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Restriction fragment length polymorphism (RFLP) an useful method for praesymptomatic diagnosis of Wilson's disease
$75
Ceil biology," molecular biology; fimction
]
[ P2C6/197 [
I P2C6/199
THE POLYMERIC IMMUNOGLOBULIN RECEPTOR IS THE MAJOR HIGH AFFINITY CALMODULIN BINDING PROTEIN IN RAT LIVER ENDOSOMES. C.Endch, K.E.Mostov, and J.R.Havel, Departamento de Bioiogia Celular y Anatomia Patol6gica. Facuitad de Medicina. Universidad de Barcelona. Barcelona. Spain. Cardiovascular Research Institute. School of Medicine. University of Califomia, San Francisco. USA.
CELL LINEAGE IN THE RAT LIVER RETROVIRAL MEDIATED GENE TRANSFER
Rat liver endosornes contain one major high affinity calmodulinbinding protein. We have identified this calmodulin-binding protein as the polymeric immunoglobulin receptor (plgR) by two methods. First highly purified endosomes were analysed by two dimensional gel electrophoresis. The resolved proteins were transferred to nitrocellulose and probed with radioiodinated calmodulin. The major calmodulin-binding protein comigrated with authentic plgR that was detected with a monoclonal antibody directed against the receptor's cytoplasmic domain. Second, endosomes were solubilized with Triton X-100 and the solubilized proteins passed over a calmodulin affinity column. Specific calmodulin-binding proteins were eluted by chelating calcium. The major eluted protein was identified as the plgR by using the same antibody. The receptor was concentrated in two endosomal Subfractions: early endosomes and especially in a receptor-recycling compartment. The receptor was also present in a sinusoidal plasma membrane fraction, but absent in fractions containing apical (bile canalicular) or lateral plasma membrane domains of the hepalocyte. These data suggest a role for calmodulin in the transcytosis of IgA and/or IgM. Finally, we have investigated the expression of the plgR during liver regeneration after a partial hepatectomy and results showed t h a t ' a significant decreased expression of plgR occurred during the pre-replicative phase. Altogether, these results seem indicate that the intracellular trafficking of IgA and IgM from blood to bile is modulated by calmodulin/Caz'.
P2C6/198 ]
USING
M.-P. Bralet, P. Arbuthnot, D. Calise, C. B r & h o t and N. Ferry, INSERM U. 370, Paris, France. The liver is often considered as a slowly renewing organ which retains a high replicativc potential in response to toxic injuries or surgical resection. During carcinogenesis, there is proliferation of hepatic cells but the exact nature of the cell at the origin of hepatocellular c a r c i n o m a remains unknown. [n order to investigate cell lineage in n o r m a l liver and during chemical c a r c i n o g e n e s i s , we h a v e used an in vivo genetic labeling procedure. A modified [3-galactosidase gene coupled to a nuclear localization signal: n l s - L a c Z gene was incorporated into a r e c o m b i n a n t rctrovirus and used as a marker gene. On liver c r y o s t a t s e c t i o n s , e x o g e n o u s [3-galactosidase activity was detected on the cell nuclei using X-Gal as substrate. In normal liver, we showed that hepatocytes are able to divide throughout the life of the animal and do not migrate in the hepatic Iobule. We thus found no evidence for streaming of hepatocytes in normal liver. In h e p a t o c e l l u l a r c a r c i n o m a i n d u c e d by e i t h e r a c e t y l a m i n o f l u o r e n e or d i e t h y l n i t r o s a m i n e , we o b s e r v e d a marked proliferation of well differentiated hepatocytes during the first six months of the treatment. This study demonstrates the fcasability of cell lineage in the liver.
