bluuebunny/crossref_metadata_2025 huggingface dataset:
Influence of Dietary Fat on the Effect of Endotoxin on Murine Hepatic Peroxisomes
Authors: DeLamatre J G , Schilleci J T , Hanson L H
Published: November 2004

This study investigates the effects of either a high–fat diet or endotoxin on peroxisome metabolism as assessed by measuring catalase activity, catalase mass, and peroxisomal β–oxidation. Three mouse strains C3H/HeJ, BALB/c, and C57BL/6J were fed either a low–fat or a high–fat diet and injected intraperitoneally with 1 microg Escherichia coli lipopolysaccharide. These parameters were not different in C3H/HeJ mice fed a high–fat diet compared with controls fed a low–fat diet. Total liver catalase activity and peroxisomal β–oxidation were higher in BALB/c mice fed high–fat diet compared with low–fat controls. Total liver catalase activity, catalase mass and peroxisomal β–oxidation increased to the greatest extent in C57BL/6J mice fed a high–fat diet. Endotoxin treatment did not alter any of the parameters in mice fed a low–fat diet. Among mice fed a high–fat diet, endotoxin did not affect hepatic catalase or peroxisomal β–oxidation in the C3H/HeJ mice, decreased catalase activity in BALB/c mice (28%), and greatly decreased both catalase (66%) and peroxisomal β–oxidation (69%) in C57BL/6J mice. The decrease in catalase activity in C57BL/6J mice was apparently because of an inactivation of the enzyme as determined by the activity/mass ratio. Thus, endotoxin is showed to inhibit both catalase and peroxisomal β–oxidation in mice and the sensitivity to endotoxin is greatest in C57BL/6J mice fed a high–fat diet.
Cyclosporine Inhibits Catabolism of Low–Density Lipoproteins in Hepg2 Cells by About 25%
Authors: Rayyes O A , Wallmark A , Florén C
Published: November 2004

The aim of this study was to elucidate the possible causes of elevated low–density lipoprotein (LDL)–cholesterol levels in transplanted patients treated with the immunosuppressant drug, cyclosporine. HepG2 cells, from a well–differentiated cell–line of hepatoma cells, were cultured and used as a model for

in vitro

hepatocytic LDL uptake. Different concentrations of cyclosporine, which were within the range of concentrations found in humans treated with cyclosporine, were added to tissue culture medium together with 125I–LDL. The results showed that cyclosporine reduced LDL uptake and degradation in HepG2 cells by about 25%. The cells were also pretreated with cyclosporine for 1 to 24 hours and then incubated with new medium containing labeled LDL for 2 hours at 4 degrees C in an LDL–binding assay. The data showed that cyclosporine reduced the subsequent LDL binding. Cyclosporine has no toxic effects on HepG2 cells, as shown by unchanged growth capacity of the cells. By means of a 50–fold excess of unlabeled LDL, a monoclonal anti–LDL receptor antibody, and dextran sulfate, we also evaluated if this inhibition of LDL binding occurred through the LDL receptor– mediated pathway, through non–LDL receptor–mediated pathways, or through both. The results show that cyclosporine reduces LDL binding and uptake by mainly inhibiting the LDL receptor–mediated pathway. We also studied the effect of the LDL–cyclosporine complex on the binding of labelled LDL. The presence of cyclosporine in the LDL particle does not influence the binding behaviour of LDL to its receptor. We also found that cyclosporine reduces the expression of the LDL receptor messenger RNA (mRNA) by about 40%. Thus, the interpretation of this study is that cyclosporine can cause an increase in LDL–cholesterol in the plasma of transplantation patients by reducing the catabolism of LDL in the liver by inhibiting mainly the LDL receptor–mediated catabolism through an effect on LDL receptor synthesis.
Stimulation of Prostaglandin Synthesis in Cultured Liver Cells by Ccl4
Authors: Johnston D E , Kroening C
Published: November 2004

The contribution of hepatocytes to liver prostaglandin synthesis is controversial, partly because hepatocytes of varying purity have been studied. In this study, prostaglandin synthesis was examined in conventional and ricin–purified rat hepatocytes that were incubated with gaseous carbon tetrachloride as a model stimulus for lipid peroxidation and prostaglandin synthesis. Hepatocytes that were incubated for 2 hours with 1 ml, of liquid CCl4/5 L gas volume showed no evidence of cell death or injury. However, twice this volume of CCl4 caused total cell death. Enzyme immunoassay (EIA) failed to detect prostaglandin E2 (PGE2) synthesis in hepatocyte cultures after the first day in culture, under a variety of cell culture conditions. Conventional hepatocyte cultures, but not ricin–purified hepatocytes, synthesized thromboxane B2 (approximately 300 pg/mg protein) and prostaglandin D2 (PGD2) (range, 1,000–6,000 pg/mg protein). Conventional hepatocyte cultures released prostaglandin F2α; (PGF2α) immunoreactivity (ranging from approximately 900 to 1,800 pg/mg protein). Ricin–purified hepatocyte cultures made at least half as much PGF2α immunoreactivity as did corresponding conventional hepatocytes. However, cyclooxygenase inhibitors did not inhibit the portion of PGF2α immunoreactivity made by ricin–purified hepatocytes. PGF2α immunoreactivity released by ricin–purified hepatocytes cochromatographed with PGF2 α, and probably represents F2–isoprostanes resulting from lipid peroxidation in hepatocytes. F2– isoprostane release was detected by immunoassay. Conventional cultures of rat hepatocytes contain Kupffer and endothelial cells, which can synthesize significant amounts of cyclooxygenase products. Highly purified hepatocytes do not produce cyclooxygenase products, even with a maximal stimulus to lipid peroxidation. CCl4 causes the release of F2–isoprostanes from hepatocytes with or without observable cell injury, as detected by Trypan blue exclusion or lactate dehydrogenase (LDH) release.
Endotoxin Stimulates Hydrogen Peroxide Detoxifying Activity in Rat Hepatic Endothelial Cells
Authors: Spolarics Z , Stein D S , Garcia Z C
Published: November 2004

