Neutral Sugar Fragments from Antibiotic K-52B

The neutral constituent sugars of antibiotic K-52B and their glycosidic linkages were examined by methylation analysis and Smith degradation. After partial acid hydrolysis of K-52B, neutral oligosaccharides I, II and III were isolated, and the constituent sugars of each oligosaccharide and their glycosidic linkages were similarly examined. K-52B was found to contain α-d-glc_p-(l → 4)-d-gal_p-(l → 4)-l-fuc and l-ara_f-(1 → 4)-d-gal-(l → as neutral sugar fragments.     

Expression profiles of MUC mucins and trefoil factor family (TFF) peptides in the intrahepatic biliary system: physiological distribution and pathological significance

Mucin secreted by mucosal epithelial cells plays a role in the protection of the mucosal surface and also is involved in pathological processes. So far, MUC1-4, 5AC, 5B, 6-8, 11-13 and 15-17 genes coding the backbone mucin core protein have been identified in humans. Their diverse physiological distribution and pathological alterations have been reported. Trefoil factor family (TFF) peptides are mucin-associated molecules co-expressed with MUC mucins and involved in the maintenance of mucosal barrier and the biological behavior of epithelial and carcinoma cells. Intrahepatic biliary system is a route linking the bile canaliculi and the extrahepatic bile duct for the excretion of bile synthesized by hepatocytes. Biliary epithelial cells line in the intrahepatic biliary system, secreting mucin and other molecules involved in the maintenance and regulation of the system. In this review, the latest information regarding properties, expression profiles and regulation of MUC mucins and TFF peptides in the intrahepatic biliary system is summarized. In particular, we focus on the expression profiles and their significance of MUC mucins in developmental and normal livers, various hepatobiliary diseases and intrahepatic cholangiocarcinoma.     

Isolation of Neonatal Extrahepatic Cholangiocytes

The intra and extrahepatic bile ducts of the liver are developmentally distinct, and may be differentially affected by certain diseases. However, differences between intra and extrahepatic cholangiocytes, and between neonatal and adult cells, are not well understood.Methods for the isolation of cholangiocytes from intrahepatic bile ducts are well established^1-4. Isolation of extrahepatic ductal cells, especially from the neonate, has not yet been described, although this would be of great benefit in understanding the differences between distinct cholangiocyte populations and in studying diseases such as biliary atresia that appear to target the extrahepatic ducts. Described here is an optimized technique to isolate both neonatal and adult mouse extrahepatic bile duct cells. This technique yields a pure cell population with minimal contamination from mesenchymal cells like fibroblasts.This method is based on the removal of the extrahepatic ducts and gallbladder, followed by meticulous dissection and scraping to remove fat and fibroblast layers. Structures are embedded in thick layers of collagen and cultured for approximately 3 weeks to allow outgrowth of cholangiocytes in monolayers, which can then be trypsinized and re plated for experimental use.     

Histopathology findings in common marmosets (<Emphasis Type="Italic">Callithrix jacchus</Emphasis> Linnaeus, 1758) with chronic weight loss associated with bile tract obstruction by infestation with <Emphasis Type="Italic">Platynosomum</Emphasis> (Loos, 1907)

Chronic weight loss in marmosets is often associated with wasting marmoset syndrome (WMS), an important disease that occurs in callitrichid colonies around the world. Even though its etiology is very difficult to determine, particular variables, such as weight loss, diarrhea and alopecia, associated or not with infestation in the pancreatic ducts with Trichospirura leptossoma (Nematoda: Thelazioidea), seem to be linked with the syndrome. This study investigated the histopathology of the lungs, duodenum, liver, gallbladder, extrahepatic bile ducts and pancreatic ducts of six common marmosets (Callithrix jacchus) suffering from severe non-diarrheic weight loss. Three individuals died naturally and the other three were euthanized. Microscopic findings showed the presence of adult flukes (Platynosomum) in the liver. These flukes, which provoke common infection in cats, were also observed inside the gallbladder as well as in the intra and extrahepatic bile ducts in common marmosets. Portal fibrosis was observed in two animals, which developed chronic fibrosing hepatopathy (biliary pattern, grade 3). The disease progresses without diarrhea and without pancreatic lesions or infestation. With the progression, the animals presented with ascending cholangitis, cholestasis and portal fibrosis, sometimes culminating in secondary biliary cirrhosis. Therefore, this infirmity, associated with chronic weight loss in common marmosets, could be another etiological factor linked with WMS.     

