PubMed Health⌕ Search

Biomedical subjects

M Arrese

Publications and source records attributed to M Arrese.

At least 19 recordsLinked to original sources
PubMed2004

Bile secretory function in the obese Zucker rat: evidence of cholestasis and altered canalicular transport function.

BACKGROUND: Obese Zucker rats (ZR) have been used as an experimental model for non-alcoholic fatty liver disease and are particularly susceptible to various types of liver injury. Bile secretory function has not been assessed in ZR. AIM: To study bile secretion and expression of the main hepatobiliary transporters in ZR. METHODS: Bile flow and biliary secretion of lipids and glutathione were determined in eight and 14 week old obese ZR and their lean controls. Protein mass and mRNA of the Na(+)/taurocholate cotransporting polypeptide (Ntcp), the bile salt export pump (Bsep), and the multidrug resistant associated protein 2 (Mrp2) were assessed by western and northern blot, respectively. The effects of administration of a tumour necrosis factor alpha inactivator (etanercept) and an insulin sensitiser (rosiglitazone) were assessed in obese ZR while leptin was given to non-obese rats to study its effect on Mrp2 expression. RESULTS: ZR exhibited increased body weight and hyperlipidaemia. Only 14 week old obese ZR has fatty liver. Decreased bile flow and biliary lipid and glutathione secretion as well as reduced hepatic transport of both taurocholate and bromosulphthalein were found in obese ZR. Hepatic Mrp2 protein mass was markedly reduced (-70%) in obese rats while Ntcp and Bsep protein levels were similar to lean rats. Downregulation of Mrp2 seems to involve both transcriptional and post-transcriptional mechanisms probably related to insulin and leptin resistance. CONCLUSIONS: Obese ZR exhibit an impaired bile secretory function with significant functional and molecular alterations consistent with mild cholestasis. A defective hepatobiliary transport capacity may be a contributory factor in rendering the obese ZR more susceptible to liver injury.

Animals↗
PubMed2000

Differential expression of canalicular membrane Ca2+/Mg(2+)-ecto-ATPase in estrogen-induced and obstructive cholestasis in the rat.

Extracellular adenosine triphosphate (ATP) may regulate hepatocyte and cholangiocyte functions, and under some conditions it may have deleterious effects on bile secretion and cause cholestasis. The canalicular membrane enzyme Ca2+/Mg2+-ecto-ATPase (ecto-ATPase) hydrolyzes ATP/adenosine diphosphate (ATP/ADP) and regulates hepatic extracellular ATP concentration. Changes in liver ecto-ATPase in estrogen-induced cholestasis were examined in male rats receiving 17alpha-ethinylestradiol (E groups) for 1, 3, or 5 days (5 mg/kg/day, sc) and compared with changes in rats subjected to obstructive cholestasis (O groups) for 1, 3, or 8 days. Activity of ecto-ATPase, protein mass in canalicular membranes and bile (estimated by Western blotting), steady state mRNA levels (by Northern blotting), and cellular and acinar distributions of the enzyme (histochemistry and immunocytochemistry) were assessed in these groups. Activity of ecto-ATPase, protein mass in isolated canalicular membranes, and enzyme mRNA levels were significantly increased in E group rats as compared with controls. In contrast, these parameters were markedly decreased in O group rats, and the enzyme protein was undetectable in bile. The ecto-ATPase histochemical reaction was markedly increased in the canalicular membrane of E group rats, extending from acinar zone 2 to zone 1, whereas it decreased in the O group. The ecto-ATPase immunocytochemical reaction was present in the canalicular membrane and pericanalicular vesicles in control and E group hepatocytes, but it decreased in obstructive cholestasis and was localized only to the canalicular membrane. Thus, significant changes in liver ecto-ATPase were apparent in 17alpha-ethinylestradiol-induced cholestasis that were opposite to those observed in obstructive cholestasis. Assuming that the alterations observed in obstructive cholestasis are the result of the cholestatic phenomenon, we conclude that changes in ecto-ATPase in 17alpha-ethinylestradiol-treated rats might be either primary events or part of an adaptive response in 17alpha-ethinylestradiol-induced cholestasis.

Adenosine Triphosphatases↗
PubMed2000

Ileal bile acid transport regulates bile acid pool, synthesis, and plasma cholesterol levels differently in cholesterol-fed rats and rabbits.

