Search PubMed⌕ Search

Biomedical subjects

D Bernuau

Publications and source records attributed to D Bernuau.

At least 37 records · Page 2Linked to original sources
PubMed1992

Cellular analysis of the kinetics of alpha-fetoprotein and nuclear oncogene activation in primary cultures of adult rat hepatocytes stimulated by epidermal growth factor.

Alpha-fetoprotein (AFP), a fetal gene normally inactivated in quiescent adult hepatocytes, is re-expressed in hepatocytes during the proliferating response induced by liver regeneration in vivo, or epidermal growth factor (EGF) in vitro. The nuclear oncogenes c-jun, c-fos and c-myc are 'immediate early' genes also activated during the proliferative response of hepatocytes. Whether AFP gene activation is linked to oncogene activation is not known. As a first step in answering this question, we have analysed the cellular kinetics of nuclear oncogene and AFP gene activation in primary cultures of adult rat hepatocytes stimulated by EGF. Gene activation was evaluated at the mRNA level by dot blot and in situ hybridization, and at the protein level by immunoperoxidase. C-jun, c-fos and c-myc mRNA steady state levels in total cellular RNA were increased from 30 min-2 h after EGF stimulation. In situ hybridization analysis showed that transcripts of the three oncogenes increased in all hepatocytes after EGF stimulation. While unstimulated cultures did not immunostain for Fos and Myc proteins. Fos immunostaining was visible in the majority of hepatocyte nuclei 1 and 2 h after EGF addition, and Myc cytoplasmic immunostaining of the majority of hepatocytes was observed at 2 h of stimulation. AFP mRNA increased in total cellular RNA 2 and 4 h after EGF stimulation, with elevated in situ hybridization signal for AFP mRNA in all hepatocytes. No hepatocytes immunostained for AFP in unstimulated cultures, but a cytoplasmic labeling of 20-30% of the hepatocytes was observed 6 h after stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Increased efficiency of gene transfection in primary cultures of adult rat hepatocytes stimulated to proliferate: a comparative study using the lipofection and the calcium phosphate precipitate methods.

Transfection of the beta-galactosidase gene in quiescent cultures of adult rat hepatocytes with the calcium phosphate precipitate or the lipofection methods gave a higher level of beta-galactosidase gene expression with the lipofection than with the calcium phosphate precipitate method, but the transfection efficiency was weak in both cases. Transfection of hepatocytes stimulated to proliferate before transfection either in vivo by partial hepatectomy or in vitro by epidermal growth factor was more efficient than transfection of quiescent hepatocytes, and the lipofection method gave better results than the calcium phosphate precipitate method.

Animals↗
PubMed1992

Functional hepatocellular heterogeneity for the production of plasma proteins.

It is now well established that hepatocytes are the main liver cells responsible for the synthesis of plasma proteins produced by the liver. That these cells are not specialized in the production of the different plasma proteins is also well established. Presently the point still debated is whether a functional hepatocellular heterogeneity exists for plasma protein synthesis as for many other hepatocyte functions. Several physiological and pathological situations suggest that this heterogeneity takes place in the hepatocytes of two opposite hepatic lobular zones, the periportal and centrilobular zones. However, this zonal difference, which supposes different regulatory mechanisms, must be confirmed by techniques other than the now classical immunocytochemistry or the in situ hybridization technique recently proposed for the demonstration of mRNAs in hepatocytes. Another hepatocellular heterogeneity, the intercellular heterogeneity, which can be observed in the same lobular zone, is more difficult to analyze, but shows that from hepatocyte to hepatocyte a variation exists in the synthesis of a given plasma protein.

Animals↗
PubMed1990

Orthotopic liver transplantation with hepatic artery anastomoses. Hemodynamics and response to hemorrhage in conscious rats.