P2 C6/200
]
RESTRICTION FRAGMENT LENGTH POLYMORPHISM (RFLP) AN USEFUL METHOD FOR PRAESYMPTOMATIC DIAGNOSIS OF WILSON'S DISEASE
MUSCAR/NIC RECEPTOR INDUCED INCREASE OF FREE CYTOSOLIC CA2+ IN RAT BILIARY EPITHELIAL CELLS
Th. Maier-Dobersber.qer°, S. Rack =, G. Granditsch+, A. Gan.ql*, Ch. Mannhalter =, P. Ferenci °. *Department of Gastroenterology and Hepatology, Internal Medicine IV, °Department of Molecularbiology, +Department of Paediatdcs, University of Vienna, Vienna, Austria. RFLP is a new diagnostic tool for screening in families with genetic diseases with one known index patient. Recently the Wilson's disease (WD) gene was identified on chromosome 13q14 and several markers up- and downstream from the gene have been described. In this report the results of RFLPs with D13S31, D13S59, D13S26, RB and ESD in two families are shown.DNA was isolated from peripheral blood mononuclear cells of pedigree members, and digested with Taql, Pvull, Ban II, Bell, EcoRI, Hphl, Hindlll, Apal and Rsal. After Southem blotting these genetic markers labeled with i-a2 . p] _ dCTP were used for hybridization.Case1 : Brother of a patient with WD, at the age of 7 years, psychiatric disturbances, caeruloplasmin 14 mg%, 24hours-urinary copper excretion 46 pg, copper content of a liver biopsy sample 172 pg per g of dry tissue weight - treatment with 50 mg of D - Penicillamine was started. RFLP analysis indicated that this patient is a heterozygous carder of WD. Treatment was stopped 3 months ago.Case 2: Sister of a girl with WD, at the age of 4 years, healthy, all biochemical parameters within the normal range, caeruloplasmin 33 mg%, urinary copper excretion 88 pg per day.By using RFLPs we found that she was homozygous for the WD gene. Therefore the diagnosis was confirmed by liver biopsy with a copper content of 1000 lag per g of dry tissue weight. Treatment with D - Penicillamine was started.These data show that by RFLPs with D13S31, D13S59, D13S26, RB and ESD the correct diagnosis of WD or WD carrier state can be made even if conventional biochemical markers give conflicting results.
C. El¢ing, A. Kassner, W. $tremmal. Div. of Gastroenterology, HeinrichHeine University D~sseldorf, FRG To evaluate whether the secretory function of biliary epithelial cells is mediated by muscarinic receptor stimulation, we analyzed the effect of the cholinergic agonist carbachol on cytosolic free Ca2+ as its second messanger. Short time cultured hepatocytes and biliary epithelial cells from normal rat livers were loaded with the Ca2+ sensitive dye Fluo3/AM. lntracellular fluorescence was monitored by confocal laser scanning microscopy. In contrast to short time cultured hepatocytes, biliary epithelial cells revealed a pulse-type increase in cytosolic free Ca2+ after exposure to carbachol beyond a threshold concentration of 0.5 laM. With increasin8 concentrations of carbachol (1-10 g.M) the latency of the Ca2+ release reaction decreased and revealed dose dependent peak values, correspondin8 to 60-90% of the maximal fluorescence intensity. Over a subsequent mean period of 200 s maximal Ca2+ levels gradually declined to baseline values, describing a sinusoidal oscillation with 0.8 to 1.5 oscillations/min. Next we analyzed whether the increase in Ca2+ during carbachol stimulation originates from intra- or extracellular sources. Therefore the I? 3 sensitive intracellular Ca2+ stores were selectively depleted with 25 ~aM tBuBHQ. Subsequent exposure to carbachol resulted in a further increase of Ca2+ by 35 % of mmdam] fluorescence intensity which is due to extracellular Ca2+ influx. On the other hand, when a Ca 2+ depleted medium was used, there was still a carbachol induced increase in intracellular fluorescence detectable originating from intracellular sources. To evaluate whether the carbachol induced influx of Ca2+ is indeed mediated by a muscarinic receptor, inhibition studies with 10 and 50 ~uM atropin were performed. This resulted in inhibition of the carbachol (10 ~.M) induced increase ot cytosolic free Ca2+ in 50 % and 90% of the cells, respectively. These data show that in BEC stimulation of muscarinic receptors by carbachol induces a cytosolic increase of free Ca2+ originating from intra- and extracellular sources.
