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Biomedical subjects

A Loranger

Publications and source records attributed to A Loranger.

17 recordsLinked to original sources
PubMed2001

Simple epithelium keratins 8 and 18 provide resistance to Fas-mediated apoptosis. The protection occurs through a receptor-targeting modulation.

Keratins 8 and 18 belong to the keratin family of intermediate filament (IF) proteins and constitute a hallmark for all simple epithelia, including the liver. Hepatocyte IFs are made solely of keratins 8 and 18 (K8/K18). In these cells, the loss of one partner via a targeted null mutation in the germline results in hepatocytes lacking K8/K18 IFs, thus providing a model of choice for examining the function(s) of simple epithelium keratins. Here, we report that K8-null mouse hepatocytes in primary culture and in vivo are three- to fourfold more sensitive than wild-type (WT) mouse hepatocytes to Fas-mediated apoptosis after stimulation with Jo2, an agonistic antibody of Fas ligand. This increased sensitivity is associated with a higher and more rapid caspase-3 activation and DNA fragmentation. In contrast, no difference in apoptosis is observed between cultured K8-null and WT hepatocytes after addition of the Fas-related death-factors tumor necrosis factor (TNF) alpha or TNF-related apoptosis-inducing ligand. Analyses of the Fas distribution in K8-null and WT hepatocytes in culture and in situ demonstrate a more prominent targeting of the receptor to the surface membrane of K8-null hepatocytes. Moreover, altering Fas trafficking by disrupting microtubules with colchicine reduces by twofold the protection generated against Jo2-induced lethal action in K8-null versus WT hepatocytes. Together, the results strongly suggest that simple epithelium K8/K18 provide resistance to Fas-mediated apoptosis and that this protection occurs through a modulation of Fas targeting to the cell surface.

Animals↗
PubMed2001

Keratin-mediated resistance to stress and apoptosis in simple epithelial cells in relation to health and disease.

Epithelial cells such as hepatocytes exhibit highly polarized properties as a result of the asymmetric distribution of subsets of receptors at unique portions of the surface membrane. While the proper targeting of these surface receptors and maintenance of the resulting polarity depend on microtubules (MTs), the Golgi sorting compartment, and different actin-filament networks, the contribution of keratin intermediate filaments (IFs) has been unclear. Recent data show that the latter cytoskeletal network plays a predominant role in providing resistance to various forms of stress and to apoptosis targeted to the surface membrane. In this context, we first summarize our knowledge of the domain- or assembly-related features of IF proteins and the dynamic properties of IF networks that may explain how the same keratin pair K8/K18 can exert multiple resistance-related functions in simple epithelial cells. We then examine the contribution of linker protein(s) that integrate interactions of keratin IFs with MTs and the actin-cytoskeleton network, polarity-dependent surface receptors and cytoplasmic organelles. We next address likely molecular mechanisms by which K8/K18 can selectively provide resistance to a mechanical or toxic stress, or to Fas-mediated apoptosis. Finally, these issues on keratin structure-function are examined within a context of pathological anomalies emerging in tissue architecture as a result of natural or targeted mutations, or posttranslational modifications at specific amino acid residues. Clearly. the data accumulated in recent years provide new and significant insights on the role of K8/K18, particularly under conditions where polarized cells resist to stressful or apoptotic insults.

Animals↗
PubMed1998

Zonal induction of mixed lineage kinase ZPK/DLK/MUK gene expression in regenerating mouse liver.

ZPK/DLK/MUK is a serine/theronine kinase believed to be involved in the regulation of cell growth and differentiation. To further explore the suggested participation of ZPK/DLK/MUK in this process, we examined the expression and cellular localization of ZPK/DLK/MUK mRNA in regenerating mouse liver following partial hepatectomy by ribonuclease protection assay and in situ hybridization. The steady-state level of APK/DLKMUK mRNA was very low in normal and sham-operated mouse livers, whereas a marked and transient increase was observed in the regenerating liver. While ZPK/DLK/MUK mRNAs were rarely detected in hepatocytes from all zones of the normal liver, hepatocytes of regenerating liver exhibit a gradient of expression ranging from low in the periportal zone, to intermediate in the mid-zone, to high in the pericentral zone. These findings demonstrate a transient stimulation of ZPK/DLK/MUK gene expression that correlates with the growth response of hepatocyte subpopulations in regenerating liver.