Orthotopic liver isotransplantation was performed in one group of Lewis rats using cuffs for the portal vein and the infrahepatic vena cava, stents for the hepatic artery and the bile duct. Three other groups were also investigated: group A, normal rats; group B, sham-transplanted rats (clamping of the vessels, washing of the liver, placing cuffs around the portal vein); and group C, sham-transplanted rats with ligature section of the hepatic artery. Blood-flow measurements were performed, 1 week after the surgical procedure, with the radioactive microsphere method in conscious animals. Transplanted rats exhibited significant (ANOVA, P less than 0.05) increase in cardiac index and decrease in mean arterial pressure and systemic vascular resistance. Blood flows of the portal territory and to the kidneys were not significantly modified. Arterial liver blood flow and arterial liver vascular resistance in rats with liver transplantation were not significantly different between normal and sham-transplanted rats but were significantly different from rats with ligature of the hepatic artery. These results confirm the validity of the method used for vascular anastomoses. Hypotensive hemorrhage (2 ml/100 g bw) induced marked hemodynamic changes, but rats with liver transplantation when compared with normal and sham-transplanted rats exhibited the following: (a) significantly lower percentage of decrease in cardiac index and in mean arterial pressure; and (b) significantly higher renal and portal tributary blood flows. Plasma catecholamine concentrations and plasma volume were higher in rats with liver transplantation than in normal rats but were not significantly different from sham-operated rats. Histologic examination of the liver revealed slight portal edema in sham-operated rats and small necrotic areas in the liver, probably corresponding to the reperfusion injury, in rats with liver transplantation. In conclusion, the method described for the four vascular anastomoses allows functional perfusion of the transplanted liver. Rats with liver transplantation exhibited a hyperkinetic circulatory syndrome and an improved tolerance to hemorrhage. Changes in plasma catecholamine concentrations and in plasma volume did not account for the hemodynamic changes.

Anastomosis, Surgical↗
PubMed1990

In situ hybridization: application to the study of gene expression during experimental hepatocarcinogenesis and human hepatocellular carcinoma.

The possibility of detecting specific mRNA directly within tumor cells offered by the in situ hybridization (ISH) technique has provided a new dimension to the study of experimental and human hepatocarcinogenesis. The combination of ISH and immunocytochemical techniques on the same or adjacent tissue sections provides a more complete picture of the various changes in gene expression. ISH may also detect posttranscriptional abnormalities of gene expression at the cellular level, such as expression of mRNA with no protein translation, or translation with decreased efficiency. Undoubtedly, the development of the ISH technique will open a new area for the understanding of the complex molecular events leading to cancer.

Albumins↗
PubMed1989

Heterogeneous lobular distribution of hepatocytes expressing acute-phase genes during the acute inflammatory reaction.

Functional heterogeneity in the lobule with regard to plasma protein synthesis is still debated. Therefore, we have localized in liver sections from normal rats and from rats with turpentine-induced AIR the mRNA and protein products of three genes with different alterations in their hepatic expression during an AIR: alpha 2M and alpha 1PI, two positively reacting acute-phase genes, and alpha 1I3, a negative acute-phase reactant. In normal liver, all hepatocytes expressed alpha 2M and alpha 1I3 mRNA, but a preferential expression of alpha 2M and alpha 1I3 mRNA and protein in the PP and ML zones was observed. During an AIR, the level of alpha 2M mRNA increased fourfold in the cytoplasm of PP and ML hepatocytes, while the level of cytoplasmic alpha 1I3 mRNA was decreased about fourfold in the same zones, with parallel variations in the expression of the corresponding proteins. In contrast, no significant modulation of the RNA and protein concentrations of both genes was detected in PV areas. alpha 1PI mRNA was expressed at the same levels in the three lobular zones in normal liver, but staining for the alpha 1PI protein was more intense in the PV zones. During the acute-phase response alpha 1PI mRNA levels were increased twofold in all three lobular zones, and alpha 1PI staining became homogeneous within the lobule. These results demonstrate that the location of a hepatocyte with the liver lobule can influence the expression of the three genes under study both at pre- and post-translational levels, in basal conditions, as well as during modulation of their expression during the inflammatory reaction.

Acute Disease↗
PubMed1989

Cellular expression of alpha-fetoprotein gene and its relation to albumin gene expression during rat azo-dye hepatocarcinogenesis.