Ceil biology," molecular biology; fimction
]
[ P2C6/197 [
I P2C6/199
THE POLYMERIC IMMUNOGLOBULIN RECEPTOR IS THE MAJOR HIGH AFFINITY CALMODULIN BINDING PROTEIN IN RAT LIVER ENDOSOMES. C.Endch, K.E.Mostov, and J.R.Havel, Departamento de Bioiogia Celular y Anatomia Patol6gica. Facuitad de Medicina. Universidad de Barcelona. Barcelona. Spain. Cardiovascular Research Institute. School of Medicine. University of Califomia, San Francisco. USA.
CELL LINEAGE IN THE RAT LIVER RETROVIRAL MEDIATED GENE TRANSFER
Rat liver endosornes contain one major high affinity calmodulinbinding protein. We have identified this calmodulin-binding protein as the polymeric immunoglobulin receptor (plgR) by two methods. First highly purified endosomes were analysed by two dimensional gel electrophoresis. The resolved proteins were transferred to nitrocellulose and probed with radioiodinated calmodulin. The major calmodulin-binding protein comigrated with authentic plgR that was detected with a monoclonal antibody directed against the receptor's cytoplasmic domain. Second, endosomes were solubilized with Triton X-100 and the solubilized proteins passed over a calmodulin affinity column. Specific calmodulin-binding proteins were eluted by chelating calcium. The major eluted protein was identified as the plgR by using the same antibody. The receptor was concentrated in two endosomal Subfractions: early endosomes and especially in a receptor-recycling compartment. The receptor was also present in a sinusoidal plasma membrane fraction, but absent in fractions containing apical (bile canalicular) or lateral plasma membrane domains of the hepalocyte. These data suggest a role for calmodulin in the transcytosis of IgA and/or IgM. Finally, we have investigated the expression of the plgR during liver regeneration after a partial hepatectomy and results showed t h a t ' a significant decreased expression of plgR occurred during the pre-replicative phase. Altogether, these results seem indicate that the intracellular trafficking of IgA and IgM from blood to bile is modulated by calmodulin/Caz'.
P2C6/198 ]
USING
M.-P. Bralet, P. Arbuthnot, D. Calise, C. B r & h o t and N. Ferry, INSERM U. 370, Paris, France. The liver is often considered as a slowly renewing organ which retains a high replicativc potential in response to toxic injuries or surgical resection. During carcinogenesis, there is proliferation of hepatic cells but the exact nature of the cell at the origin of hepatocellular c a r c i n o m a remains unknown. [n order to investigate cell lineage in n o r m a l liver and during chemical c a r c i n o g e n e s i s , we h a v e used an in vivo genetic labeling procedure. A modified [3-galactosidase gene coupled to a nuclear localization signal: n l s - L a c Z gene was incorporated into a r e c o m b i n a n t rctrovirus and used as a marker gene. On liver c r y o s t a t s e c t i o n s , e x o g e n o u s [3-galactosidase activity was detected on the cell nuclei using X-Gal as substrate. In normal liver, we showed that hepatocytes are able to divide throughout the life of the animal and do not migrate in the hepatic Iobule. We thus found no evidence for streaming of hepatocytes in normal liver. In h e p a t o c e l l u l a r c a r c i n o m a i n d u c e d by e i t h e r a c e t y l a m i n o f l u o r e n e or d i e t h y l n i t r o s a m i n e , we o b s e r v e d a marked proliferation of well differentiated hepatocytes during the first six months of the treatment. This study demonstrates the fcasability of cell lineage in the liver.