The study aimed to assess the effect of lipopolysaccharide (LPS)

in vivo

(from

Escherichia coli

, 2 mg/kg body weight intraperitoneally) on the production and elimination of hydrogen peroxide (H2O2) in rat hepatic endothelial and Kupffer cells. Twenty–two hours after the injection of LPS, hepatic cells were isolated by collagenase and pronase digestion followed by centrifugal elutriation, and cell– associated H2O2 was determined by flow cytometry analysis using 2′,7′– dichloroflorescin diacetate (DCF–diacetate). LPS treatment did not alter the basal or phorbol myristate acetate–stimulated levels of H2O2– related fluorescence in endothelial cells; however, it doubled phorbol myristate acetate–stimulated fluorescence in Kupffer cells. Administration of varying concentrations of H202 (range, 10-7 – 10-4 mol/L) in vitro caused a significantly delayed increase in fluorescence in endothelial cells from endotoxemic rats as compared with cells from saline–injected animals. The 50% effective concentration of H2O2 was found at 1.1 × 10-6 and 8.1 × 10-6 mol/L on endothelial cells after saline and LPS treatment, respectively. No differences were detected in H2O2–stimulated fluorescence between resting and LPS–stimulated Kupffer cells. Administration of varying glucose concentrations in vitro significantly decreased the H2O2– stimulated fluorescence in endothelial and Kupffer cells from LPS– injected animals. Inhibition of nitric oxide synthase by in vitro administration of NG–monomethyl–L–arginine (L–NNMMA) did not alter the H2O2– or phorbol myristate acetate–stimulated responses in endothelial and Kupffer cells. As shown earlier, LPS stimulates the gene expression of GLUT1 glucose transporter, glucose–6–phosphate dehydrogenase (G6PD), superoxide dismutases, and glutathione peroxidase in hepatic endothelial cells. The present data indicate that the LPS–induced metabolic alterations are accompanied by an increased H2O2–detoxifying capacity in hepatic endothelial cells. This may represent a protective mechanism against exogenous oxidative stress caused by activated hepatic phagocytes during inflammation. Our observations are consistent with primed production of reactive oxygen species (ROS) in LPS–activated Kupffer cells.
Mutation in Hbv Rna–Dependent Dna Polymerase Confers Resistance to Lamivudine In Vivo
Authors: Tipples G A , Ma M M , Fischer K P , Bain V G , Kneteman N M , Tyrrell D L
Published: November 2004

The (–) enantiomer of 3′–thiacytidine (lamivudine) has been found to be a potent inhibitor of hepatitis B virus (HBV) and human immunodeficiency virus (HIV) replication. Mutation of methionine to valine or isoleucine at the YMDD (tyrosine, methionine, aspartate, aspartate) motif of the HIV reverse transcriptase has been shown to be responsible for lamivudine resistance in HIV. The hepadnaviruses also have the YMDD motif in their DNA polymerase. Therefore, it is possible that hepadnaviruses could develop lamivudine resistance by a similar mutation at this motif. We analyzed the HBV from a liver transplantation patient who developed recurrent HBV viremia during lamivudine treatment. The polymerase gene was amplified by polymerase chain reaction (PCR), and the region coding for the YMDD motif was sequenced. The pretreatment HBV sequence coded for YMDD, while the lamivudine–resistant mutant HBV coded for YIDD (tyrosine, isoleucine, aspartate, aspartate). With the documented changes in the YMDD motif of lamivudine–resistant HIV, it is likely that the methionine–to– isoleucine mutation in the YMDD motif of the HBV polymerase contributes significantly to the lamivudine–resistance of HBV isolated from this patient.
Accumulation and Persistence of Hepatitis B Virus Core Gene Deletion Mutants in Renal Transplant Patients Are Associated With End–Stage Liver Disease
Authors: Günther S , Baginski S , Kissel H , Reinke P , Krüger D H , Will H , Meisel H
Published: November 2004

In renal transplant recipients, chronic hepatitis B virus (HBV) infection often leads to cirrhosis and liver failure. In this study, we investigated whether or not in these patients viral variants would emerge despite immunosuppression, and whether they are associated with a specific course of liver disease. In a population of 552 renal transplant recipients hepatitis B 24 surface antigen (HBsAg)–positive patients were available for a 2–year follow–up. By polymerase chain reaction (PCR) and DNA sequencing, HBV genomes with deletions in the middle of the core gene (C–gene) were found in 9 out of the 24 patients. Seven of the 9 patients (group I) showed either persistent or increasing amounts of these variants; all patients had cirrhosis, and 5 died of end–stage liver disease. The viral variants emerged at least 1 year before liver failure. In 2 out of the 9 patients, the core deletion variants disappeared, and no further deterioration of the liver function was observed thereafter. In the remaining 15 patients (group II) without deletion mutants detected at any time, only 3 had cirrhosis (P< .001, group I vs. II), and none died (P< .001). Between both groups, there were no statistically significant differences in the other relevant variables that were examined. These results indicate that HBV C–gene deletion mutants can accumulate in long–term immunosuppressed patients, and that their persistence is associated with progressive liver disease. The accumulation of these variants may be caused by the development of cirrhosis or could be involved in hepatopathogenesis.
Cellular Target of Cyclophosphamide Toxicity in the Murine Liver: Role of Glutathione and Site of Metabolic Activation
Authors: DeLeve L D
Published: November 2004