During hepatocarcinogenesis, alpha-fetoprotein (AFP) synthesis may be dramatically increased while albumin synthesis is frequently decreased. Therefore, a reciprocal modulation between both gene expressions has been hypothesized. In this work, we combined in situ hybridization and immunoperoxidase on parallel liver tissue sections in order to analyze at the cellular level, AFP gene expression and its relation to ALB gene expression in both early and neoplastic lesions induced by 3MeDAB in the rat. In early lesions, cell populations were heterogenous as regards AFP expression. High levels of AFP transcripts were detected both in oval type cells and in a subset of basophilic hepatocytes within preneoplastic lesions. In these two highly AFP-expressing cell populations, significant levels of ALB transcripts were concomitantly detected. In the majority of altered hepatocytes, no AFP expression was detected while the level of ALB expression was decreased. In neoplastic lesions, AFP expression was strikingly heterogenous and independent from the degree of morphological differentiation. No evidence of reciprocal modulation with ALB gene expression could be assessed. In both preneoplastic and neoplastic lesions, a few altered hepatocytes displayed significant levels of AFP transcripts while no corresponding protein could be detected; such a discrepancy was not observed for ALB. This work shows that during 3MeDAB hepatocarcinogenesis, AFP gene activation occurs in heterogenous cell populations and according to different cellular patterns. Our observations lend no support to the hypothesis of a reciprocal modulation between AFP and ALB gene expressions during rat azo-dye hepatocarcinogenesis.

Albumins↗
PubMed1989

[In situ hybridization: principles and applications].

In situ hybridization is a molecular biological technique recently introduced in histology. Its principle consists of forming stable nucleic hybrids in tissues or cells. In this review, the main steps of the technique are discussed in regard to the preparation of probes and their labelling, the fixation of tissues and cells and their permeabilization in order to facilitate the penetration of labelled probes. Conditions of hybridization and requisite control reactions are also analysed. Quantification possible when radioactive probes are used is detailed. The three current applications of in situ hybridization, the localization of a gene on a chromosome, the demonstration of viral genomes in cells and the investigation of messenger RNAs coding for a determined protein, are illustrated with specific examples.

Animals↗
PubMed1988

Albumin and alpha-fetoprotein gene expression in various nonhepatic rat tissues.

Albumin and alpha-fetoprotein (AFP), two major serum proteins, are synthesized predominantly in the liver and yolk sac of mammals. In the present paper we report on the developmental expression of the corresponding genes in nonhepatic rat tissues. Significant quantities of mature albumin and AFP mRNAs were revealed in kidney, pancreas, heart, and lung of fetal and/or newborn rats using dot blot and Northern blot assays. Very low levels of these mRNA sequences were also detected in adult kidney and pancreas using sensitive RNA-cDNA solution hybridization assays. In situ hybridization analysis revealed that the albumin and AFP gene transcripts are present in the tubular cells of the 20-day-old fetal kidney. In order to elucidate further the mechanisms governing this expression, we studied the chromatin structure and methylation pattern in the 5'-end of these two genes. A faint band, corresponding to a specific DNase I-hypersensitive site upstream from the albumin gene, was detected in the fetal and neonatal kidney nuclei but not in adult kidney. For both genes, a site CG, demethylation of which is correlated with expression in liver and hepatoma cell lines, is highly methylated in fetal kidney even though AFP and albumin genes are expressed. Taken together, these results show the presence of a cell population in the rat kidney that actively transcribes both the albumin and AFP genes. The expression of these genes may be mediated by mechanisms differing in at least some steps from those exerted in the liver.

Animals↗
PubMed1988

Model for the study of portal-systemic collateral vascular resistance in the conscious rat.

In order to obtain a model for the study of portal-systemic collateral vascular resistance, total portal vein occlusion was performed in rats 48 hr or 3 wk after partial obstruction. Four groups of conscious restrained rats were studied: a) sham-operated, b) partial portal vein ligated, c) 48 hr-total portal vein occluded, and d) 3 wk-total portal vein occluded. In comparison with the sham group, the three portal vein ligated groups had significantly higher cardiac output, portal tributary blood flow, portal pressure (7.7 +/- 0.4 versus 13.5 +/- 0.5, 13.6 +/- 0.8, and 17.7 +/- 1.1 mmHg, mean +/- SE, respectively) and hepatic arterial blood flow (5.8 +/- 0.6 versus 9.5 +/- 0.7, 8.3 +/- 0.5, and 13.9 +/- 1.9 ml/min, respectively). Cardiac output and portal tributary blood flow did not differ between the portal vein ligated groups, but portal pressure and hepatic arterial blood flow were significantly higher in the 3 wk-total portal vein occlusion group. The 3 wk-total portal vein occlusion group showed 99.1 +/- 0.3% shunting, different from the partial (29.7 +/- 16.9%, p less than 0.01) and 48 hr-total portal vein occlusion (46.5 +/- 14.7%, p less than 0.05) groups. Portography confirmed absence of portal-portal collaterals in the 3 wk-total portal vein occlusion group. It is suggested that rats with 3 wk-total portal vein occlusion are useful for the study of acute modifications of portal-systemic collateral circulation, as shunting is total and consistent in this model.

