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In situ hybridization analysis of α-fetoprotein mRNA expression in rat liver during regeneration after partial hepatectomy
GS-I 1
IN SITU HYBRIDIZATION ANALYSIS OF ~-FETOPROTEIN mRNA EXPRESSION IN RAT LIVER DURING REGENERATION AFTER PARTIAL HEPATECTOMY D. Bernuau, A. Poliard, G. Feldmann Laboratoire de Biologie Cellulaire, INSERM U 24, Facult4 de M~decine X. Bichat, Paris, FRANCE.
Liver regeneration after partial hepatectomy (PH) in the rat is accompanied by a transient increase in the liver production of ~-fetoprotein (AFP), a protein normally expressed at very low levels in the adult. The cellular basis for this modulation of gene expression is not clarified. It has been suggested that AFP synthesis is resumed in a small percentage of hepatocytes, presumably at a critical phase of the proliferative cycle. We decided to reevaluate the problem by analysing the cellular expression of AFP mRNAs by in situ hybridization. Male Sprague-Dawley rats (100-120 gm) were subjected to PH or sham operation. Liver was fixed by perfusion with paraformaldehyde and paraffin-embedded sections were in situ hybridized with an AFPspecific, 35S-labeled cDNA probe. Optical silver grain counts were made after 8-15 day exposure. On liver sections from non-hepatectomized or sham-operated control rats, all hepatocytes contained a small number of nuclear and cytoplasmic silver grains, without any zonal differences within the liver lobule. An increased number of nuclear silver grains was observed electively on periportal hepatocytes at 2h, 6h and 24h after PH. The maximal hybridization signal was recorded at 48h post-PH. By this time, all hepatocytes, whatever their situation in the liver lobule, displayed an increased number of both nuclear (2.5-3 fold) and cytoplasmic (1.52 fold) grains. These data show that modulation of AFP gene expression during liver regeneration after PH (i) involves all hepatocytes, but is first recorded in the periportal zones, (2) consists in nuclear accumulation of AFP mRNAs, with a relatively smaller elevation of the cytoplasmic transcripts, and (3) is not related to the entry of hepatocytes into the proliferative cycle, since the changes can be detected largely before the onset of DNA synthesis.
GS-I 2
CORE-GENE ENCODED PROTEINS IN HBSAG-POSITIVE SERA K. Gmelin, L. Tl~eilmann, B. Kommerell, E. Pfaff +, J. Salfeld + epartment of Internal Medicine, University of Heidelberg, and MBH, U n i v e r s i t y of H e i d e l b e r g , H e i d e l b e r g , FRG
P21 and P16 are two d i f f e r e n t p r o t e i n s encoded b y the Core-gene of the hepatitis B v i r u s (HBV). In s e r u m , P16 b e a r i n g the determinants of HBeAg appears as a free protein, whereas P21 has been found only in the core of hepatitis B v i r i o n s , thus r e p r e s e n t i n g the HBcAg. We studied the presence of both proteins in serum simultaneously by Western blot analysis. HBsAg, HBeAg, antiHBe, a n t i - H B c , and anti-HBs were determined by commercial radioimmunoassays, and HBV DNA by a dot blot assay. Antibodies against a fusion protein spanning a section of the Core-gene of HBV (Science 231 (1986) 594) as well as against a pre-S1 protein (Virology lq8 (1986) 15) were raised in rabbits. A total of 60 sera were studied; HBeAg was found in 17 sera, anti-HBe in 36, HBV DNA in 11, core proteins in 42 (P16 in 7 sera, P21 in q2 s e r a ) , and pre-S1 proteins in 20 sera. P21 core proteins were found in 10 sera positive for HBeAg, 28 sera positive for anti-HBe, 9 sera w i t h HBV DNA, and in 20 sera positive for pre-S1 proteins. Six out of 7 P16 core proteinpositive sera were H B e A g - p o s i t i v e , /4 w e r e HBV D N A - p o s i t i v e , 6 w e r e positive for pre-S1 p r o teins, and all 7 sera contained P21 core proteins. Pre-S1 proteins were found in sera positive for P16 or P21 core p r o t e i n s o n l y . Our results show that P21 core p r o t e i n s may be present both in HBeAg- and in a n t i - H B e - p o s i t i v e sera, whereas P16 core protein is expected to be eliminated in a n t i - H B e - p o s i t i v e sera. T h i s elimination does not coincide with the clearance of DNA containing hepatitis B viral particles.