Animals↗
PubMed1997

Simple epithelium keratins are required for maintenance of hepatocyte integrity.

Keratin 8 (K8)-deficient adult mice develop a severe disease of the gastrointestinal tract characterized mainly by colorectal hyperplasia and inflammation. Given that hepatocytes contain K8/K18 heteropolymers only, this animal model was used to assess the contribution of these simple epithelium keratins to hepatocyte structural and functional integrity. Homozygous mutant (HMZ), heterozygous, and wild-type (WT) mice were examined for hepatocyte structural and metabolic features and their survival to partial hepatectomy. Except for the presence of few necrotic foci, no other tissular or cellular alterations were observed in nonhepatectomized HMZ mouse livers; glycogen and lipid peroxidation levels were essentially normal, but a small reduction in bile flow was observed. In response to a single pentobarbital injection, HMZ mice had longer sleeping times than heterozygous and WT mice. After a two-thirds partial hepatectomy under pentobarbital anesthesia, all HMZ mice died within a few hours, whereas those anesthetized with ether survived for 1 to 2 days. One hour after hepatectomy after pentobarbital anesthesia, many hepatocytes contained erythrocytes and large vacuoles in the cytoplasm, which suggests damage at the plasma membrane level in response to a sudden increase in portal blood flow. In line with these findings, an uptake of trypan blue by HMZ but not WT mouse hepatocytes was observed during a 10 ml/minute perfusion via the portal vein with a dye-supplemented buffer. Subsequent cellular dispersion led to viable WT mouse hepatocytes but largely nonviable HMZ mouse hepatocytes. Better viability was obtained at lower perfusion rates. Partially hepatectomized heterozygous mice developed liver steatosis, a condition that was not associated with a change in K8 content but perhaps linked to the presence of the neo gene. Transgenic HMZ mouse rescue experiments with a full-length K8 gene confirmed that the phenotypic alterations observed in partially hepatectomized HMZ mice were caused by the disruption of the K8 gene. Taken together, these findings demonstrate that simple epithelium keratins are essential for the maintenance of hepatocyte structural and functional integrity.

Animals↗
PubMed1996

Structural and functional alterations of hepatocytes during transient phalloidin-induced cholestasis in the rat.

To study the relationship between the dynamic actin web and bile secretion, we developed an acute model of cholestasis, using phalloidin, and examined sequential morphologic and biochemical events in rat liver. Biliary function (bite flow, bile, and canalicular membrane components) and cellular integrity (release of hepatic enzymes in serum and bile, canalicular structure, and microfilaments distribution) in rats given a single iv dose of phalloidin (0.8 mg/kg body weight) were assessed at 15, 45, and 90 min, 24 hr, and 5 days postinjection. Bile flow decreased significantly at 45 and 90 min, but cholestasis was transient since bile secretion returned to control levels at 24 hr. The biliary bile acid secretion rate was not modified during the same time period, indicating that cholestasis may have been due to impairment of the bile acid independent component of bile flow. Serum alanine aminotransferase and lactate dehydrogenase as well as biliary alkaline phosphatase and alkaline phosphodiesterase-1 activities were not altered by phalloidin treatment. These data, coupled with morphologic studies, provide no evidence of cell damage. Electron microscopy revealed that the pericanalicular actin web in both centrilobular and periportal hepatocytes was increased at 90 min and further enlarged at 24 hr and 5 days after phalloidin injection. At all time periods, the canalicular structure was well preserved. Na+K+ -ATPase and Mg2+ -ATPase activities in membrane fractions enriched in bile canalicular complexes decreased significantly at 15 min and remained low up to Day 5. Mg2+ -ATPase activity returned to control levels by Day 5. The lipid constituents of liver cell membranes enriched in canalicular complexes showed no significant variations 90 min after toxin treatment but, at 24 hr, phospholipid content rose and membrane fluidity increased. These results clearly indicate that the bile flow variation after a single low dose of phalloidin can be dissociated from specific pericanalicular microfilament distribution, lending further support to the view that normal biliary function is not strictly dependent on the integrity of the actin filament network.

