bluuebunny/crossref_metadata_2025 huggingface dataset:
Sequence of Exons and the Flanking Regions of Human Bilirubin–Udp–Glucuronosyltransferase Gene Complex and Identification of A Genetic Mutation in A Patient With Crigler–Najjar Syndrome, Type I
Authors: Bosma Piter J. , Chowdhury Namita Roy , Goldhoorn Bart G. , Hofker Martin H. , Oude Elferink Ronald P.J. , Jansen Peter L.M. , Chowdhury Jayanta Roy
Published: March 2007

ABSTRPlCT

Crigler–Najjar syndrome, type I is a heterogeneous disorder that may result from mutations of various regions of the bilirubin–UDP–glucuronosyltransferase gene complex that encodes two bilirubin–UDP–glucuronosyltransferase isoforms and a phenol–UDP–glucuronosyltransferase isoform in the human liver. The two bilirubin–UDP–glucuronosyltransferase messenger RNAs and the phenol–UDP–glucuronosyltransferase messenger RNA have identical 3′ regions derived from four consecutive exons. The 5′ region of each messenger RNA is unique and is derived from distinct single exons. By screening a human genomic library with probes corresponding to various regions of the messenger RNAs, we have isolated five cosmid clones containing overlapping segments of this large gene complex that spans at least 84 kb of the human genome. To facilitate the amplification of each exon by polymerase chain reaction and their adjacent splice junctions, we have delineated the intron–exon boundaries of the four common region exons and the two single exons that encode the unique regions of the two bilirubin–UDP–glucuronosyltransferase isoforms and have described sequences of the regions flanking each exon. All exons encoding the two bilirubin–UDP–glucuronosyltransferase isoforms and their splice junctions were amplified from the DNA of two control subjects and a Crigler–Najjar syndrome, type I patient. The DNA from the Crigler–Najjar syndrome, type I patient revealed a point mutation in exon 3 (a common region exon) resulting in a stop codon. RNA blot showed that the two bilirubin–UDP–glucuronosyltransferase messenger RNAs in the liver of the Crigler–Najjar syndrome, type I patient were of normal length but were reduced in concentration. As expected, the mutation was found in the common region of both bilirubin–UDP–glucuronosyltransferase messenger RNAs in the liver of the patient. The premature stop codon is predicted to encode truncated and inactive bilirubin–UDP–glucuronosyltransferase forms. Bilirubin–UDP–glucuronosyltransferase proteins were not detectable by immunotransblot in the liver of the Crigler–Najjar syndrome, type I patient. The single mutation in a common region exon explains the simultaneous loss of activity of both bilirubin–UDP–glucuronosyltransferase isoforms and marked reduction of UDP–glucuronosyltransferase activity toward phenol. (Hepatology 1992;15:941-947).
Is Continued Enthusiasm for Ursodeoxycholic Acid Therapy for the Treatment of Primary Biliary Cirrhosis Warranted?
Authors: Wiesner Russell H.
Published: March 2007

ABSTRPlCT

Background.

In primary biliary cirrhosis the hepatic lesions may result, at least in part, from the intracellular accumulation of potentially toxic endogenous bile acids. Preliminary work suggests that the administration of ursodiol (also called ursodeoxycholic acid), a hydrophilic bile acid without hepatotoxicity, leads to improvement in the condition of patients with primary biliary cirrhosis.

Methods.

We conducted a two–year, multicenter, double–blind trial to compare the efficacy of ursodiol with that of placebo. Patients with biopsy–proved primary biliary cirrhosis were randomly assigned to receive either ursodiol (13 to 15 mg per kilogram of body weight per day) (n = 73) or placebo (n = 73). Treatment failure was defined as a doubling of bilirubin levels to more than 70 μmol per liter or the occurrence of a severe complication (ascites or variceal bleeding) or an adverse reaction.

Results.

Treatment failed in 6 patients in the ursodiol group, as compared with 13 in the placebo group (P < 0.01 by Cox regression model). A single patient in each group withdrew because of minor adverse effects. After two years of treatment, the proportion of patients with clinically overt disease decreased only in the ursodiol group (P < 0.02). The patients treated with ursodiol had significant improvements in serum levels of bilirubin, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, γ–glutamyltransferase, cholesterol, and IgM (all P < 0.001); the antimitochondrial–antibody titer (P < 0.01); and the Mayo risk score (P < 0.001). Follow–up analysis of 95 liver–biopsy specimens showed a significant improvement in the mean histologic score (P < 0.002) and in all the characteristic histologic features except fibrosis only in the group given ursodiol.

Conclusions.

Ursodiol is a safe and effective treatment for primary biliary cirrhosis.
Time Course of Hepatitis A Virus Antibody Titer After Active and Passive Immunization
Authors: Fujiyama Shigetoshi , Iino Shiro , Odoh Koichi , Kuzuhara Shoji , Watanabe Hiroaki , Tanaka Masahiko , Mizuno Kyosuke , Sato Tatsuo
Published: March 2007

