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G Feldmann

Publications and source records attributed to G Feldmann.

At least 37 records · Page 2Linked to original sources
PubMed1996

Effect of diethylmaleate on bile secretion and ultrastructural appearance of hepatocytes in normal rats and mutant rats with defective organic anion secretion.

Diethylmaleate is an organic anion secreted into bile as a glutathione conjugate. Its transport by the hepatocyte is associated with dilatation of the Golgi apparatus and the appearance of small vesicles in the pericanalicular area. It has been speculated that the Golgi apparatus could play a role in the intracellular transport and/or the biliary canalicular secretion of diethylmaleate. The purpose of this work was to determine whether the alterations in the Golgi apparatus and the pericanalicular vesicles could mediate the canalicular secretion of diethylmaleate. Diethylmaleate biliary secretion and diethylmaleate-induced bile flow were measured in Sprague-Dawley rats, and in TR- rats which have an inherited defect in the excretion into bile of organic anions, including glutathione conjugates. Livers of both Sprague-Dawley and TR-rats were examined by electron microscopy, to characterize the changes in intracellular organelles. In Sprague-Dawley rats, as previously described, diethylmaleate administration was associated with an increase in bile flow, which was parallel in time to the secretion into bile of diethylmaleate conjugates. Electron microscopic examination of the liver after diethylmaleate administration showed dilatation of the Golgi saccules. In contrast, in TR- rats, the increase in bile flow and the secretion of diethylmaleate conjugated were nearly absent. Nevertheless, electron microscopic examination showed a dilatation of the Golgi saccules similar to that observed in Sprague-Dawley rats. TR- rats, in addition to the changes in the Golgi apparatus, had marked dilatation of the endoplasmic reticulum. These results show that biliary secretion of diethylmaleate conjugates was severely impaired in TR- rats, in spite of a dilatation of the Golgi apparatus and of the endoplasmic reticulum. We conclude that it is unlikely that the alterations in the Golgi apparatus (and the endoplasmic reticulum) induced by diethylmaleate play a role in the canalicular secretion of diethylmaleate. We do not exclude the possibility that these organelles could play a role in intracellular transport of this compound. Alternatively, these alterations could be due to a "toxic" effect of diethylmaleate accumulation in hepatocytes.

Animals↗
PubMed1996

Cellular and subcellular localization of acetaldehyde-protein adducts in liver biopsies from alcoholic patients.

Acetaldehyde, the first product of ethanol in hepatocytes, can react with protein to form acetaldehyde-protein adducts (APAs). Because it has been suggested that these adducts could be involved in the pathogenesis of ethanol-induced hepatic lesions and in fibrogenesis, we performed an ultrastructural immunohistochemical study to precisely define the cellular and subcellular localization of APAs. A preembedding technique of indirect immunoperoxidase was performed in liver biopsy specimens from eight patients with alcoholic liver disease, using a specific antiserum against APAs. In all specimens, APAs were detected in the rough endoplasmic reticulum, in some peroxisomes, and in the cytosol of hepatocytes. In four patients with steatofibrosis or cirrhosis, labeling of Ito cells was also observed. In these cases, the same staining pattern was observed in the cytoplasmic processes of myofibroblasts in areas of fibrogenesis. When isolated rat Ito cells were incubated in the presence of acetaldehyde, APAs were also detected in the cytoplasm. These results show that APA formation occurs in hepatocytes at the sites of acetaldehyde production. Detection of APAs in human and rat Ito cells strongly suggests that acetaldehyde can diffuse into Ito cells and bind to cytoplasmic proteins to form local APAs. Because Ito cells are the main effector cells of liver fibrosis, detection of APAs in these cells points to their possible involvement in liver fibrogenesis.

Acetaldehyde↗
PubMed1996

The transcytotic pathway of an apical plasma membrane protein (B10) in hepatocytes is similar to that of IgA and occurs via a tubular pericentriolar compartment.

