cytotoxic effect on prostatic cancer cells and that the amount of tax01 needed is very low. Also MT-related structural changes could be observed. Thus tax01 may have an effect on the prostatic cancer cells also in vivo and may be a useful tool for examining different structural mechanisms in cancer treatment.

Intramembrane Particle Density on the Fracture Faces of the Membranes in Vinblastine-Induced Autophagic Vacuoles. E.-L. PUNNONEN,* P. HIR~IM;~KI,*,~ AND K. LOUNATMAA,* *Department of Cell Biology, University of Jyviiskyk?, SF-401 00 Jyviiskyl& Finland; TFarmos Group Ltd, Research Center, SF-20101 Turku, Finland: and *Department of Electron Microscopy, University of Helsinki, SF-00280 Helsinki, Finland. Intramembrane particle density on the P- and E-fracture faces of the limiting membranes in apparently newly formed autophagic vacuoles was determined in vinblastine-induced autophagocytosis in mouse liver (vinblastine 50 mg/kg, ip, 2 hr). The surface area of the fracture faces was measured from freeze-fracture micrographs using a set of squares (5 x 5 mm), and the number ofthe intramembrane particles on the measured areas was counted.

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At least 20 autophagic vacuoles per each fracture face were included in the analysis. The intramembrane particle density on the outer limiting membrane was 119 + 37 on the P-fracture face and 32 f 10 on the E-face. Corresponding densities on the inner limiting membrane were 21 +- 9 on the P-face and 16 + 5 on the E-face. The particle density varied considerably from one autophagic vacuole to another, and the particles on the membranes of the vacuoles were often very heterogeneously distributed. The intramembrane particle density on the membranes of apparently newly formed autophagic vacuoles seems to be much lower than on the other cellular membranes (Loss et al., 1978). This may indicate that no preexisting membrane type is used as such in autophagic vacuole formation.

Drowning: Ultrastructural Alterations of the Alveolar System. K. WSCHEL, Institute of Forensic Medicine, University of Hamburg, Butenfeld 34, D-2000, Hamburg 54, Germany. Investigations were carried out on anesthetized rats which by a tracheotomy tube actively aspirated liquids of different osmolarities covering a range from tap water to 2.9% NaCl solution. In every range of osmolarity the ultrastructural alterations show areolar limitations and different stages of development. In freshwater the influx of liquid causes a hypoxemic-dysoric alveolose. Findings: Diffise or pulvinate edematous swellings of all compartments of the blood-gas barrier, cytolysis, karyolysis, membrane disintegration, hydropic alterations of the organelles, dilatation of the drainage tracts of the alveolar interstitium, vesicular transformation caused by a dilatation of the pinocytotic system ending in endothelial and epithelial vesiculation. Saltwater drowning leads to a hypoxemic alveolose with a marked compaction of the matrix. Findings: Numerous finger-shaped protusions and constrictions of the epithelium (villous transformation), exposure of the basement membrane, erythrocyte sludge, and thorn-apple-like erythrocytes in the capillaries. The ultrastructural lesions of the alveolar wall were also investigated in freshwater and saltwater drowning victims with only a short period of autolysis (less than 24 hr). The ultrastructural alterations of the human lung correspond to the findings derived from animal experiments.

Ultrastructure and Immunohistochemistry of Pulmonary Basal Lamina Injury in a Rheumatoid Patient during Gold Treatment. PAAVO P.XXKK~,* MARKKU HAKALA,? SEPPO SUTINEN,* SISKO ANTTILA,* AND JUSSI JOUPPILA,$ *Department of Pathology, University of Oulu, Oulu; and TDepartments of Internal Medicine, Oulu University Central Hospital; and Etelii-Pohjanmaa Central Hospital, Seiniijoki, Finland. Connective tissue disorders may be complicated by pulmonary manifestations, as interstitial fibrosis and obliterative bronchiolitis, which may be de novo processes or complications of treatment. However, there are no previous reports on pulmonary basal lamina injury in gold reactions or rheumatoid lung disease. We describe a 47-year-old housewife with cough, breathlessness, and intermittent fever during gold treatment, originally prescribed for seropositive polyarthritis, which later proved to be systemic lupus erythematosus (SLE). The close temporal relationship between symptoms and treatment was suggestive of drug reaction. An open lung biopsy showed abundant interstitial edema with mononuclear inflammatory cells and some eosinophils and slight bronchiolitis but no granulomas. The picture was nonspecific but suggested hypersensitivity pneumonitis. Electron microscopy revealed some splitting and local disappearance of basal laminae.