Hepatic venoocclusive disease (HVOD) is caused by the disruption of the microcirculation by an as–yet unknown mechanism. Previous

in vitro

studies with azathioprine, monocrotaline, and dacarbazine suggested that toxins that cause HVOD initially causing HVOD target sinusoidal endothelial cells (SEC) perhaps via profound glutathione (GSH) depletion. The current study examines cyclophosphamide toxicity in SEC and hepatocytes, as well as the interplay between the two cell types. Cyclophosphamide was not directly toxic to SEC, but in coculture of SEC and hepatocytes, cyclophosphamide was significantly more toxic to SEC. Two cyclophosphamide metabolites, 4–hydroperoxycyclophosphamide and acrolein, were equally toxic to SEC, and toxicity occurred at 20–fold–lower concentrations than in hepatocytes. 4–Hydroperoxycyclophosphamide depleted GSH by greater than 95% before inducing cell death in SEC. When hepatocyte–GSH levels were sustained with supplemental methionine and serine in coculture, toxicity in both cell types was diminished. In coculture, SEC are significantly more susceptible than hepatocytes to cyclophosphamide toxicity, and this is likely caused by acrolein generated by the hepatocyte. As seen with other toxins implicated in HVOD, the profound depletion of SEC GSH precedes the onset of toxicity. The degree of cyclophosphamide toxicity induced in SEC is determined by both metabolic activation and GSH detoxification in the hepatocytes.
Increasing Hepatic Cholesterol 7α–Hydroxylase Reduces Plasma Cholesterol Concentrations in Normocholesterolemic and Hypercholesterolemic Rabbits
Authors: Xu G , Salen G , Shefer S , Ness G C , Nguyen L B , Tint G S , Parker T S , Roberts J , Batta A K , Chen T S , Zhao Z , Kong X
Published: November 2004

The effect of bile acid depletion and replacement with glycodeoxycholic acid on plasma cholesterol concentrations, hepatic low–density lipoprotein (LDL) receptor binding and messenger RNA (mRNA) levels, and hepatic activities and mRNA levels for 3–hydroxy–3–methylglutaryl coenzyme A (HMG–CoA) reductase and cholesterol 7α–hydroxylase was investigated in 19 New Zealand white (NZW) and 15 Watanabe heritable hyperlipidemic (WHHL) rabbits. Bile acid depletion was produced by external bile drainage for 5 days, which maximized cholic acid synthesis. Replacement was achieved by infusing glycodeoxycholic acid intraduodenally for 24 hours so that the hepatic bile acid flux reached prefistula levels. Plasma and liver cholesterol concentrations were 13 times and 50% greater, respectively, hepatic LDL receptor–mediated binding was 26% less, and cholesterol 7α–hydroxylase activity and mRNA levels were 62% and 86% less in WHHL than NZW rabbits. After bile drainage, plasma cholesterol concentrations decreased 29% in NZW rabbits and 40% in WHHL rabbits and were associated with a 2.1–fold increase in hepatic LDL receptor–mediated binding in the NZW rabbits, but there was no change in the WHHL rabbits. Cholesterol 7α– hydroxylase activity and mRNA levels increased three and four times in NZW and WHHL rabbits, respectively, although liver cholesterol levels remained unchanged. Replacement with exogenous glycodeoxycholic acid increased plasma cholesterol concentrations 1.7 times in NZW rabbits and decreased enhanced cholesterol 7α–hydroxylase activity 54%, mRNA levels 86%, cholic acid synthesis 38%, and hepatic LDL receptor– mediated binding 57% in NZW rabbits. Bile acid depletion stimulated cholic acid synthesis by up–regulating cholesterol 7α–hydroxylase to use cholesterol and reduce plasma concentrations substantially in both NZW and WHHL rabbits, although LDL receptors did not function in WHHL rabbits. Glycodeoxycholic acid replacement inhibited elevated cholesterol 7α–hydroxylase, cholic acid synthesis, and hepatic LDL receptor binding to reestablish baseline plasma cholesterol levels in NZW rabbits. Hypercholesterolemia in WHHL rabbits was related to the combination of dysfunctional LDL receptors and inhibited cholesterol 7α–hydroxylase. Plasma cholesterol concentrations were reduced significantly when cholesterol 7α–hydroxylase was stimulated even in the absence of LDL receptor function.
Monoclonal Antibody Against Lymphocyte Function–Associated Antigen 1 Inhibits the Formation of Primary Biliary Cirrhosis–Like Lesions Induced by Murine Graft–Versus–Host Reaction
Authors: Kimura T , Suzuki K , Inada S , Hayashi A , Isobe M , Matsuzaki Y , Tanaka N , Osuga T , Fujiwara M
Published: November 2004

Interaction between intercellular adhesion molecule 1 (ICAM–1) and lymphocyte function–associated antigen 1 (LFA–1) might be involved in the pathogenesis of liver diseases. We investigated whether monoclonal antibodies (mAbs) against these two adhesion molecules could inhibit the formation of primary biliary cirrhosis (PBC)–like lesions in an animal model using graft–versus–host reaction (GVHR) with major histocompatibility complex class II disparity. PBC–like hepatic lesions such as cellular infiltration of portal area and nonsupprative destructive cholangitis (NSDC) were generated by injecting spleen T cells of C57BL/6 (B6) mice into (B6. C–H–2bm12 X B6) F1 mice. In the liver of these mice, increased number of LFA–1–positive cells and enhanced expression of ICAM–1 on sinusoidal endothelial cells and bile duct epithelial cells were observed immunohistochemically, when compared with F1 mice without GVHR. Hepatic lesions of these mAb–treated mice were almost completely inhibited in these mice compared with GVHR mice. Furthermore, we studied to determine which anti–LFA–1 mAb or anti–ICAM–1 mAb was essential to inhibit the hepatic lesions. Mice solely treated with anti–LFA–1 mAb showed significant inhibition of hepatic lesions, whereas treatment with anti–ICAM–1 mAb could not inhibit the lesions. Despite the inhibition of hepatic lesions, induction of GVHR and production of antimitochondrial antibodies were not impaired in mAb–treated mice. We conclude that LFA–1 mediates cell infiltration into the liver in this murine model of GVHR and suggest a possible therapeutic role of mAbs to this adhesion molecule in selective autoimmune liver diseases.
Glutamine, Myo –Inositol, and Organic Brain Osmolytes After Portocaval Anastomosis in the Rat: Implications for Ammonia–Induced Brain Edema
Authors: Córdoba J , Gottstein J , Blei A T
Published: November 2004