Actin Cytoskeleton↗
PubMed1995

Specialization switch in differentiating embryonic rat liver progenitor cells in response to sodium butyrate.

Embryonic (E) 12 rat liver epithelial cells constitute a population of bipotential progenitor cells which can differentiate along the hepatocyte (Hep) or biliary epithelial cell (BEC) lineage in primary culture. In the present study, E12 cells were seeded on fibronectin-coated substratum and exposed to sodium butyrate (SB) for various exposure times, and the emergence of the Hep or BEC phenotype was monitored by following the variations in albumin production and assessing the appearance of the two surface-exposed markers HES6 and BDS7. Continuous exposure to SB resulted into a major reduction in albumin production and, at Day 9 postseeding, few cells coexpressed BDS7 and albumin. When cells were exposed to SB for 5 days and then cultured for an additional 5 days without SB, they massively express BDS7, but very little HES6. Moreover, the reverse sequence, i.e., 5 days without SB followed by 5 days with it, led to the appearance of many cells expressing both HES6 and BDS7. These results indicate that progenitors committed preferentially along the Hep lineage still have the option to switch to BECs, at a transitional stage that we refer to as a "differentiation window."

Animals↗
PubMed1995

Bile formation and hepatic plasma membrane composition in guinea-pigs and rats.

We compared bile formation, and biliary and liver plasma membrane composition in guinea-pigs and rats in an attempt to explain the observation that the bile flow rate and the bile acid independent fraction of bile flow (BAIF) in guinea-pigs is about five to seven times higher than in rats. Analysis of electrolytes in bile showed that bicarbonate was significantly [acid] higher in guinea-pigs while Cl-, phosphate and Ca2+ were markedly lower than in rats. High bile independent secretion in guinea-pigs was associated with a significantly lower concentration of total bile acid, phospholipid and cholesterol than in rats. Bile acid distribution studies showed that glycine conjugated chenodeoxycholate and ketolithocholate were the main bile acids in guinea-pigs, while taurine conjugated cholate and muricholate were the predominant bile acids in rats. Total fatty acid analysis of bile indicated that in rats the major fatty acids were palmitic acid (C16:0) and linoleic acid (C18:2, n-6). In guinea-pigs, the contribution of these fatty acids was lower than in rats and compensated with a significantly higher percentage of oleic acid (C18:1, n-9). Concentrations of anionic polypeptide fraction (APF), an acidic calcium binding apoprotein closely associated with biliary phospholipid and cholesterol secretion was also significantly lower in guinea-pigs. Canalicular plasma membrane analysis showed that as compared with rats, specific activities of Na+,K+ ATPase, and cholesterol and phospholipid content were markedly lower in guinea-pigs.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphatases↗
PubMed1993

Localization of DNA topoisomerase II in Chinese hamster fibroblasts by confocal and electron microscopy.

The localization of the 170- and the 180-kDa isoforms of the enzyme DNA topoisomerase II in growing Chinese hamster fibroblasts has been studied by confocal immunofluorescence microscopy and immunogold electron microscopy after labeling with affinity-purified isoform-specific polyclonal antibodies. Immunofluorescence and immunogold studies, together with quantitative image analysis, show that the two isoforms are present in the nucleoplasm and in the nucleolus. In the nucleoplasm both isoforms are frequently localized at the periphery of heterochromatin regions. In the nucleolus the immunofluorescence and immunogold signals relative to surface area are higher than in the nucleoplasm; both isoforms are localized predominantly in the fibrillar zones. During mitosis both isoforms remain detectable in the cytoplasm. The differential expression of the two isoforms during the cell cycle, observed in other studies, suggests that they have different functions, and their presence in both the nucleoplasm and the nucleolus suggests that these functions are required in both of these nuclear compartments.

