On the three-dimensional structure of quick-frozen hepatic Mallory bodies with special reference to the appearance of cytoplasmic vesicles.

Livers containing Mallory bodies (MBs, hyalin degenerative cytoplasmic inclusions) were examined using Heuser's and Van Harreveld's cryo-techniques. The tissues were collected from 1) a patient suffering from alcoholic hepatitis and 2) mice treated with griseofulvin (GF, an anti-mitotic drug). Normal mouse liver and isolated MBs from GF-treated mice were also analyzed by the same methods. Our results suggest that under the toxic influence of alcohol or GF on microtubular elements, MBs are generated by entanglement of elements of 10 nm filaments with microtubule elements. This in turn inhibits cellular transport processes. The reticular net of the ER-element which is usually observable in the normal tissue is changed into numerous small vesicles in the pathological and experimental tissues. The diameters of hepatocytes containing these vesicles were 1.5 to 2 times larger than control diameters. MBs have previously been described in thin sections as filamentous tangles. On replicas we found that they appear to be composed of pairs of filaments twisted in a roughly helical manner, each having a diameter less than 10 nm. The paired helical nature of the MB-filaments is reminiscent of other inclusion bodies, which are also composed of elements of 10 nm filaments, observable in various neurological diseases.     

Mallory bodies: Lesions of hepatocytes containing proteins of the keratin-myosin-epidermin group

Summary Mallory's alcoholic hyalin in hepatocytes was found also in other diseases and is now referred to as Mallory bodies. Data concerning their histochemical, immuno and electron microscopic properties are partly contradictory. In this study, early stages of Mallory bodies reacted strongly with configurational technics for myosins; affinity tended to decrease when material with the properties of keratohyalin and the matrix of stratum corneum was formed. Thus, many Mallory bodies contained histochemically distinct myoid and keratin-like proteins. Electron microscopists demonstrated thick and thin filaments resembling contractile systems in Mallory bodies; the failure of immunologists to visualize actomyosin may be due to the heterogeneity of these proteins. The currently popular term prekeratin has been applied to a variety of substances extracted from epidermis, hoof and hair under different conditions. The prekeratin of recent immunofluorescence studies seems to contain mainly epidermin and low molecular matrix proteins; both were studied extensively by chemists. Epithelial filaments, including tonofibrils and contractile fibrils regarded as a subgroup of myofibrils, were well known half a century ago, but were banished by electron microscopy. Observations in this study and data on epidermal actomyosin indicate that different proteins of the k-m-e-f group can indeed coexist in epithelial cells. The formation and resolution of Mallory bodies can be regarded as an example of the well known shifts of epithelial cells between secretory and keratinizing states.     

Mallory bodies: lesions of hepatocytes containing proteins of the keratin-myosin-epidermin group

Mallory's alcoholic hyalin in hepatocytes was found also in other diseases and is now referred to as Mallory bodies. Data concerning their histochemical, immuno and electron microscopic properties are partly contradictory. In this study, early stages of Mallory bodies reacted strongly with configurational technics for myosins; affinity tended to decrease when material with the properties of keratohyalin and the matrix of stratum corneum was formed. Thus, many Mallory bodies contained histochemically distinct myoid and keratin-like proteins. Electron microscopists demonstrated thick and thin filaments resembling contractile systems in Mallory bodies; the failure of immunologists to visualize actomyosin may be due to the heterogeneity of these proteins. The currently popular term prekeratin has been applied to a variety of substances extracted from epidermis, hoof and hair under different conditions. The prekeratin of recent immunofluorescence studies seems to contain mainly epidermin and low molecular matrix proteins; both were studied extensively by chemists. Epithelial filaments, including tonofibrils and contractile fibrils regarded as a subgroup of myofibrils, were well known half a century ago, but were banished by electron microscopy. Observations in this study and data on epidermal actomyosin indicate that different proteins of the k-m-e-f group can indeed coexist in epithelial cells. The formation and resolution of Mallory bodies can be regarded as an example of the well known shifts of epithelial cells between secretory and keratinizing states.     