Brainmyo–inositol, an organic osmolyte, is decreased in cirrhotic patients with hepatic encephalopathy but appears unchanged in fulminant hepatic failure. An osmoregulatory response to the increase in brain glutamine may explain the decrease in brainmyo–inositol; if this is the case, organic osmolytes may account for differences in the development of brain edema seen in acute or chronic liver failure. The response ofmyo–inositol and nine other organic osmolytes to the increase in brain glutamine at different time intervals after portacaval anastomosis (PCA) in the rat was studied. Organic osmolytes were measured in brain tissue and cerebrospinal fluid. Water in cerebral cortex was measured after ammonia infusion with the gravimetric method. Six weeks after PCA, despite an increase in brain glutamine (PCA, 16.4 ± 2 mmol.kg wt–1.kg wt–1; sham, 5 ± 1 mmol.L–1.kg wt–1), the content of total organic osmolytes did not increase (PCA, 44.1 ± 3; sham, 43 ± 4) because of a decrease of other osmolytes (myo–inositol, 54%; urea, 39%; taurine, 33%; and glutamate, 8%). Brainmyo–inositol was lower at 3 weeks (3.4 ± 0.5 kg wt–1) than at 1 day after PCA (4.7 ± 0.5 kg wt–1). An ammonia infusion resulted in brain edema at both time points. In conclusion, the reduction in brainmyo–inositol in PCA rats is accompanied by the decrease of other organic osmolytes, supporting the view that changes inmyo–inositol reflect an osmoregulatory response. The decrease in brainmyo–inositol is more marked as time elapses after PCA. In a model in which short–term and large doses of ammonia were infused, the decrease in brainmyo–inositol did not prevent the development of brain swelling. Understanding brain osmoregulatory mechanisms may provide new insights into hepatic encephalopathy and brain edema in fulminant hepatic failure.
Fe–Nitrilotriacetic Acid—Binding Proteins Associated With Rat Liver Plasma Membrane
Authors: Barisani D , Wessling-Resnick M
Published: November 2004

The uptake of nontransferrin–bound iron by hepatocytes is known to occur and may contribute to the deposition of iron and resulting injury during hemochromatosis. To examine the proteins that may function in the transport of nontransferrin–bound iron, the properties of FeNTA– binding to rat liver basolateral plasma membranes were characterized. The binding of 55FeNTA to purified liver basolateral plasma membranes was measured using a simple centrifugation assay. The binding activity could be solubilized with 0.1% octylglucoside; apparent molecular weight Mapp approximately 210 kd for the binding complex was determined by gel filtration chromatography. Immobilized metal affinity chromatography was used to further purify binding protein(s) from rat liver plasma membranes and at least six polypeptides were identified by silver staining. If associated in a stoichiometric complex, the molecular mass of these proteins would predict a size of approximately 227 kd in fairly close agreement with the gel filtration experiments. The characterization of FeNTA–binding proteins associated with basolateral membranes is the first step towards understanding elements responsible for the uptake of nontransferrin–bound iron by the liver.
Phylogenetic Analysis of Hepatitis C Virus Isolates and Their Correlation to Viremia, Liver Function Tests, and Histolog
Authors: Zeuzem S , Franke A , Lee J , Herrmann G , Rüster B , Roth W K
Published: November 2004

Nucleotide sequence analysis of hepatitis C virus (HCV) strains showed substantial variability leading to a classification into several genotypes and subtypes. The data correlating HCV genotypes and subtypes with hepatitis C viremia levels, demographic characteristics of patients (age, mode of transmission, duration of infection), and severity of liver disease are conflicting. The interpretation of several studies is further complicated because the molecular methods used lacked specificity for genotyping/subtyping and underestimated viremia levels, especially in patients infected with HCV genotypes 2 and 3. In the present study we investigated 97 consecutive patients with chronic hepatitis C using molecular “gold standard” methods. HCV subtyping was performed by sequence and phylogenetic analysis of the nonstructural (NS)–5 region and serum HCV–RNA concentration was assessed by a validated genotype–independent quantitative reverse– transcription–polymerase chain reaction assay using an internal RNA standard. Patients infected with subtypes HCV–1b, HCV–2a–c, and HCV–4 were older than patients infected with HCV–1a and HCV–3a. Serum HCV–RNA levels ranged from 1.5 × 104 to 1.0 × 108 copies/mL with no significant differences between median serum HCV–RNA concentrations in patients infected with different genotypes/subtypes. Although patients infected with HCV–1b were older, no biochemical or histological evidence was obtained that this subtype is associated with more severe liver disease. Furthermore, the present study showed a lack of correlation between the serum HCV–RNA concentration, biochemical parameters, and liver histology. The median serum HCV–RNA levels in patients with chronic persistent hepatitis, chronic active hepatitis, and liver cirrhosis were 5.0 × 106 copies/mL, 2.5 × 106 copies/mL, and 5.0 × 106 copies/mL, respectively. These differences were not significant. In conclusion, using optimized and validated molecular techniques, the present cross–sectional study showed no correlation between HCV genotypes/subtypes, viremia, liver function test results, and histology.
Analysis of the Tumorigenicity of the X Gene of Hepatitis B Virus in A Nontransformed Hepatocyte Cell Line and the Effects of Cotransfection With A Murine P53 Mutant Equivalent to Human Codon 249
Authors: Oguey D , Dumenco L L , Pierce R H , Fausto N
Published: November 2004