Animals↗
PubMed1992

Cytokeratin 14 expression in rat liver cells in culture and localization in vivo.

Rat liver epithelial cells (LECs) are non-parenchymal proliferating cells that readily emerge in primary culture and can be established as cell lines, but their in vivo cell(s) of origin is unclear. We reported recently some evidence indicating that the LEC line, T51B, contains two cytokeratins (CKs) equivalent to human CK8 and CK14 respectively. T51B cells also contain vimentin assembled as a network of intermediate filaments distinct from that of the CKs. In the present study, we examined the expression of CK14 gene in various LEC preparations and a Triton-resistant rat skin cytoskeletal fraction, and then assessed its usefulness as an LEC specific marker in the liver. Northern and Western blot analyses with cDNAs and antibodies for CK8, CK14, CK18 and vimentin confirmed that rat hepatocytes express CK8 and CK18 genes only, whereas T51B cells express CK8, CK14 and vimentin genes in the absence of CK18. CK14 was also present in LECs derived as primary from embryonic-day 12 rat liver and secondary cultures from 4-day-old rat liver. Primary cultures of oval cells isolated from 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) treated rat liver (an enriched source of biliary epithelial cells) contained CK14 mRNAs which were slightly shorter than those in LECs. The analyses of CK5 (the usual partner of CK14) gene expression using specific cDNA and antibody clearly demonstrated its absence in LECs. In situ double immunolocalization analyses by laser scanning confocal microscopy showed that CK14 was not present in hepatocytes (HES6+ cells) and was expressed in some biliary epithelial (BDS7+ cells). CK14-positive cells were also found in the Glisson's capsule. However, CK14-positive cells of the portal region were vimentin negative, whereas those of the Glisson's capsule were vimentin positive. Our results suggest that CK14 gene expression is part of the differentiation program of two types of LECs and that this differential CK14 gene expression can be used as a new means to type LECs in culture and in vivo.

Amino Acid Sequence↗
PubMed1990

Effects of bile acids on actin polymerization in vitro.

Bile acids are major determinants of canalicular bile secretion, and there are indications that choleretic bile acids increase bile canalicular contractions, in isolated rat hepatocytes. Therefore, we examined the influence of various bile acids on the rate of actin polymerization in vitro. The free forms of cholic acid, ursodeoxycholic acid, and chenodeoxycholic acid, as well as their taurine and glycine conjugates, were incubated with purified muscle actin, at a concentration of 100-300 nmoles/mg actin. The rate of actin polymerization was measured by viscometry and the fluorescence of the pyrene probe, linked to actin. Results showed that all bile acids slow the rate of polymerization, and that the effect was dose-dependent. However, the reduction by chenodeoxycholic acid was greater than that caused by the other bile acids. The results indicate that bile acids, particularly in high concentrations interact with actin, a finding that may be related to the increased bile canalicular contractility, and altered canalicular membrane morphology, induced by choleretic bile acids.

Actins↗
PubMed1985

Toxicity of peptides of Amanita virosa mushrooms in mice.