Immunochemical comparison of prekeratin and alcoholic hyalin intermediate filaments

Alcoholic hyalin is an hepatocellular aggregate composed of filaments apparently related to the prekeratin intermediate filament subclass. The relationship between these two filament preparations was determined immunochemically using guinea pig antisera derived against alcoholic hyalin, prekeratin, and major prekeratin polypeptides. Immunocrossreactivities were determined using sensitive solid-phase enzyme-immunoassays. These assays indicated that antisera derived against a given filament preparation reacted 10–1000 times better with that preparation than with the other system. The nature of crossreactive meterial was determined using antisera derived against the larger prekeratin polypeptides (M_r 61,000 and 51,000). When tested against these two antisera, alcoholic hyalin appeared to react better with the serum derived against the larger prekeratin component. Moreover, anti-alcoholic hyalin antiserum bound four to five times better to the 61,000 dalton component than to the 51,000 dalton polypeptide in the enzyme-immunoassay. Our results indicate that antigenic determinants related to prekeratin can be detected in alcoholic hyalin, but that these determinants are present in relatively low concentrations in purified alcoholic hyalin. In addition, it appears that the relative concentrations of prekeratin components in alcoholic hyalin do not reflect those in purified prekeratin.     

Intermediate filaments: analysis of filamentous aggregates induced by griseofulvin, an antitubulin agent

Mice fed griseofulvin, an antibiotic with antimicrotubular activity, formed hepatocellular aggregates of intermediate filaments, which resembled those associated with human alcoholic liver disease. These aggregates, termed Mallory bodies, were isolated from both human and mouse liver and the composition of these structures compared. Electrophoretic analysis indicated that the mouse filaments were composed of four major polypeptides (51,000, 47,000, 37,000, and 36,000 daltons). Human Mallory bodies possessed a similar number of components but of different molecular weights (56,000, 51,000, 50,000, and 38,000 daltons). Guinea pig antisera prepared against both whole human Mallory bodies and the major human polypeptide (56,000 daltons) crossreacted with mouse Mallory body material in both immunochemical and immunocytochemical systems. Our findings suggest that the two filament systems possess similar biochemical and immunological properties.     

Maintenance of desmosomes in mouse hepatocytes after drug-induced rearrangement of cytokeratin filament material. Demonstration of independence of desmosomes and intermediate-sized filaments

The distribution of desmosomes and cytokeratin filaments (tonofilaments) in hepatocytes of normal mice and those intoxicated with griseofulvin was studied by immunofluorescence microscopy. Treatment with griseofulvin over prolonged periods of time resulted in the dissociation of cytokeratin filaments from the plasma membrane and the inclusions of cytokeratin material in typical cytoplasmic aggregates, i.e. "Mallory bodies". However, such hepatocytes still displayed typical desmosomal arrays, including rather regularly spaced desmosomes along the bile canaliculi. These observations show that, in this tissue, desmosomes are able to maintain their characteristic positions along the plasma membrane after disconnection of the intermediate filament cytoskeleton. This indicates that maintenance of desmosomal integrity and position is independent of desmosome anchorage to tonofilaments. The results are discussed in relation to current concepts of desmosome formation and turnover.     

Intermediate filaments in hyaline material in alcoholic liver disease (Mallory bodies) and in cultured hepatoma cells.

Antibodies specific for cytoskeletal intermediate (10 nm) filaments reacted both with the hyaline deposits in alcoholic liver disease (Mallory bodies) and with perinuclear hyaline material of cultured hepatoma cells used as a model for hyaline formation in vitro. Our results suggest that disorganization and accumulation of intermediate filaments is an important step in the pathogenesis of alcoholic liver injury.     

A cell culture system for the induction of Mallory bodies: Mallory bodies and aggresomes represent different types of inclusion bodies

Mallory bodies (MBs) represent keratin-rich inclusion bodies observed in human alcoholic liver disease and in several chronic non-alcoholic liver diseases. The mechanism of their formation and their relationship to other inclusion bodies such as aggresomes is incompletely understood. We could induce keratin aggregates typical of MBs in cultured clone 9 rat hepatocytes by transgenic expression of wild-type and mutant aquaporin2 or α1-antitrypsin and under various forms of other cellular stress. By immunocytochemical analysis, p62 and poly-ubiquitin, components of classical MBs, could be demonstrated in the keratin aggregates of clone 9 hepatocytes. In addition, histone deacetylase 6, a microtubule-associated deacetylase, was identified as a novel component of the keratin aggregates. Thus, together with their ultrastructural appearance as randomly oriented, organelle-free aggregates of keratin filaments, the keratin aggregates in clone 9 hepatocytes correspond to MBs. An imbalance in keratin 8 to18 with very low levels of keratin 18 appears to be the underlying cause for their formation. The formation of MBs was microtubule-dependent although not depending on the activity of histone deacetylase 6. Forskolin-induced MBs in clone 9 hepatocytes were reversible structures which disappeared upon drug withdrawal. The MBs were not related to aggresomes since overexpressed misfolded transgenic proteins were undetectable in the keratin aggregates and no vimentin fiber cage was detectable, both of which represent hallmarks of aggresomes. Thus, cultured clone 9 hepatocytes are a useful system to study further aspects of the pathobiology of MBs.     