In hepatocytes, newly synthesized apical plasma membrane proteins are first delivered to the basolateral surface and are supposed to reach the apical surface by transcytosis. The transcytotic pathway of apical membrane proteins and its relationship with other endosomal pathways has not been demonstrated morphologically. We compared the intracellular route of an apical plasma membrane protein, B10, with that of polymeric IgA (pIgA), which is transcytosed, transferrin (Tf) which is recycled, and asialoorosomucoid (ASOR) which is delivered to lysosomes. Ligands and anti-B10 monoclonal IgG were linked to fluorochromes or with peroxidase. The fate of each ligand was followed by confocal and electron microscopy in polarized primary monolayers of rat hepatocytes. When fluorescent anti-B10 IgG and fluorescent pIgA were simultaneously endocytosed for 15-30 minutes, they both uniformly labelled a juxtanuclear compartment. By 30-60 minutes, they reached the bile canaliculi. Tf and ASOR were also routed to the juxtanuclear area, but their fluorescence patterns were more punctate. Microtubule disruption prevented all ligands from reaching the juxtanuclear area. This area corresponded, at least partially, to the localization of the mannose 6-phosphate receptor, an endosomal marker. By electron microscopy, the juxtanuclear compartment was made up of anastomosing tubules connected to vacuoles, and was organized around the centrioles. B10 and pIgA were mainly found in the tubules, whereas ASOR was segregated inside the vacuolar elements and Tf within thinner, recycling tubules. In conclusion, transcytosis of the apical membrane protein B10 occurs inside tubules similar to those carrying pIgA, and involves passage via the pericentriolar area. In the pericentriolar area, the transcytotic tubules appear to maintain connections with other endosomal elements where sorting between recycled and degraded ligands occurs.

Animals↗
PubMed1996

Cytogenetic analysis of BC2, a new human hepatoma cell line, by fluorescent in situ hybridization.

Cytogenetic analysis of a new human hepatoma cell line BC2 was performed with conventional cytogenetic techniques and fluorescent in situ hybridization. Numerical and structural abnormalities were observed by conventional cytogenetics for chromosomes 1, 2, 4, 7, 8, 9, 10, 11, 15, 17 and 20. Chromosome painting allowed to specify the translocation of chromosome 1, and to characterize 3 markers from chromosome 8 and one marker from 9, which were unrecognizable by conventional techniques. Comparison of chromosome 1 abnormalities with those reported in the literature for other human hepatoma cell lines showed that structural abnormalities of chromosome 1 were present in different regions of this chromosome. A review of the literature was done, and the results discussed, suggesting that alterations of chromosome 1 may be important in hepatocarcinogenesis.

Carcinoma, Hepatocellular↗
PubMed1996

Ultrastructural immunogold labeling of lipid-laden enterocytes from patients with genetic malabsorption syndromes.

Intestinal biopsies from patients having genetic disorders of lipoprotein assembly and secretion, such as abetalipoproteinemia (ABL) or Anderson's disease (AD), contain large amounts of lipids which are accumulated in the enterocytes. Determination of the intracellular sites in which the lipids accumulate and to which apolipoproteins the lipids are bound would help to identify the defects in these diseases and further elucidate the mechanisms by which lipoprotein assembly and secretion occur normally. Ultrastructural immunogold labeling, however, is hampered by the poor preservation of the lipids accumulated in the enterocytes of these patients. We have used routine electron microscopy (fixation and ultra-thin sectioning) along with three methods for immunogold labeling of lipid-laden enterocytes: ultrathin cryosectioning, low temperature freeze substitution with embedding in Lowicryl K4M, and ultra-low temperature freeze substitution with embedding in Lowicryl HM20, to establish a protocol for investigating the intestinal tissue from these patients. Ultracryosectioning, while preserving the overall morphology of the lipid laden enterocytes, did not preserve the lipid content and the immunogold labeling of apolipoprotein B (ApoB) appeared dislocated. Freeze substitution and low temperature embedding in Lowicryl K4M, in contrast, appeared to better preserve the lipid and lipoprotein structures; however, the antigenicity of both apoAI and apoB appeared to be lost and no specific labeling could be obtained. Freeze substitution and embedding in Lowicryl HM20 best preserved the lipid and lipoprotein structures while maintaining apoprotein antigenicity. In conclusion, immunogold labeling of apolipoproteins on lipid structures in the lipid-laden enterocytes of patients with ABL and AD is best obtained by freeze substitution and embedding in Lowicryl HM20.