A study was performed on the hepatic reaction of mice to acute intoxication with virotoxins (alaviroidin, viroisin, deoxoviroisin, viroidin, deoxoviroidin) and phalloidin, cyclic peptides isolated from Amanita virosa mushrooms. Purified fractions were administered intraperitoneally at various dosages to determine the LD50 which ranged from 1.0 to 5.3 mg/kg, with viroidin, phalloidin, and viroisin being the most potent. The virotoxins and phalloidin induced hemorrhagic necrosis of the liver. The development of hepatic lesions was followed by enhanced serum alanine aminotransferase (ALT) activity as well as by light and electron microscopic changes. In additional groups, bile ducts were cannulated and bile was collected for 2 hr after injection of the peptides (1 mg/kg) to determine their cholestatic potential. The earliest changes in hepatocytes were plasma membrane invagination and cytoplasmic vacuole formation. At later time periods, erythrocyte accumulation was evident in vacuoles and in the cytoplasm. The severity of hepatic damage, as judged by morphologic analysis, was correlated with serum ALT activity. Two of the peptides tested (viroisin and phalloidin) decreased bile flow by more than 50% over control values. Mild ultrastructural alterations in the bile canalicular pole of hepatocytes were observed during the development of cholestasis. Since virotoxins, like phalloidin, are bound to actin, it is possible that their affinity to cellular actin may be responsible for their hepatotoxicity.

Agaricales↗
PubMed1982

Effect of silybin on phalloidin-actin interactions in vitro.

The possible antagonism between silybin and phalloidin an F-actin has been examined in vitro. Phalloidin protects F-actin against denaturation by potassium iodine, cytochalasin B, DNase and pronase. Silybin does not counteract the effect of phalloidin on F-actin. However, silybin alone slowed the rate of polymerization of actin, but this effect was moderate and obtained with very high concentrations. These results indicate that the site of action in vitro of silybin is not actin.

Actins↗
PubMed1981

[Phalloidin counteracts the destruction of F-actin by osmic acid. II. Protection by phalloidin of F-actin crosslinked with aldehydes (author's transl)].

We have studied by viscometry and spectrophotometry the effects of glutaraldehyde, acetaldehyde, acrolein and formaldehyde on F-actin in vitro. Pretreatment with acrolein and, to a lesser extent, with glutaraldehyde, results in increased destruction of F-actin during the subsequent degradation with osmic acid. Formaldehyde alone disintegrates actin filaments. Acetaldehyde gives the best results and does not seem to damage F-actin. Phalloidin protects F-actin against destruction by osmic acid and this protection is also observed in the F-actin which has been pretreated with the four aldehydes mentioned.

Acetaldehyde↗
PubMedDate not supplied

Modulation of cytokeratin and actin gene expression and fibrillar organization in cultured rat hepatocytes.

Intermediate filaments of rat hepatocytes are composed of cytokeratins 8 and 18 (CK8 and CK18, respectively). Recent work from our laboratory has indicated a close relationship between the synthesis of these cytokeratins, their organization into intermediate filaments, and the promotion of growth and differentiation of cultured rat hepatocytes by insulin, epidermal growth factor, and dexamethasone. In the present study, we examined the mRNA expression, level of protein synthesis, and fibrillar distribution of cytokeratins 8 and 18 and actin in hepatocytes, isolated from normal and dexamethasone-injected rats and cultured as monolayers or spheroids in the presence of insulin, or from normal rat hepatocytes, cultured as monolayers in the presence of dexamethasone, insulin, and dimethyl sulfoxide. The CK8 mRNA level was lower in hepatocytes isolated from noninjected rats and cultured as either monolayers or spheroids, than in those from dexamethasone-injected rats. However, the CK18 mRNA level varied in a manner that was different from that of CK8 mRNA, showing that the modes of expression of the two genes were independent. The various changes in hepatocyte culture conditions led to variations in albumin mRNA levels that largely followed those observed in CK8 mRNA levels. In the case of actin, the amount of mRNAs varied from relatively high levels in hepatocyte monolayers to extremely low levels in hepatocyte spheroids, even though in both cases the cells were isolated from dexamethasone-injected rats. These changes in mRNA levels did not necessarily correlate with changes in the synthesis of cytokeratins 8 and 18, and actin. Changes in culture conditions induced a major reorganization in the distribution of cytokeratin intermediate filaments and actin filament between the region near the surface membrane and the cytoplasm. The most divergent patterns in cytokeratin intermediate filaments and actin filament distributions were observed between hepatocytes cultured as spheroidal aggregates and as monolayers in the presence of dimethyl sulfoxide. The former condition resulted in patterns of cytokeratin and actin gene expression and fibrillar organization that best matched those in situ. In the latter condition, inappropriate patterns were obtained, in spite of the fact that dimethyl sulfoxide treated hepatocytes are known to exhibit survival and functional activities equivalent to that of hepatocyte spheroids. These results demonstrate for the first time that the survival and functional activity (i.e., albumin production) of rat hepatocytes in vitro is not necessarily correlated with a particular pattern of cytokeratin and actin gene expression and fibrillar arrangement.