S17
IN SITU HYBRIDIZATION ANALYSIS OF ~-FETOPROTEIN mRNA EXPRESSION IN RAT LIVER DURING REGENERATION AFTER PARTIAL HEPATECTOMY D. Bernuau, A. Poliard, G. Feldmann Laboratoire de Biologie Cellulaire, INSERM U 24, Facult4 de M~decine X. Bichat, Paris, FRANCE.
Liver regeneration after partial hepatectomy (PH) in the rat is accompanied by a transient increase in the liver production of ~-fetoprotein (AFP), a protein normally expressed at very low levels in the adult. The cellular basis for this modulation of gene expression is not clarified. It has been suggested that AFP synthesis is resumed in a small percentage of hepatocytes, presumably at a critical phase of the proliferative cycle. We decided to reevaluate the problem by analysing the cellular expression of AFP mRNAs by in situ hybridization. Male Sprague-Dawley rats (100-120 gm) were subjected to PH or sham operation. Liver was fixed by perfusion with paraformaldehyde and paraffin-embedded sections were in situ hybridized with an AFPspecific, 35S-labeled cDNA probe. Optical silver grain counts were made after 8-15 day exposure. On liver sections from non-hepatectomized or sham-operated control rats, all hepatocytes contained a small number of nuclear and cytoplasmic silver grains, without any zonal differences within the liver lobule. An increased number of nuclear silver grains was observed electively on periportal hepatocytes at 2h, 6h and 24h after PH. The maximal hybridization signal was recorded at 48h post-PH. By this time, all hepatocytes, whatever their situation in the liver lobule, displayed an increased number of both nuclear (2.5-3 fold) and cytoplasmic (1.52 fold) grains. These data show that modulation of AFP gene expression during liver regeneration after PH (i) involves all hepatocytes, but is first recorded in the periportal zones, (2) consists in nuclear accumulation of AFP mRNAs, with a relatively smaller elevation of the cytoplasmic transcripts, and (3) is not related to the entry of hepatocytes into the proliferative cycle, since the changes can be detected largely before the onset of DNA synthesis.
GS-I 2
CORE-GENE ENCODED PROTEINS IN HBSAG-POSITIVE SERA K. Gmelin, L. Tl~eilmann, B. Kommerell, E. Pfaff +, J. Salfeld + epartment of Internal Medicine, University of Heidelberg, and MBH, U n i v e r s i t y of H e i d e l b e r g , H e i d e l b e r g , FRG
P21 and P16 are two d i f f e r e n t p r o t e i n s encoded b y the Core-gene of the hepatitis B v i r u s (HBV). In s e r u m , P16 b e a r i n g the determinants of HBeAg appears as a free protein, whereas P21 has been found only in the core of hepatitis B v i r i o n s , thus r e p r e s e n t i n g the HBcAg. We studied the presence of both proteins in serum simultaneously by Western blot analysis. HBsAg, HBeAg, antiHBe, a n t i - H B c , and anti-HBs were determined by commercial radioimmunoassays, and HBV DNA by a dot blot assay. Antibodies against a fusion protein spanning a section of the Core-gene of HBV (Science 231 (1986) 594) as well as against a pre-S1 protein (Virology lq8 (1986) 15) were raised in rabbits. A total of 60 sera were studied; HBeAg was found in 17 sera, anti-HBe in 36, HBV DNA in 11, core proteins in 42 (P16 in 7 sera, P21 in q2 s e r a ) , and pre-S1 proteins in 20 sera. P21 core proteins were found in 10 sera positive for HBeAg, 28 sera positive for anti-HBe, 9 sera w i t h HBV DNA, and in 20 sera positive for pre-S1 proteins. Six out of 7 P16 core proteinpositive sera were H B e A g - p o s i t i v e , /4 w e r e HBV D N A - p o s i t i v e , 6 w e r e positive for pre-S1 p r o teins, and all 7 sera contained P21 core proteins. Pre-S1 proteins were found in sera positive for P16 or P21 core p r o t e i n s o n l y . Our results show that P21 core p r o t e i n s may be present both in HBeAg- and in a n t i - H B e - p o s i t i v e sera, whereas P16 core protein is expected to be eliminated in a n t i - H B e - p o s i t i v e sera. T h i s elimination does not coincide with the clearance of DNA containing hepatitis B viral particles.
S17