We investigated the effect of ileal bile acid transport on the regulation of classic and alternative bile acid synthesis in cholesterol-fed rats and rabbits. Bile acid pool sizes, fecal bile acid outputs (synthesis rates), and the activities of cholesterol 7alpha-hydroxylase (classic bile acid synthesis) and cholesterol 27-hydroxylase (alternative bile acid synthesis) were related to ileal bile acid transporter expression (ileal apical sodium-dependent bile acid transporter, ASBT). Plasma cholesterol levels rose 2.1-times in rats (98 +/- 19 mg/dl) and 31-times (986 +/- 188 mg/dl) in rabbits. The bile acid pool size remained constant (55 +/- 17 mg vs. 61 +/- 18 mg) in rats but doubled (254 +/- 46 to 533 +/- 53 mg) in rabbits. ASBT protein expression did not change in rats but rose 31% (P < 0.05) in rabbits. Fecal bile acid outputs that reflected bile acid synthesis increased 2- and 2.4-times (P < 0.05) in cholesterol-fed rats and rabbits, respectively. Cholesterol 7alpha-hydroxylase activity rose 33% (24 +/- 2.4 vs. 18 +/- 1.6 pmol/mg/min, P < 0.01) and mRNA levels increased 50% (P < 0.01) in rats but decreased 68% and 79%, respectively, in cholesterol-fed rabbits. Cholesterol 27-hydroxylase activity remained unchanged in rats but rose 62% (P < 0.05) in rabbits. Classic bile acid synthesis (cholesterol 7alpha-hydroxylase) was inhibited in rabbits because an enlarged bile acid pool developed from enhanced ileal bile acid transport. In contrast, in rats, cholesterol 7alpha-hydroxylase was stimulated but the bile acid pool did not enlarge because ASBT did not change. Therefore, although bile acid synthesis was increased via different pathways in rats and rabbits, enhanced ileal bile acid transport was critical for enlarging the bile acid pool size that exerted feedback regulation on cholesterol 7alpha-hydroxylase in rabbits.

Animals↗
PubMed1999

Mutational analysis of influenza A virus nucleoprotein: identification of mutations that affect RNA replication.

The influenza A virus nucleoprotein (NP) is a multifunctional polypeptide which plays a pivotal role in virus replication. To get information on the domains and specific residues involved in the different NP activities, we describe here the preparation and characterization of 20 influenza A virus mutant NPs. The mutations, mostly single-amino-acid substitutions, were introduced in a cDNA copy of the A/Victoria/3/75 NP gene and, in most cases, affected residues located in regions that were highly conserved across the NPs of influenza A, B, and C viruses. The mutant NPs were characterized (i) in vivo (cell culture) by analyzing their intracellular localization and their functionality in replication, transcription, and expression of model RNA templates; and (ii) in vitro by analyzing their RNA-binding and sedimentation properties. The results obtained allowed us to identify both a mutant protein that accumulated in the cytoplasm and mutations that altered the functionality and/or the oligomerization state of the NP polypeptide. Among the mutations that reduced the NP capability to express chloramphenicol acetyltransferase protein from a model viral RNA (vRNA) template, some displayed a temperature-sensitive phenotype. Interestingly, four mutant NPs, which showed a reduced functionality in synthesizing cRNA molecules from a vRNA template, were fully competent to reconstitute complementary ribonucleoproteins (cRNPs) capable of synthesizing vRNAs, which in turn yielded mRNA molecules. Based on the phenotype of these mutants and on previously published observations, it is proposed that these mutant NPs have a reduced capability to interact with the polymerase complex and that this NP-polymerase interaction is responsible for making vRNPs switch from mRNA to cRNA synthesis.

Amino Acid Sequence↗
PubMed1999

[Molecular biology and medicine: basic concepts].

Through the advancements of molecular genetics, physicians and researchers are in an extraordinary period of study concerning the molecular basis of medicine. Molecular biology is making a tremendous impact on both diagnosis and treatment of diseases through the clinical introduction of molecular methods. These techniques, restricted for many years to basic biological research, include the polymerase chain reaction, DNA and protein electrophoresis, cloning of genes into viral or bacterial vectors and methods to rapidly sequence DNA and identify mutations. In this article the authors attempt to provide basic concepts on these themes for the non-trained physicians in order to help them to understand recent developments and foresee their future implications.