Chronic infection with hepatitis B virus (HBV) is associated with the development of human hepatocellular carcinoma (HCC). One of the HBV genes, HBx, may have transforming potential, but this issue is still the subject of controversy. One of the major difficulties in addressing this question is the lack of a suitable in vitro model. We used a nontransformed, differentiated murine hepatocyte cell line (AML12) to transfect the HBx gene and examine its transforming capabilities. Because mutations of the p53 gene, in particular at codon 249, have been implicated in HCC development in geographical areas with high incidence of the tumor, we also studied the putative cooperative role in transformation between HBx and mutated p53 by cotransfecting HBx with a murine p53 mutant equivalent to human ser249 (ser246p53). Transfection with HBx plasmids containing the HBx gene under the control of two different promoters resulted in fewer colonies than in control plasmids. The toxic effect of HBx on colony formation was abolished by cotransfection with 246p53, suggesting that the inhibitory effect requires functionally intact p53. Clonal cell lines that stably expressed HBx messenger RNA (mRNA) (HBX lines) were tested for their growth characteristics and their ability to grow in soft agar and form tumors in nude mice. At passages 19–27 after transfection, one of four HBx–expressing lines showed the capacity for anchorage–independent growth in soft agar and produced poorly differentiated hepatocellular carcinomas in 8 of 13 sites of injection in nude mice. HBX lines as well as clonal cell lines of cells transfected with 246p53 (246 cell lines), cotransfected with HBx and 246p53 (246x lines) or transfected with control plasmids, were analyzed by flow cytometry to determine the fraction of cells in S phase (SPF). 246 and 246X lines had similar SPFs that were approximately twofold greater than control or HBX lines. 246x lines showed morphological changes in culture such as marked cellular heterogeneity, cell crowding, and the presence of multinucleated giant cells, but their tumorigenicity was not increased compared with the HBX lines. These data show that HBx has a weak tumorigenicity in murine hepatocytes and that the addition of mutation of p53 at codon 249 to HBx expression does not increase tumorigenicity in AML12 cells.
Membrane–Bound Carbonic Anhydrase Iv Is Expressed in the Luminal Plasma Membrane of the Human Gallbladder Epithelium
Authors: Parkkila S , Parkkila A , Juvonen T , Waheed A , Sly W S , Saarnio J , Kaunisto K , Kellokumpu S , Rajaniemi H
Published: November 2004

Alkaline hepatic bile is acidified in the gallbladder to prevent calcium precipitation and gallstone formation. Because membrane–bound carbonic anhydrase (CA) isoenzyme IV participates with cytoplasmic CA II in the acidification of urine in the kidney, we studied its expression in different regions of the human biliary tract using immunohistochemical techniques. The enzyme was expressed in the apical plasma membrane of the gallbladder epithelial cells and in the endothelium of the subepithelial capillaries. In the liver, some epithelial cells of the large bile ducts showed positive staining. Its presence in the gallbladder epithelium could be confirmed by Western blotting, which showed a single 35–kd polypeptide band, corresponding in molecular weight to the intact enzyme. The majority of the enzyme was phased to Triton X–114 detergent phase. A small amount of 35–kd polypeptide was also seen in the water phase. Smaller proteolytic fragments of the enzyme were not seen, suggesting that the tissue sample was well preserved. The results show that CA IV is expressed in abundance in the human gallbladder epithelium, where it may participate together with cytoplasmic CA II and ion transporters in acidification of the gallbladder bile via bicarbonate reabsorption.
Increased Blood Manganese in Cirrhotic Patients: Relationship to Pallidal Magnetic Resonance Signal Hyperintensity and Neurological Symptoms
Authors: Spahr L , Butterworth R F , Fontaine S , Bui L , Therrien G , Milette P C , Lebrun L H , Zayed J , Leblanc A , Pomier-Layrargues G
Published: November 2004

Increasing evidence suggests that manganese deposition is responsible for the T1–weighted magnetic resonance imaging (MRI) signal hyperintensity consistently observed in pallidum of cirrhotic patients. However, the relationship between blood manganese and the etiology or severity of liver disease, as well as the neurological symptomatology in these patients, has not been well established. In the present study, blood manganese concentrations were measured by atomic absorption spectrometry together with MRI and neurological evaluation in 57 cirrhotic patients with various etiologies and severity of liver disease. Blood manganese concentrations were elevated in 67% of cirrhotic patients and were significantly higher in patients with previous portacaval anastomoses or transjugular intrahepatic portosystemic shunt (TIPS). Pallidal signal hyperintensity was observed in 88% of patients, and significant correlations were demonstrated between blood manganese and pallidal index (PI) (a measure of pallidal signal hyperintensity), as well as Child–Pugh score. Assessment of extrapyramidal symptoms using the Columbia rating scale revealed a significant incidence of tremor, rigidity, or akinesia in up to 89% of cirrhotic patients. However, there was no significant correlation between blood manganese and extrapyramidal symptoms, although severity of akinesia was significantly greater in Child–Pugh C patients. Extrapyramidal symptoms could result from a toxic effect of manganese on basal ganglia dopaminergic function. These findings further support a role for manganese in the etiology of pallidal MRI signal hyperintensity in patients with chronic liver disease.
Hepatic Regeneration Is Associated With Preservation of Microsomal Glucuronidation
Authors: Zakko W F , Green R M , Gollan J L , Berg C L
Published: November 2004