Animals↗
PubMed1988

Alpha fetoprotein and albumin gene transcripts are detected in distinct cell populations of the brain and kidney of the developing rat.

We report the cellular localization of alpha-fetoprotein (AFP) and albumin (ALB) gene transcripts in rat kidney and brain as detected by in situ hybridization on tissue sections with [35S]-labelled alpha-fetoprotein and albumin cDNA probes. Both types of mRNA were present in distinct cell populations of the developing kidney and brain. In the kidney, both gene transcripts were distributed over all developing tubular cells in the 20-day-old fetus. During the first 3 weeks of life, a gradual decrease in the expression of AFP and ALB mRNA was apparent, the rate of decrease being greater on proximal tubules than on the other tubular cells. From the 4th week onwards, a weak signal for both mRNAs persisted in the majority of the tubular cells. In the brain, all neuronal cells expressed both genes. Transcript cellular distribution was mainly cytoplasmic during fetal and early postnatal life and became predominantly nuclear at 3, 4 and 5 weeks, suggesting that posttranscriptional mechanisms are involved in the control of AFP and ALB gene expression at these stages. In the adult brain no significant signal was recorded thereafter. Coexpression of AFP and ALB transcripts by specific cell types, together with their gradual disappearance concomitant with postnatal organ maturation, suggests a possible role for these proteins in terminal differentiation processes of tubular and neuronal cells.

Aging↗
PubMed1988

Cellular analysis of alpha-fetoprotein gene activation during carbon tetrachloride and D-galactosamine-induced acute liver injury in rats.

To analyze the cellular mechanisms of alpha-fetoprotein (AFP) gene activation during liver regeneration, we have measured the steady-state level of liver AFP mRNA by dot blot and revealed AFP transcripts and protein in liver sections by in situ hybridization and immunoperoxidase. AFP gene activation was studied from rats intoxicated with carbon tetrachloride or D-galactosamine, two toxics inducing different patterns of liver necrosis and regeneration. At 48 and 72 hours of carbon tetrachloride intoxication, liver AFP mRNA steady-state levels were slightly increased. On liver sections, large necrotic centrilobular areas were apparent with a 7- to 10-fold increase of hepatocyte mitotic index as compared with controls. At the same times of intoxication, a 2-fold increase in the signal level for AFP mRNA over all nonnecrotic hepatocytes was observed, whereas the protein remained undetectable in most of the hepatocytes. In contrast, after D-galactosamine intoxication, no increase of AFP mRNA steady-state levels was detectable. Scattered necrotic hepatocytes were visible at 48 and 72 hours of intoxication, with only a 2-fold increase of hepatocyte mitotic index, as compared with controls. Moreover, while the hybridization signal level for AFP mRNA was not increased over hepatocytes, large amounts of AFP mRNA and protein were detected in proliferated oval cells and bile duct-like structures. These results suggest that at least two distinct mechanisms at the cellular level may explain AFP gene activation during chemically-induced liver regeneration: (a) a moderate increase of AFP mRNA by all the remaining hepatocytes probably linked to their reentry into the proliferative cycle, which appears the main mechanism after carbon tetrachloride intoxication, and (b) a quantitatively important AFP expression by a small number of proliferated cells with new phenotypes (oval cells and bile-duct like structures), observed in the D-galactosamine model.

Animals↗
PubMed1987

Detection of albumin mRNAs in rat liver by in situ hybridization: usefulness of paraffin embedding and comparison of various fixation procedures.

Our aim was to define optimal conditions for efficient and reproducible albumin mRNA detection in rat liver by in situ hybridization. We used an albumin-specific [3H]-labeled cDNA probe with a specific activity of 6-8.10(6) cpm/microgram DNA. In situ hybridization is as efficient on paraffin sections as on cryostat sections for detecting albumin mRNAs. Perfusion fixation with a 4% paraformaldehyde solution results in homogeneous RNA retention within tissue blocks, in contrast with immersion fixation, which yields heterogeneous RNA preservation. Comparison of immersion fixation with three different fixatives (paraformaldehyde, ethanol-acetic acid, and Bouin's fixative) shows that the highest level of hybridization signal is obtained with paraformaldehyde. Ethanol-acetic acid and Bouin's fixative appear less efficient for albumin mRNA detection. Loss of mRNAs within liver tissue blocks over time is largely although not completely prevented by paraffin embedding.