DNA↗
PubMed1999

[Molecular medicine: present and future].

The genetic background of individuals is recognized as an important clue in the analysis of classical hereditary and multifactorial acquired diseases. This new concept derives from the development and increasing use of molecular genetics in clinical medicine. The application of molecular biology techniques in biomedical investigation has encompassed the identification of the pathogenesis and etiology of diseases, prenatal diagnosis the production of new therapeutic agents, gene therapy and the development of pharmacogenetics. The impact on the fundamentals and practice of clinical medicine that will have the use of molecular biology is analyzed in this review.

Animals↗
PubMed1999

[Identification of molecular defects in liver diseases. Recent advances].

Recent molecular studies have resulted in the identification of genetic alterations underlying several hereditary disorders of the liver. Cloning of disease genes are increasing our understanding of the basic defects in liver diseases. This review focuses on selected inherited liver diseases such as hyperbilirubinemic syndromes, hemochromatosis, Wilson disease and genetic cholestatic syndromes and illustrate the knowledge gained on these disorders from molecular studies. Potential implications of the identification of disease genes such as practical applications for diagnosis, information on prognosis and the possibility to design new therapies are discussed.

Cholestasis↗
PubMed1999

[Hepatotoxicity by amoxicillin/clavulanic acid: case report].

We report a 72 years old diabetic male that, after the use of combined amoxicillin-clavulanic acid, developed pruritus and jaundice. Liver function tests showed serum total bilirubin of 4.3 mg/dL aspartate aminotransferase 140 U/l (normal < 35 U/L), alanine aminotransferase 470 U/L (normal < 40) and alkaline phosphatases of 400 U/L (normal < 100). Serology for hepatitis A, B and C viruses was negative, ERCP showed a normal biliary tree and liver biopsy disclosed a cholestatic hepatitis. Ursodeoxycholic was started to relieve pruritus. Liver function tests improved shortly thereafter, suggesting that this drug may be useful in the treatment of drug induced cholestasis.

Aged↗
PubMed1998

Endotoxin downregulates rat hepatic ntcp gene expression via decreased activity of critical transcription factors.

Sodium-dependent uptake of bile acids across the hepatic basolateral membrane is rapidly and profoundly diminished during sepsis, thus contributing to the pathogenesis of sepsis-associated cholestasis. This effect is mediated by endotoxin or effector cytokines, which reduce expression of several hepatobiliary transporters, including the sodium-dependent bile acid transporter gene, ntcp. We test here the hypothesis that endotoxin treatment leads to impaired binding activity of ntcp promoter trans-acting factors, resulting in reduction of ntcp mRNA expression. After endotoxin administration, ntcp mRNA levels reached their nadir by 16 h, and nuclear run-on assays demonstrated a marked reduction in ntcp gene transcription. At 16 h after treatment, nuclear binding activities of two key factors that transactivate the ntcp promoter, hepatocyte nuclear factor (HNF) 1 and Footprint B binding protein (FpB BP), decreased to 44 and 47% of pretreatment levels, respectively, while levels of the other known ntcp promoter transactivator, signal transducer and activator of transcription 5, were unaffected. In contrast, the universal inflammatory response factors nuclear factor kappaB and activating protein 1 were both upregulated significantly. Examination of nuclear extracts obtained at sequential time points revealed that the maximal decrease in nuclear activities of both HNF1 and FpB BP preceded the nadir of ntcp mRNA expression by 6-10 h. Furthermore, these two nuclear factors returned towards normal levels before the recovery of ntcp mRNA levels observed by 48 h. Since HNF1alpha mRNA levels were unchanged at all time points, HNF1 is likely to be regulated posttranscriptionally by endotoxin. We conclude that the downregulation of ntcp gene expression by endotoxin is mediated at the level of transcription through tandem reductions in the nuclear binding activity of two critical transcription factors. These findings provide new insight into the coordinated downregulation of hepatobiliary transporters during sepsis.

Animals↗
PubMed1998

Maternal cholestasis does not affect the ontogenic pattern of expression of the Na+/taurocholate cotransporting polypeptide (ntcp) in the fetal and neonatal rat liver.