Disturbed structural interactions between microfilaments and tight junctions in rat hepatocytes during extrahepatic cholestasis induced by common bile duct ligation

 Microfilaments in epithelial cells are important for the structural and functional integrity of tight junctions. In the present study, we examined the relationship between microfilaments and tight junctions in hepatocytes of rat liver following common bile duct ligation (CBDL) for up to 2 weeks. Actin filaments and tight junctions were studied by fluorescence microscopy using 7-nitrobenzene-2-oxa-1,3-diazole phallacidin (NBD-ph) and an anti-ZO-1 antibody, respectively. Double-stained sections were examined with confocal laser scanning microscopy (CLSM). Electron microscopy was applied for the assessment of structural alterations in microfilaments and in tight junctions with detergent-extraction and freeze-fracture preparations. Our results showed that F-actin was present at the entire plasma membrane of hepatocytes in control liver, whereas CBDL increased the amount of F-actin mainly at the bile canalicular and lateral plasma membranes. Simultaneously, the immunofluorescence of ZO-1 underwent striking changes, i.e., from a uniform to an irregular staining pattern with various fluorescence intensities. CLSM demonstrated a colocalization of ZO-1 and F-actin in control liver and its deterioration in CBDL liver. Electron microscopy showed marked alterations of microfilaments and tight junctions due to CBDL. It is concluded that actin filaments are intimately associated with tight junctions in normal hepatocytes. CBDL impairs this association by progressively diminishing the structural interaction between F-actin and ZO-1, which may in turn lead to functional disturbances of tight junctions. Accepted: 28 August 1996     

Chronic ethanol administration alters hepatic surface membranes as evidenced by decreased concanavalin A binding

The effects of chronic ethanol administration on the hepatic surface membrane were examined. The binding of the lectin, concanavalin A (Con A), to isolated hepatocytes was used to ascertain changes in the hepatic plasma membrane, especially in regard to glycoprotein composition, due to chronic ethanol feeding. Hepatocytes, isolated from rats fed ethanol for 5 to 7 weeks, had a decreased ability to bind Con A when compared to hepatocytes from either the pair-fed controls or ad libitum chow-fed rats. Since decreased Con A binding was more apparent at high Con A concentrations, reduced lectin binding likely reflected changes in the composition of surface membrane glycoproteins in the livers of the ethanol-fed rats. When ethanol (50 mM) was added to the incubation medium containing hepatocytes from ethanol-fed rats, pair-fed controls, or chow-fed rats, no effects on Con A binding were observed. These results indicate that chronic ethanol administration induces changes in the oligosaccharide chains of plasma membrane glycoproteins in the liver. Such alterations may play a role in the pathogenesis of alcoholic liver disease.     

Expression of facilitative glucose transporter in rat liver and choroid plexus. A histochemical study in native cryostat sections.

Two isoforms of facilitative glucose transporters (GLUT), namely the erythroid/brain-type GLUT 1 and the liver-type GLUT 2, were demonstrated in native cryostat sections of normal rat liver and brain by immunofluorescence and a very sensitive immunoalkaline phosphatase reaction. Fixation with 0.1% alcoholic periodic acid resulted in an excellent localization of GLUT 2 in liver and GLUT 1 in brain. GLUT 1 in liver, however, could successfully be demonstrated after fixation with 1% alcoholic formaldehyde. GLUT 2 occurred in all hepatocytes as a basolateral membrane protein with a gradient of high expression in the periportal area and a lower one in the perivenous part. The first layer of hepatocytes adjacent to the hepatic vein coexpressed GLUT 1. In addition, GLUT 1 could be detected in the smooth muscle layer of the portal vein and in the apical and lateral plasma membrane of the bile duct epithelium. In brain, GLUT 1 showed a high expression in the microvessels, the ependyma and in the basal plasma membrane of choroid plexus epithelial cells. The blood capillaries associated with the choroidal epithelium were, however, negative for GLUT 1. The importance of the new findings in this study for the physiological role of the respective facilitative glucose transport proteins is discussed.     