Significant controversy exists regarding the regulation of glucuronidation during the process of hepatic regeneration. We used a partial hepatectomy rat model to elucidate the effects of hepatic regeneration on the various components of the microsomal glucuronidation system. Hepatic microsomes were prepared by standard sucrose density centrifugation, coupled with a modified technique involving Percoll centrifugation. Microsomal uridine diphosphate (UDP)–glucuronosyltransferase (UGT) protein expression and UGT messenger RNA (mRNA) levels were measured by Western and Northern blotting. UGT enzyme activity was determined toward two prototypical aglycones,

p

–nitrophenol and estrone, in intact and digitonin–treated microsomes. Microsomal uptake of the cosubstrate for all glucuronidation reactions, UDP–glucuronic acid (UDP–GlcUA), was determined using a rapid–filtration assay. Microsomal enrichment after hepatectomy was preserved only when the Percoll method was used. Microsomal UGT protein expression and UGT mRNA levels were unaltered after hepatectomy. UGT enzyme activity toward estrone was unchanged 1 day posthepatectomy compared with sham laparotomy controls. Similarly,

p

–nitrophenol glucuronide formation was unaffected by hepatic regeneration 1, 2, and 5 days posthepatectomy when digitonin–treated microsomes were used. Glucuronidation of p–nitrophenol in intact microsomes was increased in partial hepatectomy compared with sham–operated controls at 1 and 2 days. This increase was not attributable to changes in microsomal UDP–GlcUA uptake, which was comparable in both groups. We conclude that microsomal glucuronidation, in contrast to other well characterized hepatic metabolic functions, is highly preserved during liver regeneration.
Qualitative and Quantitative Analysis of Agnor Proteins in Chemically Induced Rat Liver Carcinogenesis
Authors: Trere D , Derenzini M , Sirri V , Montanaro L , Grigioni W , Faa G , Columbano G M , Columbano A
Published: November 2004

A qualitative and quantitative analysis of silver–stained nuclear organizer regions (AgNOR) proteins was performed during hepatocarcinogenesis induced in rats initiated by diethylnitrosamine (DENA) using the resistant–hepatocyte model. Nuclear proteins from control hepatocytes, hyperplastic nodules, and hepatocellular carcinomas (HCC) separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis were transferred to nitrocellulose membranes and specifically silver–stained for AgNOR proteins. No difference was observed in the distribution pattern of the silver–stained bands among control, hyperplastic, or cancer cells. The same was true if human cirrhosis and HCC were compared. The evaluation of individual AgNOR protein amounts by computerized densitometric analysis showed that (1) the integrated optical density value of the total AgNOR proteins was greatest in cancer cells, lesser in hyperplastic hepatocytes, and lowest in control hepatocytes, and (2) the amount of the two major silver–stained proteins, nucleolin (105 kd) and protein B23 (39 kd), was always a constant percentage of total AgNOR proteins. An experiment using bromodeoxyuridine incorporation showed that, during hepatocarcinogenesis, AgNOR protein quantity progressively increased and was significantly related to the increased hepatocyte labeling index. These results show that AgNOR protein distribution changes during hepatocarcinogenesis are caused neither by the synthesis of new AgNOR proteins nor by an unbalanced synthesis of individual AgNOR proteins, but to an increased synthesis of nucleolin and protein B23, which is associated with a progressive increased hepatocyte proliferation rate.
Phospholipase D Activation in Hepatocyte Growth Factor–Stimulated Rat Hepatocytes Mediates the Expressions of C– Jun And C– Fos : Involvement of Protein Tyrosine Kinase, Protein Kinase C, and Ca2+
Authors: Adachi T , Nakashima S , Saji S , Nakamura T , Nozawa Y
Published: November 2004

Hepatocyte growth factor (HGF) activated phospholipase D (PLD) in primary–cultured rat hepatocytes, as assessed by the formation of phosphatidylbutanol (PBut), a specific and stable product of PLD activity in the presence of 0.3% butanol. PLD hydrolyzes phosphatidylcholine to choline and phosphatidic acid (PA), which is further metabolized to diacylglycerol (DG) by PA phosphohydrolase (PAP). In HGF–stimulated hepatocytes, butanol prevented the formation of PA and DG. A PAP inhibitor, propranolol, inhibited DG production with a reciprocal increase of PA, implying that PLD played a role in the formation of not only PA but DG. Inhibitors for protein kinase C (PKC), Ro31–8425, H–7, and calphostin C, reduced HGF–induced PLD activation. A protein tyrosine kinase (PTK) inhibitor, genistein but not its inactive analogue daidzein, inhibited PLD activation by HGF. Moreover, depletion of extracellular Ca2+ by omission of Ca2+ or by chelating residual Ca2+ with ethyleneglycol–bis(β–aminoethyl ether)–

N,N

,N′,N′–tetraacetic acid (EGTA) abolished HGF–induced PLD activation. HGF, phorbol myristate acetate (PMA) and a DG analog, oleylacetylglycerol (OAG), activated the expression of c–jun and c–fos messenger RNAs (mRNAs). Ro31–8425, calphostin C, and genistein, which prevented HGF–induced PLD activation, inhibited induction of these immediate early genes. Butanol and propranolol at concentrations which effectively inhibited the formation of DG, suppressed HGF–induced expression of c–

jun

and c–

fos

mRNAs. However, HGF–induced mitogen–activated protein kinase (MAPK) activation was not affected by both butanol and propranolol. These results suggest that PTK, PKC, and Ca2+ regulate HGF–induced PLD activation, and that DG produced by PLD pathway may play a role in the induction of immediate early genes, which is activated in MAPK–independent manner, in rat hepatocytes.
When Is A Carrier Not A Membrane Carrier? the Cytoplasmic Transport of Amphipathic Molecules
Authors: Weisiger R A
Published: November 2004