Albumins↗
PubMed1986

Inhibitory effect of the acute inflammatory reaction on liver regeneration after partial hepatectomy in the rat.

We evaluated the influence of an acute inflammatory reaction that triggers the synthesis of exportable proteins by hepatocytes on liver regeneration after partial hepatectomy, which induces the synthesis of proteins necessary for liver cell proliferation. In hepatectomized rats with a turpentine-induced acute inflammatory reaction, the first peaks of hepatic DNA synthesis and mitosis were significantly inhibited compared with pair-fed controls subjected to partial hepatectomy only, and the liver DNA concentration at various times after partial hepatectomy was significantly lower in the former than in the latter. Inhibition was not obtained when the acute inflammatory reaction was induced 12 h or more before partial hepatectomy, suggesting that the inhibitory effect of turpentine administration depended on early events in the acute inflammatory reaction. These data suggest that one possible mechanism responsible for inhibition of regeneration might be competition at the transcriptional or the translational level between liver syntheses of various proteins, and that, under certain conditions, liver-specific functions might take precedence over regenerative functions.

Acute Disease↗
PubMed1986

Cellular analysis by in situ hybridization and immunoperoxidase of alpha-fetoprotein and albumin gene expression in rat liver during the perinatal period.

To analyze at the cellular level the decrease in alpha-fetoprotein (AFP) gene expression during the early postnatal growth, we searched for AFP gene transcripts by in situ hybridization using a specific cDNA probe, and for the corresponding protein by immunocytochemistry, on rat liver sections at various times of the perinatal period. The relative number of mRNA sequences was evaluated by Northern blot analysis. Albumin (ALB) gene expression was studied simultaneously with the same techniques. In 17-19-d-old fetuses all hepatocytes express simultaneously, for both genes, the mRNAs and the corresponding proteins. During the first postnatal weeks, at a time when the global number of AFP mRNA molecules decreases, all hepatocytes still contain cytoplasmic transcripts and protein. A zonal heterogeneity in the level of AFP gene expression develops around the first week, a higher number of gene products being detected in perivenous than in periportal hepatocytes. This heterogeneity persists until the fourth week when AFP mRNA sequences and protein are barely detectable. All hepatocytes express the ALB gene after birth, but at around the second week, a periportal intensification of the in situ hybridization signal and immunostaining becomes apparent. Our data indicate that co-expression of the AFP and ALB genes by all hepatocytes is a normal step in liver ontogeny; the diminution of AFP gene expression after birth is not the result of the disappearance of specialized cell clones; and zonal quantitative differences in the level of AFP and ALB gene expression are observed within the maturing liver lobule.

Albumins↗
PubMed1985

All hepatocytes are involved in the expression of the albumin gene in the normal adult rat: a demonstration by in situ hybridization and immunoperoxidase techniques.

Immunoperoxidase techniques have yielded conflicting results concerning the percentage of hepatocytes engaged in albumin production in normal adult rats. In addition, the question of whether functional differences in the synthesis of plasma proteins exist within the hepatic lobule remains to be determined. To clarify these questions, we have searched for gene albumin transcripts by in situ hybridization, and for the corresponding protein by immunoperoxidase, on adjacent liver sections. We observed that all hepatocytes contain albumin transcripts as well as the albumin protein, without any detectable zonal variation within the liver lobule. Taken altogether, these results demonstrate that every hepatocyte, whatever its location in the hepatic lobule, is actively engaged in albumin gene expression.

Albumins↗
PubMed1985

Myeloid metaplasia, perisinusoidal fibrosis, and nodular regenerative hyperplasia of the liver.

We describe two patients with myeloid metaplasia in whom portal hypertension resulted, not from infiltration of the liver sinusoids by myeloid cells, but from perisinusoidal fibrosis and nodular regenerative hyperplasia of the liver. We hypothesize that myeloid metaplasia induced the development of perisinusoidal fibrosis, which resulted in heterogeneous hepatic tissue blood perfusion, with atrophy of the liver cells in the underperfused areas and nodular regenerative hyperplasia in the normally perfused areas.

Aged↗