To investigate the antibody titer necessary to prevent hepatitis A virus infection, either 15 or 7.5 mg/kg of immune serum globulin was injected into 10 antihepatitis A virus negative volunteers and their serum antihepatitis A virus titers were observed for 28 wk. In addition, antibody titers were observed for 96 wk in a phase 1 clinical trial of a hepatitis A vaccine. The two studies were then compared to assess the immunogenicity of the vaccine and the persistence of the antibody. Serum–neutralizing antibody titers that were greater than or equal to 4 (considered as positive) persisted for 18 wk and 14 wk after the injection of 15 and 7.5 mg/kg of globulin, respectively. Hepatitis A virus vaccine recipients showed adequate neutralizing antibody titers, with the groups receiving 1, 0.5 and 0.25 μg/dose showing titers of 45.5, 44.7 and 44, respectively, at 18 mo after the third inoculation. These findings suggested that effective blood antibody titers were likely to be retained in the 1.0 μg or 0.5 μg/dose groups for at least several years. Moreover, the serum antihepatitis A virus titers demonstrated by a modified radioimmunoassay changed in parallel with the neutralizing antibody titers in the volunteers injected with globulin. (Hepatology 1992;15:983–988).
Role of Extracellular Matrix in Regulating Fenestrations of Sinusoidal Endothelial Cells Isolated From Normal Rat Liver
Authors: McGuire Richard F. , Bissell Montgomery D. , Boyles Janet , Roll Joseph F.
Published: March 2007

Open fenestrations are a conspicuous feature of sinusoidal endothelial cells and allow free movement of plasma into the space of Disse. In hepatic fibrosis, the number of fenestrations decreases as interstitial collagen increases in the liver, a change that correlates with deposition of extracellular matrix in the space of Disse. In this study, the possibility of a causal relationship between altered fenestral morphology and perisinusoidal matrix has been examined by culturing rat sinusoidal endothelial cells on individual matrix proteins or on a native matrix consisting of human amniotic membrane with interstitial collagen (types I and III) on one side and basement membrane proteins (collagen types IV and V and laminin) on the other. Under culture conditions, individual components of the extracellular matrix failed to maintain fenestrations. A basement–membranelike gel matrix derived from the Engelbreth–Holm–Swarm tumor was similarly ineffective. Fenestral density and porosity (percentage of cell surface occupied by fenestrations) were significantly enhanced, however, when endothelial cells were cultured on the basement–membrane side of human amnion. These data suggest that support of endothelial fenestrations requires a complex matrix. In particular, physiologically derived basement membrane maintains fenestrations, whereas interstitial collagen matrix does not. The loss of fenestrations associated with hepatic fibrosis may be related in part to an accumulation of interstitial collagens in the space of Disse. (Hepatology 1992;15:989–997).
Absence of Hepatitis B Virus Precore Mutants in Patients With Chronic Hepatitis B Responding to Interferon–α
Authors: Xu Jianye , Brown David , Harrison Tim , Lin Yue , Dusheiko Geoffrey
Published: March 2007

Precore defective HBV mutants may gradually prevail because of immune selection and explain spontaneous seroconversion from HBeAg to anti–HBe in HBV carriers. We have analyzed whether the presence of precore HBV mutants is a determinant of responsiveness to interferon–α therapy. Fifteen carriers (nine responders and six nonresponders)who were treated with interferon–α were examined. Serum samples were collected before and after therapy. After extraction of DNA, the precore region was amplified by the polymerase chain reaction, and the product was identified by gel electrophoresis and ethidium bromide staining and then Southern blotting and molecular hybridization. The amplified products in all patients were asymmetrically amplified by a modified polymerase chain reaction, and the precore region was directly sequenced. All patients were HBV DNA positive initially. Circulating HBeAg–negative mutants were not identified before treatment in either responders or nonresponders. All nine responders were negative for HBV DNA in serum by dot blot during or after treatment, but seven remained positive by polymerase chain amplification and Southern–blot hybridization. All of the nonresponders remained positive for HBV DNA by dot blot. A silent mutation involving the substitution of an A for G at position 1888 was found in seven carriers; however, no HBeAg–negative mutants were detected in the follow–up of either responders or nonresponders to interferon–α. These results suggest that HBV DNA is detectable by polymerase chain reaction for a period after clearance of HBsAg. A predominance of HBeAg–negative HBV virions cannot be considered a pivotal determinant of response to treatment. The role of other mutations in the precore region in determining response to therapy is uncertain. (Hepatology 1992;15:1002-1006).
Long–Term Follow–Up of Patients With Chronic Hepatitis C Treated With α–Interferon
Authors: Shindo Michiko , di Bisceglie Adrian M. , Hoofnagle Jay H.
Published: March 2007

We reanalyzed the results of a pilot study of recombinant α–interferon therapy for chronic non–A, non–B hepatitis in light of the recent discovery of the hepatitis C virus and the development of diagnostic assays for this agent. Stored serum samples from 10 patients treated between 1984 and 1986 were tested for antibody to hepatitis C virus and hepatitis C virus RNA before, during and after therapy. In addition, the current clinical, serum biochemical and virological statuses of these patients were evaluated to determine the long–term effects of interferon therapy. All patients had evidence of hepatitis C virus infection, with hepatitis C viral RNA, antibody to hepatitis C virus or both markers detectable in serum. Serum hepatitis C virus RNA was found to disappear in seven of eight patients whose aminotransferase levels became normal with interferon therapy but remained present in two patients who did not respond to therapy. Levels of hepatitis C virus RNA decreased and disappeared when serum aminotransferases fell to normal levels but rose with subsequent elevation of aminotransfrase levels in two patients who had relapses in disease when interferon was stopped. During a follow–up of 3 to 6 yr, hepatitis C virus RNA remained undetectable in the six patients whose serum aminotransferase levels remained normal after interferon therapy. However, neither initial titers of hepatitis C virus RNA nor disappearance of viral RNA from serum during treatment predicted a sustained response. Thus long–term beneficial responses to α–interferon can occur in patients with chronic hepatitis C and are associated with sustained loss of hepatitis C virus RNA from serum. (Hepatology 1992;15:1013-1016).
Terlipressin Vs. Somatostatin in Bleeding Esophageal Varices: A Controlled, Double–Blind Study
Authors: Walker Siegfried , Kreichgauer Heinz-Peter , Bode Christian J.
Published: March 2007

Fifty episodes of bleeding from esophageal or gastric varices in 33 patients with cirrhosis were randomized to treatment with either intravenous terlipressin (2 mg initially and 1 mg every 4 hr for 24 hr together with bolus injection and continuous infusion of placebo) or with somatostatin (250 μg as a bolus and continuous infusion of 250 μg/hr somatostatin for 24 hr and placebo injections). Standard therapy with transfusions, fluid and electrolyte correction and lactulose was administered in both groups.