After entering the cell, small molecules must penetrate the cytoplasm before they are metabolized, excreted, or can convey information to the cell nucleus. Without efficient cytoplasmic transport, most such molecules would efflux back out of the cell before they could reach their targets. Cytoplasmic movement of amphipathic molecules (e.g., long–chain fatty acids, bilirubin, bile acids) is greatly slowed by their tendency to bind intracellular structures. Soluble cytoplasmic binding proteins reduce this binding by increasing the aqueous solubility of their ligands. These soluble carriers catalyze the transport of hydrophobic molecules across hydrophilic water layers, just as membrane carriers catalyze the transport of hydrophilic molecules across the hydrophobic membrane core. They even display the kinetic features of carrier–mediated transport, including saturation, mutual competition between similar molecules, and countertransport. Recent data suggest that amphipathic molecules cross the cytoplasm very slowly, with apparent diffusion constants 102 to 104 times smaller than in water. By modulating the rate of cytoplasmic transport, cytosolic binding proteins may regulate transport and metabolism of amphipathic molecules. Storage diseases may cause hepatocellular dysfunction by disrupting normal cytoplasmic transport.
Treatment of Nodular Hepatocellular Carcinoma Larger Than 3 Cm With Ultrasound–Guided Percutaneous Acetic Acid Injection
Authors: Ohnishi K , Yoshioka H , Ito S , Fujiwara K
Published: November 2004

To assess the efficacy of ultrasound–guided percutaneous acetic acid (15%, 20%, 30%, 40%, and 50% in concentration) injection for a large nodular hepatocellular carcinoma (HCC), percutaneous acetic acid injection was performed using 15% to 50% acetic acid in 28 patients who had one single nodular HCC larger than 3 cm or one main tumor with fewer than two HCCs smaller than 3 cm during the past 7.5 years; the main tumor was slightly hypervascular in nine patients and was highly hypervascular, showing viable cancer cells despite transcatheter arterial embolization in 19 patients. During the series of treatment sessions for each patient, the same concentration of acetic acid was used. Eight (89%) of nine slightly hypervascular main tumors, 18 (95%) of 19 highly hypervascular main tumors, and all 14 small tumors were treated successfully. No serious complications occurred as a direct sequela to acetic acid injection. The 1–, 2– and 3–year recurrence rate from the original main tumor was 0%, 20%, and 20%, respectively, in patients treated with percutaneous acetic acid injection alone and 12%, 24%, and 24% in patients treated with percutaneous acetic acid injection after transcatheter arterial embolization (

P

= NS). The 1–, 2–, 3–, 4–, and 5–year survival rates were 100%, 80%, 80%, 80%, and 0%, respectively, in the former and 90%, 81%, 81%, 61%, and 61% in the latter (

P

= NS). Percutaneous acetic acid injection (15% to 50%) may be an effective therapy for slightly hypervascular nodular HCCs larger than 3 cm and highly hypervascular nodular HCCs larger than 3 cm that remain partly viable after transcatheter arterial embolization.
Prothrombin Time in Liver Failure: Time, Ratio, Activity Percentage, or International Normalized Ratio
Authors: Robert A , Chazouilleres O
Published: November 2004

Prothrombin time (PT) is a universal indicator of liver disease severity. However, variability in thromboplastin reagents leads to large interlaboratory differences in PT results. The aim of this study was to determine whether the use of the international normalized ratio (INR) or other modes of expression might achieve PT standardization in patients with liver failure. PT was measured with seven thromboplastin reagents with different sensitivities in plasmas from 27 patients with miscellaneous chronic and acute liver failure and, as a control population, 29 patients on oral anticoagulation therapy. PT was expressed in seconds, ratio, activity percentage, and INR. In patients with liver failure, only activity percentage expression eliminated variability in PT results obtained with the seven thromboplastins while INR, seconds, and ratio values remained significantly different (

P

< .01). In patients on oral anticoagulant therapy, only INR normalized PT results. We conclude that, in patients with liver failure, INR fails to yield a PT expression independent of the thromboplastin used and only activity percentage expression may provide a common international scale of PT reporting.
Ascitic Fluid Adenosine Deaminase Insensitivity in Detecting Tuberculous Peritonitis in the United States
Authors: Hillebrand D J , Runyon B A , Yasmineh W G , Rynders G P
Published: November 2004

Tuberculous peritonitis, although common in Third World countries, remains an uncommon cause of ascites in the United States. Ascitic fluid adenosine deaminase (ADA) activity has been proposed as a useful diagnostic test. The aim of this retrospective study was to determine the clinical utility of ascitic fluid ADA activity in diagnosing tuberculous peritonitis in a U.S. patient population. A total of 368 ascitic fluid specimens from a well–characterized ascitic fluid bank, including tuberculous peritonitis (n = 7), tuberculous peritonitis in the setting of cirrhosis (n = 10), and consecutive specimens of widely varied etiologies (n = 351) were analyzed for ADA activity by ultraviolet spectrophotometry at 265 nm. The overall sensitivity of the ADA determination in diagnosing tuberculous peritonitis was only 58.8%, and the specificity was 95.4%. The accuracy of ADA determination (93.8%) compared favorably with that of the common ascitic fluid tests of white blood cell (WBC) count (>500/mm3), total protein (>2.5 g/dL), and combined WBC count and total protein (45.8%, 74.4%, and 81.3%, respectively). However, ADA was only 30% sensitive in detecting tuberculous peritonitis in the setting of cirrhosis, and cirrhosis was present in 59% of the tuberculous peritonitis patients in our population. In addition, malignancy–related ascites (13%) and bacterial peritonitis specimens (5.8%) occasionally yielded false–positive results. In conclusion, our results indicate that the ascitic fluid ADA activity has good accuracy but poor sensitivity and imperfect specificity in a U.S. patient population in which the prevalence of tuberculosis is low and underlying cirrhosis is common.
Autoreactive Cd4+ Lkm–Specific and Anticlonotypic T–Cell Responses in Lkm–1 Antibody–Positive Autoimmune Hepatitis
Authors: Lohr H F , Schlaak J F , Lohse A W , Bocher W O , Arenz M , Gerken G , Buschenfelde K M
Published: November 2004

