Phenotypic changes induced by expression of beta-galactoside alpha2,6 sialyltransferase I in the human colon cancer cell line SW948

Beta-galactoside alpha2,6 sialyltransferase (ST6Gal.I), the enzyme which adds sialic acid in alpha2,6-linkage on lactosaminic termini of glycoproteins, is frequently overexpressed in cancer, but its relationship with malignancy remains unclear. In this study, we have investigated the phenotypic changes induced by the expression of alpha2,6-sialylated lactosaminic chains in the human colon cancer cell line SW948 which was originally devoid of ST6Gal.I. Clones derived from transfection with the ST6Gal.I cDNA were compared with untransfected cells and mock transfectants. The ST6Gal.I-expressing clones show (1) increased adherence to fibronectin and collagen IV but not to hyaluronic acid. Treatment with Clostridium perfrigens neuraminidase reduces the binding to fibronectin and collagen IV of ST6Gal.I-expressing cells but not that of ST6Gal.I-negative cells; (2) accumulation and more focal distribution of beta1 integrins on the cell surface; (3) different distribution of actin fibers; (4) flatter morphology and reduced tendency to multilayer growth; (5) improved ability to heal a scratch wound; (6) reduced ability to grow at the subcutaneous site of injection in nude mice. Our data suggest that the presence of alpha2,6-linked sialic acid on membrane glycoconjugates increases the binding to extracellular matrix components, resulting in a membrane stabilization of beta1 integrins, further strengthening the binding. This mechanism can provide a basis for the flatter morphology and the reduced tendency to multilayer growth, resulting in a more ordered tissue organization. These data indicate that in the cell line SW948, the effect of ST6Gal.I expression is consistent with the attenuation of the neoplastic phenotype.     

Identification and functional expression of a second human beta-galactoside alpha2,6-sialyltransferase, ST6Gal II.

BLAST analysis of the human and mouse genome sequence databases using the sequence of the human CMP-sialic acid:beta-galactoside alpha-2,6-sialyltransferase cDNA (hST6Gal I, EC2.4.99.1) as a probe allowed us to identify a putative sialyltransferase gene on chromosome 2. The sequence of the corresponding cDNA was also found as an expressed sequence tag of human brain. This gene contained a 1590 bp open reading frame divided in five exons and the deduced amino-acid sequence didn't correspond to any sialyltransferase already known in other species. Multiple sequence alignment and subsequent phylogenic analysis showed that this new enzyme belonged to the ST6Gal subfamily and shared 48% identity with hST6Gal-I. Consequently, we named this new sialyltransferase ST6Gal II. A construction in pFlag vector transfected in COS-7 cells gave raise to a soluble active form of ST6Gal II. Enzymatic assays indicate that the best acceptor substrate of ST6Gal II was the free disaccharide Galbeta1-4GlcNAc structure whereas ST6Gal I preferred Galbeta1-4GlcNAc-R disaccharide sequence linked to a protein. The alpha2,6-linkage was confirmed by the increase of Sambucus nigra agglutinin-lectin binding to the cell surface of CHO transfected with the cDNA encoding ST6Gal II and by specific sialidases treatment. In addition, the ST6Gal II gene showed a very tissue specific pattern of expression because it was found essentially in brain whereas ST6Gal I gene is ubiquitously expressed.     

60例活动性肝硬变患者血清载脂蛋白AI与B的临床研究

目的：为了探讨活动性肝硬变患者的血清载脂蛋白AI（APOAI）、载脂蛋白B（APOB）与肝细胞损害的严重程度及其预后的关系。方法：采和速度比浊法检测60例活动性肝硬变患者的血清APOAI、APOB水平,并与其他肝脏生化试验进行比较。结果：活动肝硬变代偿性与失代偿性两组APOAI,APOB均有着显著性差异（P＜0．01,P＜0．05）。活动性肝硬变患者的APOAI与总胆红素（TB）、凝血酶原时间（PT）呈负相关性（P＜0．01,P＜0．01,与白蛋白（ALB）,胆碱酯酶（CHE）、纤维蛋白原（FIB）呈显著正相关性（P＜0．01,P＜0．01,P＜0．05）。APOX仅与FIB呈显著相关性（P＜0．05）,好转组与死亡组两组APOAI有显著性差异（P＜0．05）,APOB无显著性差异,结论：检测活动性肝硬变患者血清APOAI,APOB水平有助于判断肝硬变的严重程度,且APOAI比APOB更敏感、可靠。     