Expression of facilitative glucose transporter in rat liver and choroid plexus

Summary Two isoforms of facilitative glucose transporters (GLUT), namely the erythroid/brain-type GLUT 1 and the liver-type GLUT 2, were demonstrated in native cryostat sections of normal rat liver and brain by immunofluorescence and a very sensitive immunoalkaline phosphatase reaction. Fixation with 0.1% alcoholic periodic acid resulted in an excellent localization of GLUT 2 in liver and GLUT 1 in brain. GLUT 1 in liver, however, could successfully be demonstrated after fixation with 1% alcoholic formaldehyde. GLUT 2 occurred in all hepatocytes as a basolateral membrane protein with a gradient of high expression in the periportal area and a lower one in the perivenous part. The first layer of hepatocytes adjacent to the hepatic vein coexpressed GLUT 1. In addition, GLUT 1 could be detected in the smooth muscle layer of the portal vein and in the apical and lateral plasma membrane of the bile duct epithelium. In brain, GLUT 1 showed a high expression in the microvessels, the ependym and in the basal plasma membrane of choroid plexus epithelial cells. The blood capillaries associated with the choroidal epithelium were, however, negative for GLUT 1. The importance of the new findings in this study for the physiological role of the respective facilitative glucose transport proteins is discussed.     

Cytologic, ultrastructural and immunologic features of intracytoplasmic hyaline bodies in fine needle aspirates of hepatocellular carcinoma

Intracytoplasmic hyaline bodies in malignant cells from an aspirate of a liver mass are suggestive of hepatocellular carcinoma. Such inclusions were studied by light and electron microscopy and by immunocytochemistry in fine needle aspirates from five cases of hepatocellular carcinoma. Seen by light microscopy, the inclusions were round or ovoid and were surrounded by a prominent halo. By both light and electron microscopic immunocytochemistry, the hyaline bodies showed negative staining for alpha-fetoprotein, alpha-1-antitrypsin and cytokeratin. Ultrastructurally, they were not membrane bound and were composed of filamentous, finely granular material, resembling the early stages of Mallory bodies.     

Dense bodies of the contractile system of cardiac muscle in Venus mercenaria

Dense bodies in the heart muscle of Venus mercenaria exist in two forms, free and attached. Free dense bodies morphologically consist of fascicles of thin filaments in parallel array and bound together by a dense, amorphous proteinaceous material. The binding of dense bodies to the cell membrane is effected via connecting filaments of the amorphous material of the dense body which join a condensation of morphologically similar material attached to the inner osmiophilic layer of the unit membrane. This composite of dense body, connecting filaments, membrane condensation and unit cell membrane has been termed collectively the attachment plaque. The attachment plaque is part of an extensive network on the cell surface which obligates that surface to a role in the contractile process. Moreover, this set of attachment plaques imposes an organization and an orientation to most thin filaments of the cell and preserves the contractile axis of the cell.     

SK HEP-1: A human cell line of endothelial origin

Summary SK-HEP-1 is an immortal, human cell line derived from the ascitic fluid of a patient with adenocarcinoma of the liver. We have determined that these cells are of endothelial origin. Despite the location of the tumor from which SK HEP-1 was derived, the cell line does not have properties of hepatocytes. Northern blot analysis of total cellular RNA shows no messenger RNA for the hepatic-specific proteins albumin, alpha-fibrinogen, or gamma-fibrinogen. Endothelial characteristics are seen by transmission electron microscopy. These features include numerous pinocytotic vesicles, electron dense granules consistent with Weibel-Palade bodies, and abundant intermediate filaments, identified immunocytochemically as vimentin. Cultures grown on plastic dishes grow in bundles of polygonal to spindle-shaped cells. Proteins characteristic for endothelial cells are identified by immunocytochemistry. Addition of basement membrane material (Matrigel) or type I collagen to the cultures induces these cells to organize into a tubular network.     

The influence of plectin deficiency on stability of cytokeratin18 in hepatocellular carcinoma

Intermediate filaments are important in building the cellular architecture. Previously we found cytokeratin18 was modulated in human hepatocellular carcinoma. Plectin is a cross-linking protein that organizes the cytoskeleton into a stable meshwork, which can maintain the uniform size and shape of hepatocytes. Because the cells of hepatocellular carcinoma were morphologically different from the hepatocytes, we speculated that expression of plectin and organization of intermediate filament might play roles in the pleomorphism of hepatocellular carcinoma cells. In this paper, we studied the plectin expression of hepatocellular carcinoma and liver tissues by immunohistochemistry and immunoblot. The results revealed that plectin was deficient and cytokeratin18 was modulated in hepatocellular carcinoma. Furthermore, we knockdown the plectin mRNA in Chang cells, the result revealed the plectin was deficient and the organization of cytokeratin18 was altered. Conclusively, this study offers a hypothesis that plectin deficient might play an important role in the tumorigenesis of hepatocellular carcinoma. Dr. Yi-Hsiang Liu contributed equally as the first author.     