Peripheral blood mononuclear cells (PBMC) of patients with autoimmune hepatitis (AIH) and controls were studied for their proliferative response to six overlapping synthetic peptides covering the 33–amino acid immunodominant region of cytochrome P450IID6, the main target antigen of LKM–1 antibody–positive type II AIH. PBMC from 8 of 8 type II AIH patients (100%), 6 of 12 LKM–1 antibody–negative type I AIH patients (50%), but only 4 of 31 patients with chronic hepatitis C (12.9%) reacted with a 23–amino acid LKM peptide and mainly with a shorter 18–amino acid LKM peptide. Follow–up showed that LKM–specific T–cell responses decreased after immunosuppression had started. Fine specificity, HLA restriction, and cytokine release of LKM–specific T cells were analyzed with 16 CD4+ peptide–specific T–cell lines and 21 CD4+ T–cell clones isolated and expanded from blood and liver tissue of six AIH patients. Activated LKM–specific T cells released interferon gamma (IFN–γ) but no or little interleukin–4. In three AIH patients, PBMC showed specific recognition of autologous LKM–specific T cells, suggesting the presence of a regulatory T–cell network. These T cells also showed the CD4+ phenotype and secreted large amounts of IFN–γ. Furthermore, it was assessed that the regulatory T–cell response is clonotypic. To conclude, we describe a major T–cell epitope in AIH that was recognized by Th1 helper cells isolated from blood and liver tissue. This autoreactive T–cell response correlated widely with disease activity and LKM–1 antibody status and seemed to be regulated by anticlonotypic T cells.
Urinary Excretion of Urodilatin in Patients With Cirrhosis
Authors: Salo J , Jimenez W , Kuhn M , Gines A , Gines P , Fernandez-Esparrach G , Angeli P , Claria J , Bataller R , Arroyo V , Forssmann W , Rodes J
Published: November 2004

Cirrhotic patients with ascites show increased plasma levels of natriuretic peptides from cardiac origin (i.e., atrial natriuretic peptide [ANP] and brain natriuretic peptide [BNP]). Urodilatin is a unique member of the natriuretic peptide family because it is exclusively synthesized in the kidney acting on a paracrine fashion in the regulation of sodium excretion. To investigate the renal production of urodilatin in cirrhosis and its relationship with other natriuretic peptides and sodium retention, urodilatin excretion and plasma levels of ANP were measured in 21 healthy subjects, 13 cirrhotic patients without ascites and 23 cirrhotic patients with ascites. Urine urodilatin was measured with a highly specific radioimmunoassay using a polyclonal antibody against human urodilatin. Patients with ascites had marked sodium retention (UNa 7 ± 2 mEq/d) as compared to patients without ascites and healthy subjects (29 ± 3 mEq/d and 34 ± 5 mEq/d, respectively,

P

< .001). Patients with cirrhosis and ascites had urine urodilatin excretion similar to patients without ascites and healthy subjects (82 ± 8 pmol/g, 95 ± 10 pmol/g, and 89 ± 9 pmol/ g of creatinine, respectively; not significant). In addition, immunoreactive urodilatin from cirrhotic patients with ascites and healthy subjects showed a similar chromatographic pattern. By contrast, plasma ANP levels were increased significantly in patients with ascites (29 ± 3 fmol/mL) as compared with patients without ascites or healthy subjects (14 ± 3 fmol/mL and 6 ± 1 fmol/mL, respectively;

P

< .01). In conclusion, urine urodilatin excretion is normal in patients with cirrhosis even in the presence of marked sodium retention. The coexistence of increased ANP levels and normal urodilatin excretion suggests that in cirrhosis both natriuretic peptides are regulated independently.
Cholesterol 7α–Hydroxylase Activities From Human and Rat Liver Are Modulated In Vitro Posttranslationally by Phosphorylation/Dephosphorylation
Authors: Nguyen L B , Shefer S , Salen G , Chiang J Y , Patel M
Published: November 2004

Purified cholesterol 7α–hydroxylases (C7αH) from human and rat liver microsomes, and from transformed Escherichia coli expression systems, were incubated with 0.3 mmol/L [gamma–32P] adenosine triphosphate (ATP) in the presence and absence of bacterial alkaline phosphatase (AP) or rabbit muscle adenosine 3′,5′–cyclic monophosphate (cAMP)–dependent protein kinase. The amounts of 32P incorporation after separation of human and rat C7αH proteins by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) were related to C7αH catalytic activities (determined by a radioisotope incorporation method) and enzyme protein mass (determined by Western blotting and laser densitometry). Both human and rat C7αH activities significantly decreased after dephosphorylation by AP (–57% – –72%) and increased up to twofold with phosphorylation by rabbit muscle cAMP–dependent protein kinase. The increases in C7αH activities were proportional to the amounts of cAMP–dependent protein kinase used, and were coupled to 32P incorporation into the purified enzymes. Both the activation of C7αH and the amounts of 32P incorporation were time–dependent and reached a maximum after 1 hour of incubation with 5 U of cAMP–dependent protein kinase. In a second set of experiments, purified human and rat liver C7αH were dephosphorylated by 30–minute incubation with AP, followed by inactivation of the phosphatase by the inhibitor NaF, and rephosphorylation of C7αH by 30–minute incubation with rabbit muscle cAMP–dependent protein kinase or bovine heart cAMP–independent protein kinase. Rephosphorylation of the dephosphorylated C7αH proteins by cAMP–dependent protein kinase increased C7αH catalytic activities up to fourfold, and the stimulation in catalytic activities paralleled the increases in 32P incorporation into the purified enzymes. Bovine heart protein kinase was as potent as rabbit muscle cAMP–dependent protein kinase in stimulating catalytic activity and 32P incorporation into the human C7αH protein. Because the protein mass of these purified enzymes did not change, the short–term regulation or catalytic efficiency of C7αH (activity per protein mass unit) is modulated, in vitro, posttranslationally by a phosphorylation/dephosphorylation mechanism in both the human and the rat enzymes.