In the terlipressin group, 22 of 25 bleeding episodes (88%) were initially stopped by the vasoactive drugs, and in the somatostatin group 19 of 25 bleeding episodes (76%) were initially stopped by the vasoactive drugs. Two of the three bleeding episodes not arrested by terlipressin and five of the six bleeding episodes not arrested by somatostatin were controlled by balloon tamponade. In one patient in each group variceal bleeding initially could not be stopped, and the patients died. The failure rate of the vasoactive treatment alone, including rebleeding episodes within the study period, was 20% in the terlipressin group and 32% in the somatostatin group. The control rate, including balloon tamponade, was 96% in both groups. The hospital mortality rate was 16% (4 of 25) in the terlipressin group and 24% (6 of 25) in the somatostatin group. Blood transfusions, use of balloon tamponade and duration of bleeding did not differ significantly.

This interim analysis of the ongoing study indicates that a large proportion of bleeding episodes from esophageal and fundic varices can be stopped initially (82%) and definitively controlled (74%) by vasoactive drugs alone. Differences in the effectiveness of terlipressin and somatostatin were not observed. (Hepatology 1992;15:1023–1030).
Bleeding From Staple Line Erosion After Esophageal Transection: Effect of Omeprazole
Authors: Kaye Graham L. , McCormick Aiden P. , Siringo Sebastiano , Hobbs Kenneth E.F. , McIntyre Neil , Burroughs Andrew K.
Published: March 2007

Esophageal staple transection effectively controls acute variceal bleeding, but up to 50% of these patients will have recurrent upper gastrointestinal bleeding. In our experience, most of these bleeding episodes are caused by total or partial circumferential ulceration at the level of the staple transection: staple line erosion. It caused rebleeding in 29 (40%) of our patients. Whereas the pathogenesis of this lesion is unknown, acid reflux is a consequence of transection surgery. Assuming that staple line erosion could be healed by acid suppression therapy, thereby preventing recurrent bleeding, an acid suppression regimen was evaluated prospectively in 24 patients. Only six (25%) healed with daily standard (300 mg) or high–dose (1,200 mg) ranitidine combined with sucralfate (4 gm). The remaining 18 (75%) healed after omeprazole administration (40 mg/day) for 1 mo. Maintenance ranitidine alone (300 mg/day) was introduced, but 11 (48%) had relapse of erosions. All 11 healed with omeprazole (40 mg/day) for 2 mo, but again on maintenance ranitidine, 10 relapsed. All healed with further omeprazole and healing persisted with long–term administration (20 mg/day). Fifteen rebleeding episodes occurred in eight patients on maintenance ranitidine. Whereas relapse of staple line erosions did occur in the absence of rebleeding, all rebleeding episodes were associated with the relapse of staple line erosion.

Omeprazole is more effective than ranitidine alone and combined with sucralfate in healing staple line erosion. Omeprazole prevents rebleeding, which may enhance the long–term benefits of staple transection for acute variceal bleeding. (Hepatology 1992;15:1031-1035).
Transjugular Liver Biopsy in Children
Authors: Furuya Katryn N. , Burrows Patricia E. , Phillips James M. , Roberts Eve A.
Published: March 2007

Transjugular liver biopsy is widely used in adult patients with liver disease when transthoracic needle liver biopsy is contraindicated by severe coagulopathy or ascites. It has not been used extensively in children. We report our experience with 30 consecutive transjugular liver biopsy procedures performed in 27 young patients at the Hospital for Sick Children in Toronto. Patient weights ranged from 14 to 91 kg (median = 42 kg); most patients had not yet attained adult size. A 9F catheter was used except in very small children, for whom we developed a 7F biopsy needle catheter. Most procedures were done with patients under general anesthesia. Specimens were obtained in all patients, and the procedure was tolerated well. A major complication occurred only once: perforation of the inferior vena cava, which was later repaired at liver transplantation. Minor complications included subcapsular extravasation of contrast agent in five patients and a small intrahepatic hematoma in one. Results of transjugular liver biopsy changed the diagnosis in 30% of patients and added valuable information about the disease process in most patients. Transjugular liver biopsy was an important component of emergency assessment for liver transplantation. Our results indicate that transjugular liver biopsy can be performed safely in children with liver disease if a skilled interventional radiologist and a well–equipped angiography suite are available. Histological studies in these patients enhance our understanding of the natural history of pediatric liver diseases. (Hepatology 1992;15:1036-1042).
Effects of Ursodeoxycholic Acid in Patients With Intrahepatic Cholestasis of Pregnancy
Authors: Palma Joaquin , Reyes Humberto , Ribalta Jose , Iglesias Joaquin , Gonazalez Manuel C. , Hernandez Ismael , Alvarez Celia , Molina Claudina , Danitz Ana Maria
Published: March 2007