Mechanisms of human hepatocarcinogenesis

The major risk factors and etiological agents responsible for development of hepatocellular carcinoma in humans have been identified and characterized. Among these are chronic infection with hepatitis B virus or hepatitis C virus, exposure to aflatoxin B1, and cirrhosis of any etiology (including alcoholic cirrhosis and cirrhosis associated with genetic liver diseases). Both chronic hepatitis and cirrhosis represent major preneoplastic conditions of the liver as the majority of hepatocellular carcinomas arise in these pathological settings. Hepatocarcinogenesis represents a linear and progressive process in which successively more aberrant monoclonal populations of hepatocytes evolve. Regenerative hepatocytes in focal lesions in the inflamed liver (chronic hepatitis or cirrhosis) give rise to hyperplastic hepatocyte nodules, and these progress to dysplastic nodules, which are thought to be the direct precursor of hepatocellular carcinoma. In most cases, the neoplastic transformation of hepatocytes results from accumulation of genetic damage during the repetitive cellular proliferation that occurs in the injured liver in response to paracrine growth factor and cytokine stimulation. Hepatocellular carcinomas exhibit numerous genetic abnormalities (including chromosomal deletions, rearrangements, aneuploidy, gene amplifications, and mutations), as well as epigenetic alterations (including modulation of DNA methylation). These genetic and epigenetic alterations combine to activate positive mediators of cellular proliferation (including cellular proto-oncogenes and their mitogenic signaling pathways) and inactivate negative mediators of cellular proliferation (including tumor suppressor genes), resulting in cells with autonomous growth potential. However, hepatocellular carcinomas exhibit a high degree of genetic heterogeneity, suggesting that multiple molecular pathways may be involved in the genesis of subsets of hepatocellular neoplasms. Continued investigation of the mechanisms of hepatocarcinogenesis will refine our current understanding of the molecular and cellular basis for neoplastic transformation in liver, enabling the development of effective strategies for prevention and/or more effective treatment of hepatocellular carcinoma.     

Helicobacter infection induces podosome assembly in primary hepatocytes in vitro

Helicobacter pylori (H. pylori) infection may contribute to many extragastric diseases including liver cirrhosis and hepatocellular carcinoma. However, the exact mechanism by which H. pylori induces the liver damage is largely unknown. We used cultured mouse primary hepatocytes as an in vitro model to investigate different aspects of liver physiology and pathology. In this study, we show that primary hepatocytes are able to assemble actin-based cytoskeletal structures called podosomes at the ventral plasma membrane. These structures are positive for podosome markers such as cortactin, vinculin and integrins and comprise proteolytic potential. Infection with the pathogen H. pylori further stimulates the formation of podosomes in primary hepatocytes. The use of pharmacological inhibitors reveals that this response is mediated, at least in part, by TGFβ, a cytokine known to regulate podosome formation in endothelial cells. Similar results are obtained with the hepatoma cell line Huh7. Podosome formation is associated with increased hepatocyte degrading capacities but also with reduced cell motility. Therefore, podosome assembly translates into hepatocyte malfunction. Our study supports the hypothesis that hepatocytes can also assemble podosomes under pathological conditions in vivo.     

The organization of actin in spreading macrophages : The actin-cytoskeleton of peritoneal macrophages is linked to the substratum via transmembrane connections

The cytoskeleton of murine peritoneal macrophages has been examined by a combination of morphological techniques, including phase-contrast light microscopy, scanning electron microscopy (SEM), and several transmission electron microscopic (TEM) methods. The cytoskeleton of cells spreading on glass, Formvar-carbon, and polystyrene substrata was exposed by brief extraction with non-ionic detergent, and stabilized by exposure to heavy meromyosin, myosin subfragment-1 or tropomyosin. In the spreading lamellae and lamellipodia the cytoskeleton is principally composed of filamentous actin, which appears as dense foci, interconnected by radiating filaments and filament bundles. The actin of the foci, as well as individual actin filaments, are connected to the substratum by transmembrane linkages which appear as filaments that pass through the plane of the (extracted) plasma membrane. Thus, the results of this study indicate that the adhesion of macrophages to substrata for the purposes of spreading and motility may be a function of transmembrane elements which link actin to substrata. Further, the formation of actin foci may serve to stiffen and stabilize the cytoskeleton, conditioning it to function in cell adhesion, spreading and locomotion.     

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.