Hepatic ABCG5/G8 overexpression reduces apoB-lipoproteins and atherosclerosis when cholesterol absorption is inhibited

We previously reported that liver-specific overexpression of ABCG5/G8 in mice is not atheroprotective, suggesting that increased biliary cholesterol secretion must be coupled with decreased intestinal cholesterol absorption to increase net sterol loss from the body and reduce atherosclerosis. To evaluate this hypothesis, we fed low density lipoprotein receptor-knockout (LDLr-KO) control and ABCG5/G8-transgenic (ABCG5/G8-Tg)xLDLr-KO mice, which overexpress ABCG5/G8 only in liver, a Western diet containing ezetimibe to reduce intestinal cholesterol absorption. On this dietary regimen, liver-specific ABCG5/G8 overexpression increased hepatobiliary cholesterol concentration and secretion rates (1.5-fold and 1.9-fold, respectively), resulting in 1.6-fold increased fecal cholesterol excretion, decreased hepatic cholesterol, and increased (4.4-fold) de novo hepatic cholesterol synthesis versus LDLr-KO mice. Plasma lipids decreased (total cholesterol, 32%; cholesteryl ester, 32%; free cholesterol, 30%), mostly as a result of reduced non-high density lipoprotein-cholesterol and apolipoprotein B (apoB; 36% and 25%, respectively). ApoB-containing lipoproteins were smaller and lipid-depleted in ABCG5/G8-TgxLDLr-KO mice. Kinetic studies revealed similar 125I-apoB intermediate density lipoprotein/LDL fractional catabolic rates, but apoB production rates were decreased 37% in ABCG5/G8-TgxLDLr-KO mice. Proximal aortic atherosclerosis decreased by 52% (male) and 59% (female) in ABCG5/G8-TgxLDLr-KO versus LDLr-KO mice fed the Western/ezetimibe diet. Thus, increased biliary secretion, resulting from hepatic ABCG5/G8 overexpression, reduces atherogenic risk in LDLr-KO mice fed a Western diet containing ezetimibe. These findings identify distinct roles for liver and intestinal ABCG5/G8 in modulating sterol metabolism and atherosclerosis.     

Delineation of molecular changes in intrahepatic cholesterol metabolism resulting from diminished cholesterol absorption

The absorption of cholesterol by the small intestine is a major route for the net entry of cholesterol into the body and can therefore affect the plasma low density lipoprotein-cholesterol (LDL-C) concentration. These studies used ezetimibe, a potent inhibitor of cholesterol absorption, to delineate the biochemical and molecular changes in intrahepatic metabolism and biliary lipid secretion when there is a major reduction in chylomicron cholesterol delivery to the liver. In female LDL receptor (LDLR)-deficient (LDLR-/-) mice fed a basal diet containing ezetimibe (0-10 mg/day/kg body weight), cholesterol absorption was reduced up to 91%, fecal neutral sterol excretion was increased up to 4.7-fold, and plasma total cholesterol concentrations decreased by up to 18%. Blocking cholesterol absorption prevented the accumulation of very low density lipoproteins and LDL in the circulation of LDLR-/- mice fed a lipid-rich diet. In female LDLR+/+ mice fed the lipid-rich diet with ezetimibe, the relative mRNA level for the LDLR in the liver was 2-fold greater than in matching mice given the lipid-rich diet alone. We conclude that in the mouse the reduction in plasma LDL-C levels induced by blocking cholesterol absorption reflects both a diminished rate of LDL-C production and a modest increase in hepatic LDLR expression.     

奥曲肽辅助十二指肠镜治疗急性水肿型胆源性胰腺炎疗效及对炎症反应的影响

目的:探讨奥曲肽辅助十二指肠镜治疗急性水肿型胆源性胰腺炎疗效及对炎症反应的影响。方法:选择急性水肿型胆源性胰腺炎患者100例,随机分为研究组与对照组各50例,两组患者均给予禁食、抗感染、营养支持、酶抑制剂治疗,对照组同期给予十二指肠镜治疗,研究组同期给予奥曲肽辅助十二指肠镜治疗,比较两组腹痛缓解时间,白细胞(WBC)、体温恢复正常时间,治疗前(T0)、治疗3d(T1)、治疗7d(T2)血、尿淀粉酶水平与炎症因子CRP、IL-6、TNF-α水平的变化。结果:(1)研究组腹痛缓解时间、WBC、体温恢复正常时间均较对照组明显缩短(P0.05);(2)两组患者T1、T2血、尿淀粉酶水平均较T0明显降低,且T2血、尿淀粉酶水平低于T1(P0.05),研究组T1、T2血、尿淀粉酶水平均低于对照组(P0.05);(3)两组T1、T2血清CRP、IL-6、TNF-α水平均较T0明显降低,且T2低于T1(P0.05),研究组T1、T2时血清CRP、IL-6、TNF-α水平均低于对照组(P0.05)。结论:奥曲肽辅助十二指肠镜治疗急性水肿型胆源性胰腺炎可提高早期治疗效果,减轻炎症反应。     