P2 C6/200
]
RESTRICTION FRAGMENT LENGTH POLYMORPHISM (RFLP) AN USEFUL METHOD FOR PRAESYMPTOMATIC DIAGNOSIS OF WILSON'S DISEASE
MUSCAR/NIC RECEPTOR INDUCED INCREASE OF FREE CYTOSOLIC CA2+ IN RAT BILIARY EPITHELIAL CELLS
Th. Maier-Dobersber.qer°, S. Rack =, G. Granditsch+, A. Gan.ql*, Ch. Mannhalter =, P. Ferenci °. *Department of Gastroenterology and Hepatology, Internal Medicine IV, °Department of Molecularbiology, +Department of Paediatdcs, University of Vienna, Vienna, Austria. RFLP is a new diagnostic tool for screening in families with genetic diseases with one known index patient. Recently the Wilson's disease (WD) gene was identified on chromosome 13q14 and several markers up- and downstream from the gene have been described. In this report the results of RFLPs with D13S31, D13S59, D13S26, RB and ESD in two families are shown.DNA was isolated from peripheral blood mononuclear cells of pedigree members, and digested with Taql, Pvull, Ban II, Bell, EcoRI, Hphl, Hindlll, Apal and Rsal. After Southem blotting these genetic markers labeled with i-a2 . p] _ dCTP were used for hybridization.Case1 : Brother of a patient with WD, at the age of 7 years, psychiatric disturbances, caeruloplasmin 14 mg%, 24hours-urinary copper excretion 46 pg, copper content of a liver biopsy sample 172 pg per g of dry tissue weight - treatment with 50 mg of D - Penicillamine was started. RFLP analysis indicated that this patient is a heterozygous carder of WD. Treatment was stopped 3 months ago.Case 2: Sister of a girl with WD, at the age of 4 years, healthy, all biochemical parameters within the normal range, caeruloplasmin 33 mg%, urinary copper excretion 88 pg per day.By using RFLPs we found that she was homozygous for the WD gene. Therefore the diagnosis was confirmed by liver biopsy with a copper content of 1000 lag per g of dry tissue weight. Treatment with D - Penicillamine was started.These data show that by RFLPs with D13S31, D13S59, D13S26, RB and ESD the correct diagnosis of WD or WD carrier state can be made even if conventional biochemical markers give conflicting results.
C. El¢ing, A. Kassner, W. $tremmal. Div. of Gastroenterology, HeinrichHeine University D~sseldorf, FRG To evaluate whether the secretory function of biliary epithelial cells is mediated by muscarinic receptor stimulation, we analyzed the effect of the cholinergic agonist carbachol on cytosolic free Ca2+ as its second messanger. Short time cultured hepatocytes and biliary epithelial cells from normal rat livers were loaded with the Ca2+ sensitive dye Fluo3/AM. lntracellular fluorescence was monitored by confocal laser scanning microscopy. In contrast to short time cultured hepatocytes, biliary epithelial cells revealed a pulse-type increase in cytosolic free Ca2+ after exposure to carbachol beyond a threshold concentration of 0.5 laM. With increasin8 concentrations of carbachol (1-10 g.M) the latency of the Ca2+ release reaction decreased and revealed dose dependent peak values, correspondin8 to 60-90% of the maximal fluorescence intensity. Over a subsequent mean period of 200 s maximal Ca2+ levels gradually declined to baseline values, describing a sinusoidal oscillation with 0.8 to 1.5 oscillations/min. Next we analyzed whether the increase in Ca2+ during carbachol stimulation originates from intra- or extracellular sources. Therefore the I? 3 sensitive intracellular Ca2+ stores were selectively depleted with 25 ~aM tBuBHQ. Subsequent exposure to carbachol resulted in a further increase of Ca2+ by 35 % of mmdam] fluorescence intensity which is due to extracellular Ca2+ influx. On the other hand, when a Ca 2+ depleted medium was used, there was still a carbachol induced increase in intracellular fluorescence detectable originating from intracellular sources. To evaluate whether the carbachol induced influx of Ca2+ is indeed mediated by a muscarinic receptor, inhibition studies with 10 and 50 ~uM atropin were performed. This resulted in inhibition of the carbachol (10 ~.M) induced increase ot cytosolic free Ca2+ in 50 % and 90% of the cells, respectively. These data show that in BEC stimulation of muscarinic receptors by carbachol induces a cytosolic increase of free Ca2+ originating from intra- and extracellular sources.