The efficacy and safety of ursodeoxycholic acid in the treatment of intrahepatic cholestasis of pregnancy was investigated in an open pilot study. Five patients received 1 gm/day of ursodeoxycholic acid during 20 days and another three patients received two identical periods of treatment separated by a 14–day interval free of the drug. Pruritus and serum levels of total bile salts and glutamic–pyruvic transaminase improved significantly during treatment with ursodeoxycholic acid. In the three patients who received two periods of treatment with ursodeoxycholic acid, pruritus and the laboratory alterations relapsed in the second week after the drug was discontinued, but they improved again when ursodeoxycholic acid was readministered. No adverse reactions were detected in the mothers or in their babies. All newborns were thriving normally during a follow–up period that lasted 5 mo after delivery. It is concluded that UDCA appears to be safe when administered in late pregnancy; its promising efficacy in the treatment of intrahepatic cholestasis of pregnancy should now be confirmed in controlled clinical trials. (Hepatology 1992;15:1043-1047).
Altered Zonal Expression of the Cd26 Antigen (Dipeptidyl Peptidase Iv) in Human Cirrhotic Liver
Authors: Matsumoto Yorio , Bishop Alex G. , McCaughan Geoffrey William
Published: March 2007

Dipeptidyl peptidase IV is a cell surface ectopeptidase with widespread tissue distribution. Recently it was shown to display extracellular matrix—binding properties; therefore its role in cirrhosis is of interest. The aim of this study was to use monoclonal antibodies directed against the human CD26 antigen (which has been shown to be dipeptidyl peptidase IV) to study the distribution of this molecule in normal human and cirrhotic liver. Identical staining was obtained with the three monoclonal antibodies (TaI, 1F7 and TS145) and enzyme histochemistry. In normal liver (n = 11) intense staining of hepatic acinar zones 2 and 3 was present, but little staining was seen in zone I. Hepatocyte staining was confined to the bile canalicular domain. In cirrhotic livers (n = 23) obtained at transplantation, staining of regenerating nodules without a zonal pattern was present. In addition, we saw staining of the lymphoid cell infiltrate and proliferating bile ductules. In a minority of cirrhotic biopsy specimens (four) staining of the basolateral hepatocyte domain in regenerating nodules was seen. Biopsy specimens from hepatic allografts (n = 28) were used as disease controls. These samples all showed preferential staining of zones 2 and 3, similar to that in normal biopsy speclimens. Eleven of these samples showed staining of the basolateral and bile canalicular domains. In conclusion, the normal acinar distribution of dipeptidyl peptidase IV (zones 2 and 3) is lost in cirrhotic nodules. Furthermore, the altered membrane distribution of this molecule in cirrhosis and allograft rejection may allow increased hepatocyte extracellular matrix interactions during organ remodeling. (Hepatology 1992;15:1048-1053.
Electron Microscopic Study of Brain Capillaries in Cerebral Edema From Fulminant Hepatic Failure
Authors: Kato Masataka , Hughes Robin D. , Keays Richard T. , Williams Roger
Published: March 2007

Cerebral edema is a serious complication of the encephalopathy in fulminant hepatic failure. It is a major cause of death. The mechanisms responsible for its formation are unclear, and the aim of this study was to investigate the ultrastructural appearance of brain capillaries by scanning electron microscopy. Samples of cerebral cortex were obtained immediately after death from nine patients with fulminant hepatic failure (seven cases due to acetaminophen overdose, one caused by hepatitis B and one caused by non–A, non–B hepatitis) by needle biopsy at the site of insertion of an extradural pressure transducer to monitor intracranial pressure. The intercellular tight junctions between capillary endothelial cells were intact. The endothelial cells were swollen, with increased numbers of vesicles and vacuoles. The basement membranes were enlarged and vacuolized and the pericytes had increased numbers of vesicles and vacuoles, indicative of passage of fluid by this route. Marked intracellular swelling of the perivascular astroglial foot processes was present. Thus mainly cytotoxic mechanisms, with cellular swelling, and to a lesser extent vasogenic mechanisms, with altered blood–brain barrier permeability, appear to be involved in the cerebral edema of fulminant hepatic failure. (Hepatology 1992;15:1060-1066).
Effects of Vasopressor Agents and Epoprostenol on Systemic Hemodynamics and Oxygen Transport in Fulminant Hepatic Failure
Authors: Wendon Julia A. , Harrison Phillip M. , Keays Richard , Gimsson Alexander E. , Alexander Graeme J.M. , Williams Roger
Published: March 2007

Hypotension is a serious complication in patients with fulminant hepatic failure, because it is associated with tissue hypoxia and a further compromise to end–organ function. In this study we investigated the effects of epinephrine and norepinephrine on hemodynamics and oxygen transport variables in 30 patients with fulminant hepatic failure. All had a mean arterial pressure of less than 60 mm Hg, despite adequate intravascular filling pressures.

Both epinephrine (n = 15) and norepinephrine (n = 15) improved mean arterial pressure (p < 0.001 epinephrine and norepinephrine), although this was not associated with a rise in oxygen delivery. Oxygen consumption fell (p<0.05 epinephrine, p<0.001 norepinephrine) because of a lower oxygen extraction ratio (p<0.01 epinephrine and norepinephrine). The addition of epoprostenol, a microcirculatory vasodilator, in 10 patients from each group led to an increase in oxygen consumption (p<0.001 epinephrine and norepinephrine) because of a rise in oxygen delivery (p<0.05 epinephrine, p<0.01 norepinephrine) and oxygen extraction ratio (p<0.01 epinephrine, p<0.001 norepinephrine), without a fall in mean arterial pressure.