Abetalipoproteinemia↗
PubMed1995

Direct solution hybridization of guanidine thiocyanate-solubilized cells for quantitation of mRNAs in hepatocytes.

The sensitivity of direct solution hybridization of hepatocytes solubilized in guanidium thiocyanate (GuSCN) for detecting alpha 1-acid glycoprotein and albumin mRNAs was studied. The sensitivity of detection was inversely correlated with the DNA concentration. Raising the hybridization temperature from 20 to 37 or 50 degrees C (with formamide) increased the hybridization efficiency three- to fourfold in cell lysates with a high DNA concentration (1 microgram/microliter), whereas the hybridization efficiency was already maximal at 20 degrees C in diluted samples. It was most important to normalize all hybridization reactions with an internal standard, such as sense mRNA, because of the great variation in hybridization efficiency from one cell preparation to another depending on the DNA concentration. Direct hybridization of GuSCN cell lysates labeled in vivo with [6-14C]orotic acid was more efficient than hybridizing equivalent amounts of purified [6-14C]-labeled RNA, perhaps because of greater mRNA integrity and/or better recoveries of mRNA in GuSCN cell lysates. Therefore, direct solution hybridization of GuSCN-solubilized hepatocytes, which avoids the problem of RNA purification, appears to be a rapid, sensitive, and reliable method for quantifying mRNA in hepatocytes.

Albumins↗
PubMed1995

The c-jun proto-oncogene down-regulates the rat alpha-fetoprotein promoter in HepG2 hepatoma cells without binding to DNA.

The effects of a phorbol ester (TPA) and of members of the Jun and Fos oncoprotein family on the activity of the rat alpha-fetoprotein (AFP) promoter were checked by using transient expression experiments in HepG2 hepatoma cells. TPA blocked the activity of the rat AFP promoter in a dose-dependent manner. Overexpression of c-Jun specifically repressed the rat AFP promoter but not the albumin promoter. JunB and JunD were poorer inhibitors. c-Fos expression did not potentiate the negative effect of Jun. The Jun-induced repression does not require binding of c-Jun to the AFP promoter. DNase 1 footprinting experiments did not display any high affinity binding site for Jun on the AFP promoter. Integrity of the c-Jun DNA binding domain is not required for the c-Jun protein to block the AFP promoter. The N-terminal part of Jun, which contains the activating domain, is responsible for the repression as shown by using Jun-Gal4 chimera. Jun likely exerts its negative control on the AFP promoter via protein-protein interactions with a not yet identified trans-activating factor within the -134 to +6 region or with a component of the general machinery of transcription. Jun proteins can thus be key intermediates in regulatory cascades which result in the differential modulation of the AFP and albumin gene expression in the course of liver development and carcinogenesis.

Animals↗
PubMed1995

Distribution of albumin, alpha 1-inhibitor 3 and their respective mRNAs in periportal and perivenous rat hepatocytes isolated by the digitonin-collagenase technique.