Misregulation of membrane trafficking processes in human nonalcoholic steatohepatitis

Nonalcoholic steatohepatitis (NASH) remodels the expression and function of genes and proteins that are critical for drug disposition. This study sought to determine whether disruption of membrane protein trafficking pathways in human NASH contributes to altered localization of multidrug resistance‐associated protein 2 (MRP2). A comprehensive immunoblot analysis assessed the phosphorylation, membrane translocation, and expression of transporter membrane insertion regulators, including several protein kinases (PK), radixin, MARCKS, and Rab11. Radixin exhibited a decreased phosphorylation and total expression, whereas Rab11 had an increased membrane localization. PKCδ, PKCα, and PKA had increased membrane activation, whereas PKCε had a decreased phosphorylation and membrane expression. Radixin dephosphorylation may activate MRP2 membrane retrieval in NASH; however, the activation of Rab11/PKCδ and PKA/PKCα suggest an activation of membrane insertion pathways as well. Overall these data suggest an altered regulation of protein trafficking in human NASH, although other processes may be involved in the regulation of MRP2 localization.     

Primary biliary cirrhosis sera recognize not only gp210 but also proteins of the p62 complex bearing N-acetylglucosamine residues from rat liver nuclear envelope

We have recently observed reactivity of primary biliary cirrhosis (PBC) sera with several proteins bearing N-acetylglucosamine residues from rat liver nuclear envelopes. The aim of this study was to characterize the reactive antigens. Sera from 31 patients with PBC, 30 with rheumatoid arthritis (RA) and 30 with Sjögren's syndrome (SS) were examined. Rim-like immunofluorescence staining was observed in 15 of 31 (48%) sera from patients with PBC, in 1 of 30 with RA and in 1 of 30 with SS. Upon immunoblotting using preparations of whole rat liver nuclear envelopes and their Triton X 100-KCl extract as antigen souces, a 200 kDa protein band was observed in 9 of sera with PBC. Furthermore, upon immunoblotting using the wheat germ aggulutinin-bound fraction of rat liver envelope as antigen, 62, 60 and 54 kDa protein bands corresponding to components of the p62 complex in the nuclear pore complex (Kita et al. Biochem. 113, 377–382) were observed in 7, 5 and 6 samples respectively, of the 31 PBC sera. Our data suggest that PBC sera recognize not only the 210 kDa protein but also the p62 complex proteins.Abbreviations ANA antinuclear antibody - AMA anti-mitochondrial antibodies - IF immunofluorescence - LAP2 lamina-associated polypeptide 2 - LBR lamin B receptor - anti-NBP 60 anti-nuclear localization signal binding protein 60 - NE nuclear envelope - NPC nuclear pore complex - PBC primary biliary cirrhosis - RA rheumatoid arthritis - SLE systemic lupus erythematosus - SS Sjögren's syndrome - WGA wheat germ agglutinin     

Long-term primary culture of epithelial cells from rainbow trout (Oncorhynchus mykiss) liver

Summary Long-term primary cultures of epithelial cells from rainbow trout (Oncorhynchus mykiss) liver have been established. Nearly homogenous (>97%) populations of hepatocytes were placed into primary culture and remained viable and proliferative for at least 70 d. In addition to hepatocytes, proliferative biliary cells persisted in the cultures for at least 30 d. Finally, a third type of epithelial cell, which we have termed a “spindle cell,” consistently appeared and proliferated to confluence in these cultures. The confluent cultures of spindle cells were successfully subcultured and passaged. The initial behavior, growth, and optimization of serum and media requirements for these cells is described. All three cell types proliferated as measured by thymidine incorporation, autoradiography, proliferating cellular nuclear antigen analysis, and propidium iodine staining. Further efforts to characterize the cells included western blotting and immunohistochemical staining with antibodies to cytokeratins previously reported in fish liver. From these data, it appears that all three cell populations are epithelial in nature. Furthermore, significant changes in actin organization, often indicative of transformation or pluripotent cells, were observed with increased time in primary culture.