小型猪肝硬化模型肝脏病理及生化指标的变化

目的探讨小型猪肝硬化模型制备过程中肝脏病理学及生化指标的变化。方法选用同种系巴马小型猪50头,随机分为实验组40头,对照组10头,雌雄不限,4月龄,体重15-20kg。实验组予四氯化碳复合因素造模法喂养,正常组予饲料及清水喂养。于造模的0、4、8、12、16、20、24周末进行肝脏穿刺活检及耳缘静脉抽血进行数据采集,对比分析肝纤维化一肝硬化制模过程中的肝脏病理学及生化指标的变化。结果24周末,对照组获得数据S0期70例次;实验组获得数据轻度肝纤维化（S1＋S2期）82例次,重度肝纤维化（S3＋S4期）62例次,肝硬化共39例次。从正常组到肝硬化组丙氨酸转氨酶、天冬氨酸转氨酶、总胆红素、血清肌酐及球蛋白呈逐渐升高的趋势,而白蛋白,A／G比值呈逐渐下降的趋势,凝血酶原时间则逐渐延长,统计学分析,组间存在明显差异（P〈0．01）。结论四氯化碳复合因素造模法可以成功制备出小型猪肝硬化模型,肝纤维化不同阶段肝脏病理学和生化指标均发生明显变化。     

The role of exosomes in hepatitis,liver cirrhosis and hepatocellular carcinoma

Exosomes are small vesicles that were initially thought to be a mechanism for discarding unneeded membrane proteins from reticulocytes. Their mediation of intercellular communication appears to be associated with several biological functions. Current studies have shown that most mammalian cells undergo the process of exosome formation and utilize exosome‐mediated cell communication. Exosomes contain various microRNAs, mRNAs and proteins. They have been reported to mediate multiple functions, such as antigen presentation, immune escape and tumour progression. This concise review highlights the findings regarding the roles of exosomes in liver diseases, particularly hepatitis B, hepatitis C, liver cirrhosis and hepatocellular carcinoma. However, further elucidation of the contributions of exosomes to intercellular information transmission is needed. The potential medical applications of exosomes in liver diseases seem practical and will depend on the ingenuity of future investigators and their insights into exosome‐mediated biological processes.     

Embryonic liver fodrin involved in hepatic stellate cell activation and formation of regenerative nodule in liver cirrhosis

Transforming growth factor (TGF) β(1) plays a critical role in liver fibrosis. Previous studies demonstrated embryonic liver fodrin (ELF), a β-spectrin was involved in TGF-β/Smad signalling pathway as Smad3/4 adaptor. Here we investigate the role of ELF in pathogenesis of liver cirrhosis. In carbon tetrachloride (CCl(4))-induced mice model of liver cirrhosis, ELF is up-regulated in activated hepatic stellate cells (HSCs), and down-regulated in regenerative hepatocytes of cirrhotic nodules. In activated HSCs in vitro, reduction of ELF expression mediated by siRNA leads to the inhibition of HSC activation and procollagen I expression. BrdU assay demonstrates that down-regulation of ELF expression does not inhibit proliferation of activated HSCs in vitro. Immunostaining of cytokeratin 19 and Ki67 indicates that regenerative hepatocytes in cirrhotic liver are derived from hepatic progenitor cells (HPC). Further study reveals that HPC expansion occurs as an initial phase, before the reduction of ELF expression in regenerative hepatocytes. Regenerative hepatocytes in cirrhotic liver show the change in proliferative activity and expression pattern of proteins involved in G1/S transition, which suggests the deregulation of cell cycle in regenerative hepatocytes. Finally, we find that ELF participates in TGF-β/Smad signal in activated HSCs and hepatocytes through regulating the localization of Smad3/4. These data reveal that ELF is involved in HSC activation and the formation of regenerative nodules derived from HPC in cirrhotic liver.     