The expression of albumin and alpha 1-inhibitor 3 genes was investigated in rat cell suspensions enriched in periportal (n = 10) and perivenous (n = 10) hepatocytes obtained by the digitonin-collagenase technique. The degree of enrichment of the cell suspensions was assessed: (1) by enzymic assays for the periportal marker alanine aminotransferase and for the perivenous marker glutamine synthetase; and (2) by their content of mRNAs for the periportal marker hepatic glutaminase and for glutamine synthetase. The existence of an antegrade intra-lobular gradient for albumin and alpha 1-inhibitor 3 mRNAs was demonstrated, with periportal:perivenous ratios of 2.33 and 3.80, respectively. However, no gradient was demonstrated for the respective protein contents with corresponding ratios of 0.98 and 1.21. A certain degree of overlap existed between periportal and perivenous suspensions for their content in albumin and alpha 1-inhibitor 3 mRNAs. A morphometrical analysis of the surface of digitonin-permeabilized hepatic tissue revealed that this overlap could be explained by a variable extent of permeabilization of the mediolobular zone from one rat to another and from one lobule to another in a given animal. These results suggest that while the digitonin-collagenase technique is well suited for studies in vitro of proteins expressed in sharp intra-lobular gradients or restricted to an intra-lobular compartment, it is not completely reliable for proteins distributed in continuous moderate intra-lobular gradients, such as albumin and alpha 1-inhibitor 3.

Acute-Phase Proteins↗
PubMed1995

Cytochrome P4502B follows a vesicular route to the plasma membrane in cultured rat hepatocytes.

BACKGROUND/AIMS: Autoantibodies against cytochrome P450 are found in some forms of autoimmune hepatitis. Cytochrome P450 is synthesized and mainly located in the endoplasmic reticulum but may also be expressed on the plasma membrane of hepatocytes. Vesicles migrate from the endoplasmic reticulum to the Golgi apparatus and then to the plasma membrane along microtubules. We determined the route followed by cytochrome P4502B to reach the plasma membrane. METHODS: Rat hepatocytes were cultured for 2 hours after plating with various inhibitors of cellular trafficking. Detached, uncut, nonpermeabilized hepatocytes were then exposed to a monoclonal antibody specific for cytochrome P4502B and studied by flow cytometry and confocal microscopy. RESULTS: The plasma membrane expression of cytochrome P4502B was markedly decreased after 2 hours of culture with cycloheximide (an inhibitor of protein synthesis), caffeine at 20 degrees C (conditions that decrease vesicular transport from the endoplasmic reticulum to the Golgi apparatus), brefeldin A (which redistributes Golgi components back to the endoplasmic reticulum), monensin (an inhibitor of Golgi functions), and colchicine, vinblastine, or nocodazole (three microtubule inhibitors). CONCLUSIONS: Part of cytochrome P4502B follows a microtubule-dependent vesicular route from the endoplasmic reticulum to the plasma membrane in cultured rat hepatocytes.

Animals↗
PubMed1995

Focal nodular hyperplasia of the liver: composition of the extracellular matrix and expression of cell-cell and cell-matrix adhesion molecules.

We studied by immunohistochemistry 25 cases of focal nodular hyperplasia (FNH) to evaluate the composition of the extracellular matrix and the expression and distribution of endothelial cell-cell adhesion molecules and integrin receptors. The extracellular matrix of FNH retained the overall organization of that of normal liver. The matrix of central scars resembled that of portal tracts. The main difference was the presence of large vitronectin deposits, which might indicate the existence of local hemodynamic disturbances. The matrix lining the sinusoid-like vessels running in the hyperplastic parenchyma retained characteristic features of the normal perisinusoidal matrix, such as the presence of tenascin. In the zone surrounding the central scars, it contained large amounts of laminin, von Willebrand factor, and thrombospondin, suggesting the development of perisinusoidal fibrosis. Laminin deposition was accompanied by the induction of cell-cell adhesion molecules on adjacent endothelial cells and by the up-regulation of specific integrin receptors on both hepatocytes and sinusoidal endothelial cells. In conclusion, our study: (1) reinforces the hypothesis that FNH is merely a hyperplastic response of liver parenchyma to local vascular abnormalities, and (2) shows that the lesions of perisinusoidal fibrosis associated with FNH are accompanied by the induction of integrin receptors on hepatocytes and sinusoidal endothelial cells.

Adult↗
PubMed1995

Behaviour of the small GTP-binding protein rab6 in the liver of normal rats and rats presenting an acute inflammatory reaction.