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293

This injury was confirmed by immunohistochemical staining for type IV collagen and laminin, the major components of basal laminae. This type of injury to basal lamina may be associated with hypersensitivity reaction, but we found no deposits in the basal laminae which would support the possibilty of activation of SLE. In most macrophages there was lysosomal electron dense granular material, i.e., aurosomes, which gave the spectrum of gold in electron microprobe analysis, indicating the presence of antigenic material in the lung tissue. After stopping gold treatment symptoms gradually decreased and no permanent lung disease remained.

Ciliary Orientation: Reproducible Measurement from Electron Micrographs of Respiratory Cilia. M. RAUTIAINEN, Y. COLLAN, AND J. NUUTINEN, Departments of Otolaryngology and Pathlogy, University of Kuopio, SF-70210 Kuopio, Finland.

Changes in the orientation of the cross sections of respiratory cilia in electron micrographs have traditionally been related to disturbances of ciliary function. However, this approach has been based on subjective estimation of ciliary orientation, and no standardized method for estimating ciliary orientation has been available. We have estimated ciliary orientation by estimating the angle between the plane defined by the cross sections of the central tubules and a reference line. Selection of the reference line is critical, and it must be chosen so that the majority of the measurements fall about the middle of the O-180” range. At best the distribution is at zero level at both ends of the range. It should be noted that cross sections do not allow perfect estimation of the beat direction because distinction cannot be made between the directions d and d + 180”, i.e., between the effective stroke and the ineffective recovery stroke. However, uniform orientation will result in a distribution with one peak. Random orientation, on the other hand, will result in a flat distribution. Ciliary populations with several main beat directions will show distribution with several peaks if the directions are not complementary (d and d + 180”). We made measurements along the above principles by applying a completely manual method with glass angle measure. We also tried a semiautomatic image analyzer (IBAS I). The latter approach was faster and more reproducible. Measurements were made from samples of 10 healthy nonsmokers. Four micrographs were used, each containing 9-l 10 cilia. The standard deviation of the beat directions varied from 15.0 to 4 1.2”. The results suggested that variation within a standard deviation of 40” can be considered normal (within 98% probability). Only one of the microscopic fields tested showed standard deviation values above 40”. Whether samples of functionally abnormal cilia show larger variation remains to be shown in future studies.

Effects of Griseofulvin and Nocodazole on Accumulation of Autophagic Vacuoles in Ehrlich Ascites Tumor Cells: A Morphometric Study. H. REUNANEN,* M. MARTTINEN,* AND P. HIRSIM&I,*$ *Department of Cell Biology, University of JyvZskylZ, SF-40100

Jyviiskylii, Finland, and TFarmos Group Ltd, Research Center, SF-20101 Turku, Finland. Accumulation of autophagic vacuoles (AVs) has been observed in a variety of cells treated with the microtubule inhibitor vinblastine and it has been proposed that vinblastine prevents the fusion of lysosomes and autophagosomes resulting in the accumulation of the latter. The aim of the present study was to resolve if the antimicrotubular drugs, nocodazole and griseofulvin, also induce accumulation of AVs. Ehrlich ascites tumor cells were incubated in vitro for 2 hr in a modified Krebs-Ringer phosphate buffer. Griseofulvin and nocodazole were dissolved in DMSO and used at a concentration of 50 I.lg/ml (griseo-

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fulvin) and 20 &ml (nocodazole). Controls were incubated without addition or with 2.5 ~1 DMSO/ml. Samples for morphometrical electron microscopical analysis were collected after 3-min, 30-min, I-hr, and 2-hr incubation periods. The volume densities of AVs remained in control values in all incubation periods tested. We conclude that intact microtubules are not needed in the fusion of autophagosomes and lysosomes and that vinblastine accelerates the rates of AV formation.