Actin Cytoskeleton↗
PubMedDate not supplied

Cytokeratin expression, fibrillar organization, and subtle function in liver cells.

Cytokeratins (CKs) constitute a diverse group of intermediate filament (IF) proteins, expressed as pairs in keratinized and nonkeratinizing epithelial cells. Much is known now about the expression, assembly, and function of CKs in keratinized epithelial cells, the main features being the tight coupling between CK pair switch and cell terminal differentiation (protection barrier) and the vital role of CK IFs in cell mechanical integrity. However, the picture about nonkeratinizing epithelia, like the hepatic tissue, remains quite unclear. The liver forms a multicellular system, where parenchymal cells (i.e., hepatocytes) exert diverse metabolic function(s) and nonparenchymal epithelial cells (e.g., biliary epithelial cells) usually serve structural (or accessory) purposes. In terms of differential CK gene expression, the data accumulated so far demonstrated that parenchymal cells can contain as few as one single CK pair, whereas nonparenchymal cells contain more than two CKs, one of them being a representative of those found in epidermis. Moreover, the distribution of the CK IF networks present in the different cell types varies a lot and can often be linked to the cell specialization. However, the function(s) played by these IF proteins in this multicellular tissue remains a major issue. The use of new experimental approaches, largely based on gene transfer technology, indicates that it is quite subtle.

Actins↗
PubMedDate not supplied

Biliary secretion and actin-cytokeratin filament distribution in rat hepatocytes during phalloidin-induced cholestasis.

The relationship between bile secretion (bile flow, bile acids, phospholipids, and cholesterol) and distribution of actin microfilaments (MFs) and cytokeratin (CK) intermediate filaments (IFs) was examined in hepatocytes of rats injected with a single low dose of phalloidin. This treatment induced a transient cholestasis characterized by a rapid development period (0-90 min postinjection) and a slow recovery period (24 h and 5 days postinjection). No significant changes were observed in bile acid secretion during the 5-day period. The phospholipid output dropped to less than 25% at 90 min and was back to the normal value at 24 h postinjection. In a parallel way, the cholesterol secretion dropped to 30% but came back to only 60% of the control level. Nile Red staining demonstrated a concomitant accumulation of lipids both in the cytoplasm and at the surface membrane. Immunostaining of the actin MFs and CK IFs showed that, in contrast with controls where both cytoskeletal networks were preferentially and uniformly localized at the surface membrane (i.e., sinusoidal, basolateral, and canalicular regions), the toxin treatment led to a major targeting of actin to the pericanalicular region at 24 h and a massive accumulation of well-preserved CK IFs in the cytoplasm at 5 days. Interestingly, this accumulation of CK IFs was not linked to any significant variations in CK isoforms. Together, these data indicate that a selective binding of the toxin to sinusoidal membrane actin at the time of injection triggers a sequence of events that culminate in delayed accumulation of actin MFs at the canalicular pole and of CK IFs in the cytoplasm. Moreover, the reversible perturbation of the bile secretory activity implies a functional adaptation of the hepatocytes that parallels the phalloidin-induced reorganization of both cytoskeleton networks.

Actin Cytoskeleton↗