The fall in oxygen consumption after the institution of vasopressor therapy could exacerbate tissue hypoxia and thus contribute to further organ damage in an already susceptible patient. In patients with fulminant hepatic failure who are given vasopressor support, the addition of epoprostenol may prevent the development of tissue hypoxia. (Hepatology 1992;15:1067–1071).
Hepatic Allograft Cyclosporine Concentration Is Independent of the Route of Cyclosporine Administration and Correlates With the Occurrence of Early Cellular Rejection
Authors: Sandborn William J. , Lawson George M. , Krom Ruud A.F. , Wiesner Russell H.
Published: March 2007

This study compares the hepatic–tissue cyclosporine concentrations in liver biopsy specimens from patients with and without cellular rejection after orthotopic liver transplantation. Nine patients without cellular rejection were studied, including five patients receiving intravenous cyclosporine who had biopsies on posttransplant day 7 and four patients receiving oral cyclosporine who had biopsies on day 21. In these patients no significant differences were seen in blood cyclosporine concentrations (mean ± S.D.) (310 ± 162 vs. 273 ± 310 ng/ml) and hepatic–tissue cyclosporine concentrations (3,451 ± 1,262 vs. 3,545 ± 456 ng/gm). These nine patients without cellular rejection were then compared with eight patients with cellular rejection who were receiving intravenous cyclosporine, including seven patients who had biopsies on posttransplant day 7 and one patient who had a biopsy on day 14. In patients with and without cellular rejection blood cyclosporine concentrations were 330 ± 64 vs. 294 ± 146 ng/ml, and hepatic–tissue cyclosporine concentrations were 1,879 ± 998 vs. 3,493 ± 936 ng/gm, p<0.01. We conclude that hepatic–tissue cyclosporine concentrations are independent of the route of cyclosporine administration and that low hepatic–tissue cyclosporine concentrations correlate with early cellular rejection after orthotopic liver transplantation. (Hepatology 1992;15:1086-1091).
Changes of Hepatic Fatty Acid Metabolism Produced by Chronic Thioacetamide Administration in Rats
Authors: Nozu Fumihiro , Takeyama Naoshi , Tanaka Takaya
Published: March 2007

Hepatic mitochondrial functions related to fatty acid metabolism, including the respiratory control ratio, fatty acid oxidative capacity and carnitine palmitoyltransferase I activity, were studied in vitro with mitochondria isolated from rats treated with thioacetamide for up to 12 wk. The levels of ketone bodies, carnitine, carnitine esters and malonyl-coenzyme A were also determined in liver extracts. Polarography of mitochondrial respiration from succinate or glutamate plus malate showed a lower respiratory control ratio in thioacetamide–treated rats, whereas uncoupled oxygen consumption was not altered. This suggests that the mitochondrial respiratory chain capacity remained intact in the thioacetamide–treated rats. The oxygen consumption associated with palmitoyl-coenzyme A and palmitoyl–L–carnitine oxidation by isolated liver mitochondria was increased by thioacetamide treatment on both a per-mitochondrial protein and a per-total liver basis. The carnitine palmitoyl–transferase I activity; the tissue levels of ketone bodies, carnitine and carnitine esters; and the β–hydroxybutyrate/acetoacetate ratio were all higher in the livers of thioacetamide–treated animals than in control livers, whereas the hepatic malonyl-coenzyme A level was decreased by thioacetamide. These results indicate the increased diversion of cytosolic longchain acyl–coenzyme As into the mitochondria for β–oxidation rather than their esterification and use in lipogenesis. These intrahepatic metabolic changes induced by chronic thioacetamide administration may reflect the whole–body catabolic state and can be seen as adaptive for maintaining energy homeostasis under conditions of impaired glucose tolerance. (Hepatology 1992;15:1099-1106).
Adenosine Receptor Blockade Reduces Splanchnic Hyperemia in Cirrhotic Rats
Authors: Lee Samuel S. , Chilton Lisa E. , Pak Jung-Min
Published: March 2007

To explore a possible role for adenosine in the pathogenesis of the splanchnic hyperemia of cirrhosis, we administered 8–phenyltheophylline, a specific adenosine receptor antagonist, to rats with biliary cirrhosis caused by bile duct ligation and to control shamoperated rats. Micro–Doppler flow studies showed that a 10–μmol/kg dose of 8–phenyltheophylline completely abolished the superior mesenteric hyperemic response to infusions of exogenous adenosine in both cirrhotic and control rats. Analysis of regional blood flows by radioactive microspheres demonstrated that this dose of 8–phenyltheophylline in cirrhotic rats significantly increased portal tributary vascular resistance by 60% and decreased portal tributary blood flow by 26%. This decrease was entirely the result of a 42% reduction in the intestinal blood flow. 8–phenyltheophylline did not affect cardiac output, arterial pressure or any other extrasplanchnic hemodynamic variables in cirrhotic rats. No detectable effect of 8–phenyltheophylline was seen in sham–operated rats. These results suggest that adenosine may be involved in the genesis of splanchnic hyperemia in cirrhotic rats. (Hepatology 1992;15:1107-1111).
Immunolocalization, Quantitation and Cellular Heterogeneity of Apolipoprotein B in Rat Hepatocytes
Authors: Corsetti James P. , Way Barbara A. , Sparks Charles E. , Sparks Janet D.
Published: March 2007