While it is known that the small GTP-binding protein rab6 is localized in vitro to the Golgi apparatus of several mammalian cells, its behaviour in vivo has not yet been investigated. The aim of this work was to compare by immunocytochemistry and immunoblotting the distribution of rab6 in hepatocytes from normal rats and from rats with an acute inflammatory reaction, a circumstance where the synthesis and secretion of plasma proteins by the hepatocytes is increased and which is accompanied by several changes in the Golgi apparatus. Our results show that in normal rats, rab6 was present in all hepatocytes irrespective of the location of the cell in the hepatic lobule. At the ultrastructural level, rab6 was mainly visible on the three Golgi saccules, but in some cells it appeared to be absent in saccules corresponding to the cis or the trans saccule. The inflammatory reaction was accompanied by an increase of the immunocytochemical labelling at the light and electron microscopy levels. However, by immunoblotting, no differences in the total amount of rab6, nor in its subcellular distribution were found in liver cells after acute inflammatory reaction. These results demonstrate that rab6 is restricted in vivo to the Golgi apparatus and that no significant redistribution occurs during an acute inflammatory reaction.

Animals↗
PubMed1995

Effects of nicotinamide on hepatocyte viability and secretion of albumin and alpha 1-acid glycoprotein by adult rat hepatocytes in primoculture. Comparison with dexamethasone and recombinant human interleukin-6.

The effects of nicotinamide on hepatocyte viability and secretion of albumin and alpha 1-acid glycoprotein were studied in the absence or presence of dexamethasone and/or recombinant human interleukin-6 either after cell attachment (2 h) or after 24, 48, and 72 h of culture. The evolution of hepatocyte survival during the culture was appreciated by measurement of total DNA content. The secretion of albumin and alpha 1-acid glycoprotein was measured after a 4-h period following cell attachment or after 24, 48 and 72 h of culture. The important decrease of DNA content, mRNA levels and secretion of albumin and alpha 1-acid glycoprotein in control cultures after 2-3 days was not prevented by the addition of nicotinamide. In contrast, dexamethasone alone or with recombinant human interleukin-6 improved DNA content and albumin secretion with no additional effect of nicotinamide. The secretion of alpha 1-acid glycoprotein was largely induced by dexamethasone alone or dexamethasone and recombinant human interleukin-6. The increase of alpha 1-acid glycoprotein secretion was not modified by the addition of nicotinamide and averaged respectively 27- and 60-fold for dexamethasone alone and dexamethasone and recombinant human interleukin-6 after 48 h. These observations suggested that nicotinamide, at least in the conditions tested here, is unable to prevent alterations of hepatocyte viability and gene expression of cultured hepatocytes.

Animals↗
PubMed1995

Effects of female sex hormones on mitochondria: possible role in acute fatty liver of pregnancy.

Acute fatty liver of pregnancy occurs in some women. As other cases of microvesicular steatosis are due to impaired mitochondrial oxidation of fatty acids, we investigated the effects of female sex hormones on liver mitochondria in female mice. Three hours after administration of both estradiol (36 mumol/kg) and progesterone (150 mumol/kg), the in vitro beta-oxidation of [U-14C]palmitic acid and the activity of the tricarboxylic acid cycle decreased 49 and 54%, whereas the in vivo oxidation of [U-14C]palmitic acid decreased 38%. One week of treatment with both sex hormones produced ultrastructural lesions of mitochondria, decreased the recovery of mitochondrial proteins by 34%, increased state 4 respiration by 54-77%, and decreased the activities per gram of liver of several enzymes involved in the activation, mitochondrial uptake, and oxidation of fatty acids by 34-54%. We conclude that female sex hormones have deleterious effects on liver mitochondria and suggest that these effects, together with other factors, may contribute to the development of acute fatty liver of pregnancy in some women.

Acute Disease↗
PubMed1994

Renal and microvascular effects of an aldose reductase inhibitor in experimental diabetes. Biochemical, functional and ultrastructural studies.