To assess the effects of cholestasis during pregnancy on fetal and neonatal mRNA expression, protein mass, and function of the Na+/taurocholate cotransporting polypeptide (Ntcp), common bile duct ligation (BDL) was performed in pregnant rats on day 14 of pregnancy (maternal cholestasis [MC] group), and livers were harvested at days 20 and 21 of fetal life, as well as at days 4, 7, 14, 21, and 28 after birth. Sham-operated rats and their litters were used as controls. Ntcp steady-state mRNA levels, protein mass, and function were determined by Northern blotting, immunoblotting, and taurocholate (TC) transport studies in isolated short-term cultured hepatocytes, respectively. In addition, protein mass and function of the organic anion transporting polypeptide (Oatp1), another sinusoidal bile acid transporter, were studied at 4 weeks of age. The majority of pregnant cholestatic rats (94%) were able to carry pregnancy to term. Body and liver weights of the offspring from the MC group were lower than those from sham-operated animals at all postnatal time points. Ntcp steady-state mRNA levels, protein mass, and function were unaffected by MC. The ontogenic pattern of expression was identical in offspring from MC and controls with detection of the Ntcp mRNA at day 21 of fetal life. There was a significant increase in mRNA postnatally, reaching adult levels by 7 days of age. Protein mass and function of Ntcp as well as of Oatp1 were similar in offspring from MC and control groups at 4 weeks of age. In conclusion, maternal obstructive cholestasis during the last third of pregnancy does not affect the fetal/neonatal expression of the basolateral bile acid transporters, Ntcp and Oatp1. This suggests that the impaired bile acid excretion described in this experimental model is not related to altered uptake of bile acids in the affected neonate.

Animals↗
PubMed1998

Neither intestinal sequestration of bile acids nor common bile duct ligation modulate the expression and function of the rat ileal bile acid transporter.

The regulatory responses of bile acid (BA) transport in the terminal ileum to perturbations in BA homeostasis are complex, and conflicting results have been reported by different investigators. These studies were designed to examine the response of this system to a reduction in ileal bile salt concentrations at both a functional and molecular level. Common bile duct ligation (BDL) or feeding of a novel bile acid-binding compound, GT31-104HB, for 7 days were used to reduce ileal apical membrane bile salt flux. Apical bile acid transport function was assessed by examining sodium-dependent uptake of [3H]-taurocholate (TC) into brush border membrane vesicles (BBMV). Expression of the apical sodium-dependent bile acid transporter (ASBT) and the ileal lipid-binding protein (ILBP) were assessed by Western blotting with quantitation using [125I]-labeled secondary antibody and a phosphorimager. Neither common BDL nor intestinal sequestration of BA led to a change in ileal bile acid transport function or the expression of the ASBT or the ILBP. These results indicate that a reduction in presentation of bile salts to the apical surface of the terminal ileum does not modulate the expression of the genes involved in their transport.

Allylamine↗
PubMed1998

Molecular analysis of the adaptive response of intestinal bile acid transport after ileal resection in the rat.

BACKGROUND & AIMS: The apical sodium-dependent bile acid transporter is critical for intestinal reclamation of bile salts. Its expression and activity, along with the ileal lipid-binding protein, were studied before and after intestinal resection in the rat. METHODS: The effects of surgical resection and bile acid feeding on the expression of ileal bile acid transport were assessed by a combination of functional (taurocholate uptake into crude brush border membrane vesicles) and molecular assays (Northern and Western blotting). RESULTS: Transport, apical sodium-dependent bile acid transporter and ileal lipid-binding protein messenger RNA and protein expression were restricted to the distal 30 cm of ileum. After resection, transport and expression were limited to the remaining portions of this segment. Limited ileal resection increased protein mass and, therefore, transport in the terminal 5 cm of ileum without a specific increase in transporter gene expression. Increased bile acid presentation to the terminal ileum did not induce ileal hyperplasia. Eighty-five percent intestinal resection led to ileal hypertrophy and a specific repression in bile acid transport activity. CONCLUSIONS: Native and compensatory bile acid transporter gene expression occur predominantly in the terminal 30 cm of ileum. The specific ileal responses to intestinal resection are dependent on the extent of resection.

Adaptation, Physiological↗
PubMed1998

Role of glutathione in hepatic bile formation during reperfusion after cold ischemia of the rat liver.