Hepatocyte autofluorescence represents a major problem in immunofluorescence studies with fluorescein conjugates because of significant spectral overlap. We describe a method for immunostaining hepatocytes with R–phycoerythrin (a fluorochrome with minimal overlap with autofluorescence) with paraformaldehyde fixation and Triton X–100 permeabilization for better antibody penetration. This method produced both perinuclear (presumed Golgi apparatus) and dispersed, reticular staining (presumed endoplasmic reticulum) in rat hepatocytes in culture stained with a monoclonal antibody to rat apolipoprotein B. Treatment with brefeldin A resulted in loss of apolipoprotein B perinuclear staining and increased reticular immunofluorescence consistent with known properties of brefeldin A (inhibition of protein transport within the secretory pathway by dissolution of Golgi bodies). This suggests that apolipoprotein B epitopes are present in both Golgi bodies and endoplasmic reticulum. To demonstrate the utility of the technique for quantitative studies, static cell cytofluorometry of brefeldin A—treated cells was performed, demonstrating increases in specific immunofluorescence of apolipoprotein B corresponding closely to results estimated by monoclonal antibody radioimmunoassays of cellular homogenates. The technique was then used with flow cytometry of single–cell suspensions of control rat hepatocytes derived from immunostained primary cultures to reveal cell–to–cell heterogeneity of apolipoprotein B epitope expression manifested as apolipoprotein B—negative and positive populations. Results for brefeldin A—treated cells revealed even clearer delineation of heterogeneity as indicated by frank bimodality of the populations, along with not only higher mean apolipoprotein B levels but also a significantly higher proportion of apolipoprotein B—positive cells than in the control. (Hepatology 1992;15:1117-1124).
Effects of Partial Hepatectomy on Hepatic Insulinlike Growth Factor Binding Protein–1 Expression
Authors: Ghahary Aziz , Minuk Gerald Y. , Luo Jiangming , Gauthier Tony , Murphy Liam J.
Published: March 2007

Insulinlike growth factor binding proteins modulate the action of the insulinlike growth factors in various bioassays and may regulate the bioavailability of the insulinlike growth factors in vivo. Because the insulinlike growth factors may influence hepatic regeneration, we have examined the effect of partial hepatectomy on serum insulinlike growth factor binding proteins and on the abundance of insulinlike growth factor binding protein–1 messenger RNA in the liver. All rats were fasted before and after partial hepatectomy or sham operation to avoid the confounding effects of difference in food intake. Using a conventional protocol, 70% of the liver was removed, and groups of four or five rats were killed at different intervals after partial hepatectomy. Sham–operated rats served as controls. Pooled sera from each group of rats were analyzed by ligand blotting with 125I–insulinlike growth factor–I. Liver RNA from individual rats was analyzed by slot–blot and Northern–blot hybridization. A small decrease in the 39– to 42–kD insulinlike growth factor binding protein was apparent in sera from both the sham–operated and partial hepatectomized rats. In contrast, a dramatic increase (fivefold) in the 29–kD serum insulinlike growth factor binding protein (insulinlike growth factor binding protein–1) was apparent only in the partial hepatectomized rats. Hepatic insulinlike growth factor binding protein–1 messenger RNA abundance was significantly increased (1.99 ± 0.18–fold; p<0.05) at 1 hr, reached a peak of 2.32 ± 0.22–fold (p<0.01) at 3 hr after partial hepatectomy and returned to basal levels over the subsequent 6 to 12 hr. Interestingly, the abundance of insulinlike growth factor binding protein–1 messenger RNA was also significantly increased in the kidneys of partially hepatectomized rats at 3 hr (1.47 ± 0.23–fold; p<0.05), and peaked at 6 hr (2.50 ± 0.39–fold; p<0.05) after partial hepatectomy. No significant change in hepatic or renal insulinlike growth factor binding protein–1 messenger RNA abundance was apparent in sham–operated rats. For comparison, the abundance of the growth hormone–dependent binding protein insulinlike growth factor binding protein–3 and insulinlike growth factor–I messenger RNAs was also examined. A decrease in both hepatic insulinlike growth factor binding protein–3 and insulinlike growth factor–1 messenger RNA was observed in shamoperated and hepatectomized rats. These data demonstrate that partial hepatectomy is associated with increased expression of insulinlike growth factor binding protein–1 in both the liver and kidney. The increase in tissue and circulating insulinlike growth factor binding protein–1 may modulate the mitogenic effects of insulinlike growth factor–I during liver regeneration. (Hepatology 1992;15:1125-1131).
Cytokines Tumor Necrosis Factor and Interleukin–6 in Experimental Biliary Obstruction in Mice
Authors: Bemelmans M. H.A. , Gouma D. J. , Greve J. W. , Buurman W. A.
Published: March 2007

The putative role of the cytokines interleukin–6 and tumor necrosis factor in the pathophysiology of the complications and mortality after surgery in jaundiced patients was studied in a murine model. Cytokine serum levels were determined in mice with experimental biliary obstruction. As an indicator of the activation status of macrophages, cytokine release by mononuclear phagocytes obtained from such mice was assessed.

Following surgery, interleukin–6 levels increased to 2 to 3 ng/ml after 3 to 4 hr, but declined rapidly afterward to levels of 60 pg/ml after 10 days, After 12 days, substantial interleukin–6 levels were observed in jaundiced mice (100 pg/ml), whereas levels in sham mice further decreased (p<0.001). The cytokine tumor necrosis factor was frequently present in the serum of jaundiced mice. After 22 days, when killed, all jaundiced mice showed significant tumor necrosis factor levels (p<0.001). This was in contrast to sham mice in which tumor necrosis factor was never detected.