Effects of 8 hr Running and Prior Training on Triglyceride Accumulation and Aut+ phagocytosis in Mouse Liver: Morphometric and Biochemical Results. ARI RIUTTO, MARKKU KIHLSTR~M, PIRKKO HIRSIM&,* AND VEIKKO VIHKO, Department ofCellBi-

ology, University of Jyvliskyli, 40100 Jyv~&ky/Z, Finland, and *Farmos Oy, 20100 Turku, Finland. Morphological EM studies have shown that physical stress may cause ultrastructural changes, e.g., increased autophagocytosis and fat accumulation, in the hepatocytes. We were intrested to see whether prior endurance training might affect such phenomena. Two separate experiments were performed with 4-month-old male NMRI-mice. In the morphometric experiments mice were made to run for 8 hr at a speed of 13.5 m/min and TEM samples from liver central lobe were taken after 4 and 8 hr running and 4 and 20 hr after the cessation of exercise. In the biochemical experiment mice were trained on the mill for 0, 3, 10, and 20 days (1 hr/day, speed 25 m/min) before the 8-hr prolonged exertion and liver samples for biochemical assays were taken immediately and 20 hr after running. Morphological determinations showed increasing volume density of triacylglycer01 (TG) vacuoles during running, the increase resulting from both the number and the size of the vacuoles. The slight increase in the volume density of autophagic vacuoles originated from their increased size, No marked changes were observed in morphometrical parameters characterizing peroxisomes or residual bodies. Biochemical results showed a strong training-induced adaptation in liver fat metabolism. Prior endurance training for 3 days decreased TG accumulation markedly and after 10 days training no increase in TG was observed. The total activities of cathepsin D and /Lglucuronidase and liver protein concentrations, all suggested that probably proteolytic changes occur in mouse liver after prolonged exertion even after 20 days prior training.

Zymogen Granule Ultrastructure in Parotid Gland of Chronically Reserpinized Rats. GODFRIED M. ROOMANS AND R. MARGARETA MUELLER,Department ofUltrastructureRe-

search, Wenner-Gren Institute, University of Stockholm, S-106 91 Stockholm, Sweden. The chronically reserpinized rat has been used as an animal model for the disease cystic fibrosis, a generalized exocrinopathy. The zymogen granules in the parotid gland of chronically reserpinized rats have been shown to have an abnormal ultrastructure and are characterized by a heterogeneous electron density. Heterogeneously dense zymogen granules have also been noted in the parotid gland of patients with cystic fibrosis. Since the chronic reserpine treatment is carried out over a period of 7 days, in the present study the time course of the ultrastructural changes was followed from day to day. In the parotid gland of control animals, most cells display the normal, electron dense (“dark”) granules; some cells display less electron dense (“light”), probably immature, granules. After 1 to 3 days of reserpine treatment, the proportion of cells containing “light” granules is much larger than that of cells containing “dark’ granules. However, as in control animals, cells generally either contain “light” or “dark” granules. After 4 to 5 days, the parotid acinar

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cells contain both “light” and “dark” granules, i.e., both granule types occur mixed in a single cell. At this stage the granules generally display homogeneous electron density. After 6 to 7 days, the zymogen granules are heterogeneously electron dense in various ways (“light” with “dark” nucleus, “light” with “dark” rim, or spotted). Correlation of ultrastructural abnormalities in zymogen granules with changes in cell metabolism in the animal model may provide a clue to metabolic abnormalities in the human disease.

Collagen Proliferation in Experimental ological Significance of Ultrastructural

Liver Injury and in Liver Disease: ClinicopathChanges. F. STENB~~CK AND E. A. SOTANIEMI,

Department of Pathology and Nordic Council for Arctic Medical Research, and Internal Medicine Research Unit, University of Oulu, SF-90220 Oulu, Finland. The amount and distribution of collagens was determined from carbon tetrachloride (CCL,)- and dimethyhritrosamine (DMN)-induced liver injury in rats and biopsies from patients with alcohol-induced liver disease by light and transmission electron microscopy and immunohistochemistry, and the results compared to biochemical indicators of cell function and clinical condition of the patient. The purpose was to determine the development and progression of tissue injury and the significance and characteristics of early lesions. In Ccl,-exposed rats elongated liver cells with irregular mitochondria and lysosomes as well as abundant cytoplasmic fat droplets and basement membrane (BM) formation in sinusoidal spaces and in thickened septa were seen. DMN treatment caused cell necrosis, sinusoidal hemorrhages, and inflammatory aggregates. BM accumulations resulted in capillarization of sinusoidal spaces. Specimens from patients with alcoholic liver disease also showed BM accumulation in sinusoidal spaces and in septa extending into parenchyma as well as interstitial collagens in septa, around bile ducts and vascular structures and in Disse’s space. When comparing morphological, biochemical, and clinical findings biochemical indicators of decreased cell metabolism in experimental injury were associated with sinusoidal and septal collagen deposits. Clinical liver disease in early stages was reflected in sinusoidal BM deposits preventing cell function and transport.