Aldose reductase inhibitors, and particularly sorbinil, have been reported to prevent glomerular basement membrane thickening (GBMT) and albuminuria development in diabetic rats, but contradictory observations have been published. The aim of this study was to answer the following questions (i) is the corrective effect of sorbinil on GBMT, if confirmed, associated with an effect on collagen metabolism alterations? (ii) Is it associated with an effect on microvascular functional alterations? We therefore studied the influence of sorbinil on glucosyl-galactosyl-hydroxylysyl-glucohydrolase activity (GGHG; EC 3.2.1.107 which is involved in the catabolism of collagen disaccharide units), 3- and 4-hydroxyproline content and GBMT by ultrastructural morphometry in the kidney cortex of streptozotocin-diabetic rats after 5 months of disease. In parallel, the effects on albumin renal clearance and another functional alteration, the microvascular response to norepinephrine, were evaluated. We confirmed a corrective effect of sorbinil on both renal albumin clearance and GBMT. In the diabetic rats, sorbinil diminished the 3-hydroxyproline (but not the 4-hydroxyproline) content, whether expressed per mg protein or per total kidney cortex relative to body weight. Sorbinil reduced GGHG activity measured in the dialysed 10,000 g supernatant whether expressed per mg protein or per total kidney cortex; this activity has been shown to be increased in diabetes. Sorbinil also corrected the microvascular response to norepinephrine which is altered in diabetes.

Albuminuria↗
PubMed1994

Glucagon administration in vivo stimulates hepatic RNA and protein breakdown in fed and fasted rats.

Liver RNA and protein breakdown rates were measured simultaneously in fed and in 24 h-fasted rats during a short-term cyclic perfusion, 1 h after an intraperitoneal injection of glucagon or of saline. RNA was labelled in vivo by an intraperitoneal injection of [6-14C]orotic acid, 60 h before the start of the perfusion. The accumulation of radioactive cytidine and valine in the perfusion medium for 15 min was used to determine RNA breakdown and proteolysis respectively. The portal glucagon/insulin ratio was significantly higher in the fasted glucagon-treated rats than in their fed counterparts. Although glucagon administration significantly increased RNA and protein degradation rates in the fasted and in the fed groups, the effect was greater after 24 h of starvation. The relationship between these biochemical changes and the alterations of the hepatocyte lysosomal system was investigated by determining the fractional cytoplasmic volume of lysosomal structures (autophagic vacuoles and dense bodies) by morphometry in the fasted glucagon-treated rats and in their controls. Hyperlucagonaemia significantly enhanced the relative volume of autophagic vacuoles without affecting that of dense bodies. The results showed that hyperglucagonaemia induced in vivo stimulated both liver RNA and protein breakdown and that this effect was modulated by the nutritional status of the rats.

Animals↗
PubMed1994

Expression of complement-regulatory proteins in normal and UW-preserved human liver.

BACKGROUND/AIMS: Somatic cells are protected against complement-mediated injury by specialized membrane proteins, known as complement-regulatory proteins (CRP). The knowledge of the pattern of CRP expression in the liver is important to evaluate the role of complement-mediated injury in graft rejection. METHODS: We determined the distribution of four main CRP: membrane cofactor protein (MCP), decay accelerating factor (DAF), protectin, and complement receptor 1 (CR1) in 30 histologically normal livers, 13 samples from University of Wisconsin cold-storage solution (UW)-preserved tissue and 17 postoperative biopsies of UW-preserved allografts. RESULTS: In normal liver, hepatocytes expressed only MCP. Bile duct cells were reactive for MCP and protectin. Sinusoidal endothelial cells expressed MCP and protectin but displayed no or faint expression of DAF. Endothelial cells of portal vessels and centrilobular veins expressed high levels of DAF, MCP, and protectin. No expression of CR1 was observed. No change in CRP expression was usually detected after UW preservation, except for protectin, induced on hepatocytes in 9 samples of UW-preserved liver tissue and in 9 allografts. CONCLUSIONS: Hepatocytes and sinusoidal endothelial cells, which have a defective expression of CRP, might be at risk for complement-mediated injury. However, this risk is not aggravated after UW preservation.

Adenosine↗