The presence of an activated state of macrophages in jaundiced mice was concluded from the observed high spontaneous cytokine release and significantly higher release after stimulation (p<0.05). The presence of circulating cytokines was discussed in the context of the postoperative complications observed in jaundiced patients. (Hepatology 1992;15:1132-1136).
Modification of the Amount of Cholesterol in Hepatic Steatosis Induced in Susceptible and Resistant Mice Infected With Mhv3: A Biochemical and Ultrastructural Study
Authors: Bingen Annick , Martin Jean-Pierre , Klein François , Pessah Marcia
Published: March 2007

A mouse hepatitis virus–3 strain subcultured in our laboratory is a unique experimental model in which to study virus–induced liver steatosis. This strain produces massive lipid deposition not only in sensitive adult BALB/c mice but also (though less extensive) in virus–resistant adult A/J mice. Biochemical determinations have shown that this steatosis is characterized by an increased amount of neutral lipids (sterols and triglycerides) in infected livers of BALB/c mice and by a smaller increase in those of A/J mice. However, the relative percentage of cholesterol and triglycerides is similar in both strains. Liver phospholipid content was significantly decreased in both strains of mice. To discriminate between cytoplasmic and membrane cholesterol content in different types of liver cells, an ultrastructural study was performed with filipin, a specific cholesterol marker. This study shows on one hand an important increase in the cholesterol in the hepatocytes of BALB/c mice and a smaller increase in those of A/J mice, in agreement with biochemical data. However, marked cholesterol decrease and abnormal cholesterol distribution were observed in the endothelial liver cells of infected BALB/c mice. This decreased cholesterol content probably led to higher fluidity of these membranes, which could be related to the important drop in the number of endothelial cell fenestrae observed after mouse hepatitis virus–3 infection. Because in A/J infected mice neither a decrease in the amount and distribution of cholesterol nor decreased fenestration were observed in endothelial liver cells, these findings could be correlated with the resistance of these mice to the infection. (Hepatology 1992;15:1137-1146).
Defective Biliary Excretion of Epinephrine Metabolites in Mutant (Tr-) Rats: Relation to the Pathogenesis of Black Liver in the Dubin–Johnson Syndrome and Corriedale Sheep With An Analogous Excretory Defect
Authors: Kitamura Tsuneo , Alroy Joseph , Gatmaitan Zenaida , Inoue Masayasu , Mikami Takashi , Jansen Peter , Arias Irwin M.
Published: March 2007

Dubin–Johnson patients, mutant Corriedale sheep and TR- and EHBR mutant rats have recessively inherited defective bile canalicular secretion of many nonbile acid organic anions. The human and ovine mutants have black livers and lysosomal pigment accumulation. The livers in TR- and EHBR mutant rats are not black, and sparse lysosomal pigment accumulation is seen. Previously, we postulated that the unidentified pigment in the Dubin–Johnson syndrome results from the accumulation of tyrosine, phenylalanine and tryptophan metabolites, such as metanephrine, which are normally secreted in bile as organic anions. We tested this hypothesis in TR- rats. H–epinephrine was injected intravenously; control rats secreted 2.80% ± 0.52% of the injected dose in bile as compared with 0.19% ± 0.07% in TR- rats. From 82% to 90% of biliary radioactivity was due to polar conjugates in control rats and mutant rats. TR- rats retained more of the injected dose in the liver, particularly in lysosomes, and secreted more in urine than did control rats. After feeding control and TR- rats for 4 mo with a rat chow diet supplemented with 4% tyrosine, tryptophan and phenylalanine, the liver did not become grossly black; however, histological and electron microscopic study revealed dense lysosomal pigment accumulation in TR- rats. Intraportal injection of metanephrine resulted in the appearance of black liver in TR- rats that persisted for at least 2 hr and was not associated with pigment accumulation by light or electron microscopic examination. After mating TR- males with EHBR females, all 38 offspring had bilirubinuria, which indicates that TR- and EHBR are allelic mutants. We propose that defective canalicular secretion of anionic metabolites may be responsible for the black lysosomal pigment that accumulates in the Dubin–Johnson syndrome, mutant Corriedale sheep and in TR- rats that were fed a diet enriched in aromatic amino acids. (Hepatology 1992;15:1154-1159).
Reduced Activity of the Electron Transport Chain in Liver Mitochondria Isolated From Rats With Secondary Biliary Cirrhosis
Authors: Krähenbühl Stefan , Stucki Jörg , Reichen Jürg
Published: March 2007

Mitochondrial metabolism was studied in liver mitochondria isolated from rats with secondary biliary cirrhosis induced by bile duct ligation for 5 wk. State 3 oxidation rates were decreased in mitochondrial preparations from bile duct—ligated rats as compared with sham–operated control rats by 63% and 42% using β–hydroxybutyrate and succinate as substrates, respectively. In contrast, when the substrate was ascorbate/N, N, N′, N′–tetramethyl–p–phenylenediamine state 3 oxidation rates were not affected by bile duct ligation. Oxidation rates after uncoupling with dinitrophenol were decreased for both β–hydroxybutyrate and succinate as substrates in mitochondria from bile duct—ligated rats. The phosphate potential was reduced in mitochondria from bile duct—ligated rats (12.5 ± 0.5 vs. 13.6 ± 0.2 kcal in control and bile duct—ligated rats, respectively; p<0.05). The inner mitochondrial membrane of liver mitochondria from rats with secondary biliary cirrhosis contained three times more cholesterol as compared with control rats, whereas the phospholipid composition was essentially unchanged. Mitochondrial protein content expressed per liver (calculated on the basis of activities of mitochondrial enzymes determined in liver homogenate and in isolated mitochondria) was increased by 50% in bile duct—ligated rats as compared with control rats. In conclusion, the function of the electron transport chain in liver mitochondria isolated from rats with secondary biliary cirrhosis is impaired. This decrease could be related to altered lipid composition of the inner mitochondrial membrane. (Hepatology 1992;15:1160–1166).
Stereological and Functional Analysis of Liver Mitochondria From Rats With Secondary Biliary Cirrhosis: Impaired Mitochondrial Metabolism and Increased Mitochondrial Content Per Hepatocyte
Authors: Krähenbühl Stefan , Krähenbühl-Glauser Sonja , Stucki Jörg , Gehr Peter , Reichen Jürg
Published: March 2007