Arc Welders’

Siderosis: Origin

of Endogenous

Iron in Lung Tissue. SEPPO SUTINEN,

AND SEPPO J. SIVOIWN, Department of Pathology and Institute of Electron Optics, University of Oulu, Oulu, and Institute of Occupational Health, Helsinki, Finland. Arc welders’ siderosis is a pneumoconiosis characterized by a micronodular radiographic picture, accumulation of iron pigment and phagocytes, but very little reaction in lung tissue. It has been described in welders exposed to manual metal arc mild steel welding, the most common welding method until the present time. According to previous studies, most of the iron in arc welders’ lung is bound to hemosiderin, i.e., endogenous in nature. This raised the question on the origin of endogenous iron in arc welders’ siderosis. Lung tissue of a mild steel welder was studied by specimen radiography, light microscopy, transmission electron microscopy, and X-ray microanalysis. The structure and composition of fume particles in lung tissue of the welder were compared with those of the same type of particles in fume samples generated in the laboratory, and in lung tissue of exposed rats. Two main particle types were found both in fume samples and in rat lung: One metal rich type and another containing also light elements. Most fume particles seemed to dissolve very rapidly in rat lung and ferritin appeared around the partly degraded particles. In lung tissue of the welder only a few particles of the metal rich type and none of the SISKO ANTTILA,

AALE

GREKULA,

PIRKKO-LIISA

KALLIOMKKI,

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other type were preserved. These metallic, originally round, particles were irregular in shape and always surrounded by abundant hemosiderin. The results suggest that iron is released from particles and bound to hemosiderin in macrophages, also explaining the lack of tissue reaction to mild steel.

Ultrastructure of the Porcine Myometrium during the Estrous Cycle. G. THILANDER, M. EKWALL, AND H. RODRIGUEZ-MARTINEZ, Department of Anatomy and Histology, Faculty of Veterinary Medicine, Swedish University of Agricultural Sciences, S-750 07 Uppsala, Sweden. Thin sections of selected areas of the myometrium from normally cycling gilts were studied by conventional electron microscopy to determine possible variations in the ultrastructure and the types and number of cell-to-cell contacts between smooth muscle cells. Peripheral plasma levels of ovarian steroids were monitored through the cycle. Among the four types of cell contacts observed, intermediate junctions and simple appositions were numerous, whereas interdigitations occurred to a limited extent, and nexuses (gap junctions) were few. The number of thick filaments varied with the stage of the estrous cycle, being more numerous during the follicular (estrogen dominated) phase, suggesting that an assembly/disassembly mechanism was involved.

Ultrastructural Characteristics of Neuronal Differentiation in F9 Teratocarcinoma Cells. J. WARTIOVAARA, I. REIMA, AND M. T~YNJZL& Department of Electron Microscopy, University of Helsinki, 00280 Helsinki, Finland. Neuronal differentiation can be induced in monolayer cultures of F9 teratocarcinoma cells by use of retinoic acid, dibuturyl cyclic AMP, and nerve growth factor. This model system was used to further study the ultrastructure of neuronal development in SEM and with thin sectioning and freeze-fracture. After 5 to 7 days induction F9 cells had acquired neuronal morphology and formed elaborate networks through cellular extensions as seen in SEM. Axonal growth cones were visible but synaptic contacts could not be resolved with this technique. In thin sections the differentiated cells had a rounded cell body with rich endoplasmic reticulum and numerous mitochondria, the latter being also found in cell processes along with extensive neurotubular-type structures. At sites aggregates of synaptic vesicle-like structures were seen. Specialized membrane junctions between cell processes although not true synapses could be detected. Freeze-fracture replicas prepared by double-replica techique using Freon 22 freezing often revealed well-developed intracellular vesicular areas with equal-sized (ca. 50-100 nm) vesicles. At sites membrane fractures were seen with aggregated intramembranous particles suggestive of membrane contact areas. The results indicate a typical neuronal differentiation in the ultrastructure of the F9 cells during culture under the described conditions. Other evidence like the appearance of neurotransmitters and -peptides under similar conditions promote the use of this model in the study of neurodifferentiation and synaptogenesis in vitro.