BACKGROUND/AIMS: Liver reperfusion following cold ischemia is frequently associated with diminished bile flow in patients undergoing liver transplantation. Glutathione is a major determinant of bile-acid independent bile flow, and the effects of cold ischemia on biliary glutathione excretion are unknown. METHODS: We examined the effects of cold ischemia (University of Wisconsin solution (4 degrees C), 24 h) with subsequent reperfusion (100 min) on biliary glutathione excretion in a recirculating system. Since glutathione might represent an important antioxidant within the biliary tract and oxidative stress in the biliary tract during reperfusion could contribute to the pathogenesis of bile duct injury after liver transplantation, we also assessed bile duct morphology in reperfused livers of mutant TR- -rats, in whom biliary excretion of glutathione is already impaired. RESULTS: Hepatic bile formation was diminished in reperfused Wistar rat livers after cold ischemia. Biliary glutathione concentrations and output were significantly decreased and correlated with postischemic changes in bile secretion. An increased biliary oxidized glutathione/glutathione ratio, indicating oxidative stress, was detected only immediately after the onset of reperfusion. Basal bile flow rates in TR- -rat livers which were already markedly reduced in control-perfused livers, decreased further during the early but not the later reperfusion period. Reperfusion of both Wistar and TR- -rat livers was not associated with electron microscopic evidence of bile duct damage. CONCLUSIONS: We conclude that impaired biliary excretion of glutathione contributes to decreased bile flow after cold ischemia. The absence of biliary glutathione does not appear to promote ultrastructural evidence of bile duct injury during reperfusion in the isolated perfused rat liver.

Adenosine↗
PubMed1998

Hepatobiliary transport: molecular mechanisms of development and cholestasis.

The secretion of bile requires the vectorial transport of organic and inorganic solutes from sinusoidal blood to the canalicular lumen. Hydrostatic forces cannot account for biliary secretion, because secretory pressures within bile ducts exceed that of blood within the sinusoidal space. Instead, the process of bile formation requires active transport across the basolateral membrane, transcellular movement through a variety of mechanisms, and then active transport into the canalicular space between hepatocytes. Separate hepatic and ductular transport mechanisms allow for rapid regulation of bile volume and composition required for changing physiologic needs. The array of transport proteins localized to both poles of the hepatocyte have been characterized physiologically and during development. Many have now been cloned and studied further in transgenic models. The recent identification and characterization of several genes that are mutated in inherited forms of cholestatic liver disease have provided new insight into the normal physiology of bile secretion, the pathophysiology of intrahepatic cholestasis, and an unexpected major role for a novel group of P-type ATPases in human biology and disease.

Adult↗
PubMed1997

The rat canalicular conjugate export pump (Mrp2) is down-regulated in intrahepatic and obstructive cholestasis.

BACKGROUND & AIMS: The excretion of various organic anions into bile is mediated by an adenosine triphosphate-dependent conjugate export pump, which has been identified as the canalicular isoform of the multidrug resistance protein (Mrp2). Mrp2 function is impaired in various experimental models of intrahepatic and obstructive cholestasis, but the underlying molecular mechanisms are unclear. The aim of this study was to investigate these molecular mechanisms. METHODS: The effects of endotoxin, ethinylestradiol, and common bile duct ligation (CBDL) on Mrp2 protein, messenger RNA (mRNA) expression, and Mrp2 tissue localization were determined in rat livers by Northern blotting, Western analysis, and tissue immunofluorescence. To assess whether changes were specific for Mrp2, we also examined the expression of canalicular ecto-adenosine triphosphatase (ecto-ATPase) and mdr P-glycoproteins (P-gp). RESULTS: All three cholestatic models resulted in a marked decrease in Mrp2 protein (P < 0.01) and its tissue localization at the canalicular membrane. Mrp2 mRNA levels diminished profoundly after endotoxin (P < 0.0005) and CBDL (P < 0.05), but did not change after ethinylestradiol. In contrast to Mrp2, protein expression of ecto-ATPase and P-gp remained unchanged in endotoxin- and ethinylestradiol-treated animals, whereas P-gp levels increased after CBDL (P < 0.05). CONCLUSIONS: Down-regulation of Mrp2 expression may explain impaired biliary excretion of amphiphilic anionic conjugates in these models of cholestasis.

ATP-Binding Cassette Transporters↗