Mitochondrial and cytosolic functions were studied in vivo and in perfused livers from rats with secondary biliary cirrhosis induced by bile duct ligation for 5 wk and in sham–operated controls. The livers were stereo–logically analyzed, and mitochondrial and cytosolic functions were related to liver structure. Oxygen consumption by perfused livers expressed per stereo–logically determined mitochondrial volume was decreased by 49% in bile duct—ligated rats compared with control rats. Glucose production (expressed per mitochondrial volume) was reduced by more than 90% in bile duct ligation, whereas urea production was not affected. Lactate production, a cytosolic function, was increased fivefold in bile duct ligation, and both the lactate/pyruvate and the β–hydroxybutyrate/acetoacetate ratios were increased in the liver perfusate of bile duct—ligated rats. In comparison with control rats, the stereologically determined mitochondrial volume fraction per hepatocyte was increased by 28% in bile duct—ligated rats. Activities of mitochondrial enzymes expressed per area of mitochondrial membrane or per mitochondrial volume were either unchanged (ATPase, cytochrome c oxidase and glutamate dehydrogenase) or decreased (monoamine oxidase) in bile duct ligation. Thus in comparison with control rats, mitochondrial metabolism is impaired in perfused livers from bile duct—ligated rats; increased mitochondrial volume per hepatocyte may represent a strategy to maintain hepatic energy metabolism in rats with secondary biliary cirrhosis. (Hepatology 1992;15:1167–1172).
The Touch That Hepatocytes Seem to Like
Authors: Zaret Kenneth S.
Published: March 2007

We have identified the liver–regulating protein (LRP), a cell surface protein involved in the maintenance of hepatocyte differentiation when cocultured with rat liver epithelial cells (RLEC). LRP was defined by immunoreactivity to a monoclonal antibody (mAb L8) prepared from RLEC. mAb L8 specifically detected two polypeptides of 85 and 73 kD in immunoprecipitation of both hepatocyte– and RLEC–iodinated plasma membranes. The involvement of these polypeptides, which are integral membrane proteins, in cell interaction–mediated regulation of hepatocytes was assessed by evaluating the perturbing effects of the antibody on cocultures with RLEC. Several parameters characteristic of differentiated hepatocytes were studied, such as liver–specific and house–keeping gene expression, cytoskeletal organization and deposition of extracellular matrix (ECM). An early cytoskeletal disturbance was evidenced and a marked alteration of hepatocyte functional capacity was observed in the presence of the antibody, together with a loss of ECM deposition. By contrast, cell–cell aggregation or cell adhesion to various extracellular matrix components were not affected. These findings suggest that LRP is distinct from an extracellular matrix receptor. The fact that early addition of mAb L8 during cell contact establishment was necessary to be effective may indicate that LRP is a novel plasma membrane protein that plays an early pivotal role in the coordinated metabolic changes which lead to the differentiated phenotype of mature hepatocytes.
What Are the Intracellular Signals for Agonist–Activated Calcium Entry Into Hepatocytes?
Authors: Anwer Sawkat M.
Published: March 2007

The Ca2+ signal observed in individual fura–2–loaded hepatocytes stimulated with the α1–adrenergic agonist phenylephrine consisted of a variable latency period, a rapid biphasic increase in the cytosolic free Ca2+, followed by a period of maintained elevated cytosolic Ca2+ (plateau phase) that depended on the continued presence of both agonist and external Ca2+, Microinjection of guanosine–5′–O–(3–thiophosphate) elicited a Ca2+ transient with the same basic features. The Ca2+ transient resulting from microinjecting inositol 1,4,5–trisphosphate (Ins–1,4,5–P3) occurred with essentially no latency period and consisted of a rapid spike that decayed back to preinjection levels within 15 s. Microinjection of inositol 1,4–5–trisphosphorothioate (thio–IP3), a nonmetabolizable analog of Ins–1,4,5–P3, elicited a Ca2+ transient that was initially identical to that observed with Ins–1,4,5–P3, except that the cytosolic Ca2+ remained elevated. The maintained thio–IP3–induced Ca2+ increase was dependent on the presence of external Ca2+, suggesting an activation of Ca2+ influx. Reintroduction of external Ca2+ in the presence of 5 μM phenylephrine to Ca2+–depleted cells resulted in a 2–fold greater rate of rise in the cytosolic Ca2+ compared to the rate observed upon Ca2+ addition to cells Ca2+–depleted by preatement with thapsigargin. The rate of Ca2+ rise upon Ca2+ addition to cells microinjected with thio–IP3 was similar to that observed with phenylephrine. Coinjection of the cells with thio–IP3 plus heparin reduced the rate of Ca2+ rise upon Ca2+ addition to that observed in thapsigargin–treated cells. These data indicate that the mechanism responsible for receptor–mediated stimulation of Ca2+ entry into hepatocytes involves not only capacitative Ca2+ entry but also an additional component mediated directly by Ins–1,4,5–P3.