Protective role of melatonin in early‐stage and end‐stage liver cirrhosis

The liver is composed of hepatocytes, cholangiocytes, Kupffer cells, sinusoidal endothelial cells, hepatic stellate cells (HSCs) and dendritic cells; all these functional and interstitial cells contribute to the synthesis and secretion functions of liver tissue. However, various hepatotoxic factors including infection, chemicals, high‐fat diet consumption, surgical procedures and genetic mutations, as well as biliary tract diseases such as sclerosing cholangitis and bile duct ligation, ultimately progress into liver cirrhosis after activation of fibrogenesis. Melatonin (MT), a special hormone isolated from the pineal gland, participates in regulating multiple physiological functions including sleep promotion, circadian rhythms and neuroendocrine processes. Current evidence shows that MT protects against liver injury by inhibiting oxidation, inflammation, HSC proliferation and hepatocyte apoptosis, thereby inhibiting the progression of liver cirrhosis. In this review, we summarize the circadian rhythm of liver cirrhosis and its potential mechanisms as well as the therapeutic effects of MT on liver cirrhosis and earlier‐stage liver diseases including liver steatosis, nonalcoholic fatty liver disease and liver fibrosis. Given that MT is an antioxidative and anti‐inflammatory agent that is effective in eliminating liver injury, it is a potential agent with which to reverse liver cirrhosis in its early stage.     

Expression and distribution of the prolactin receptor in normal rat liver and in experimental liver cirrhosis.

Recent results have suggested a role for prolactin (PRL) as a regeneration factor in the liver. In order to investigate the involvement of prolactin in the pathogenesis of liver cirrhosis, we studied the expression of the prolactin receptor (PRLR) and PRL during the development of cirrhosis in an animal model. 30 male rats were exposed to CCl4 by inhalation. Phenobarbitone was added to the drinking water to accelerate the formation of toxic metabolites by enzyme induction. Two control groups of 30 animals each were treated with phenobarbitone only or received no treatment. 10 animals of each group were sacrificed 35, 55, and 70 days after initiation of treatment. Liver tissue was subjected to histological examination, which demonstrated fibrosis of different grades and cirrhosis in the CCl4-treated rats. Expression of PRLR mRNA was investigated by mRNA extraction, RT-PCR and computer-supported densitometric evaluation. Compared to control liver, PRLR mRNA was expressed at a higher level in fibrotic and cirrhotic liver specimens. In normal tissue, immunohistochemical staining showed a high concentration of PRLR around the central vein and in the epithelium of the bile ducts. This pattern of distribution was lost in fibrosis and cirrhosis. An accumulation of PRLR was demonstrated within the damaged cells. Neither PRL nor PRL mRNA was detectable in normal, fibrotic, or cirrhotic liver. We conclude that PRLR is distributed in normal rat liver in a typical pattern which is lost with increasing fibrosis. PRL is not produced by rat liver, indicating that PRL does not act through autocrine or paracrine mechanisms.     

Functions of autophagy in normal and diseased liver

Autophagy has emerged as a critical lysosomal pathway that maintains cell function and survival through the degradation of cellular components such as organelles and proteins. Investigations specifically employing the liver or hepatocytes as experimental models have contributed significantly to our current knowledge of autophagic regulation and function. The diverse cellular functions of autophagy, along with unique features of the liver and its principal cell type the hepatocyte, suggest that the liver is highly dependent on autophagy for both normal function and to prevent the development of disease states. However, instances have also been identified in which autophagy promotes pathological changes such as the development of hepatic fibrosis. Considerable evidence has accumulated that alterations in autophagy are an underlying mechanism of a number of common hepatic diseases including toxin-, drug- and ischemia/reperfusion-induced liver injury, fatty liver, viral hepatitis and hepatocellular carcinoma. This review summarizes recent advances in understanding the roles that autophagy plays in normal hepatic physiology and pathophysiology with the intent of furthering the development of autophagy-based therapies for human liver diseases.     

Amino acid composition of rat and human liver microsomes in normal and pathological conditions

The amino acid composition of proteins from liver microsomes has been studied in rats and in human subjects with normal liver, with obstructive jaundice or liver cirrhosis. The pattern of the amino acid composition of microsomes appeared to be species-specific. Phenylalanine, threonine, serine, proline, histidine and [aspartic acid plus asparagine] were increased, while alanine, tyrosine, glycine and arginine were decreased in the human compared to the rat microsomes. In patients with obstructive jaundice of short duration (less than two months) only a slight decrease in leucine and phenylalanine could be noticed, while in the case of liver cirrhosis amino acid composition was markedly changed.     

The expression of soluble and cell-bound alpha 2,6 sialyltransferase in human colonic carcinoma CaCo-2 cells correlates with the degree of enterocytic differentiation.

alpha 2,6 sialyltransferase towards the N-acetyllactosaminyl sequence (alpha 2,6 ST, E.C. 2.4.99.1) is one of the major sialyltransferases in human colonic cells; it strongly increases in human colorectal tumors and is largely expressed in fetal and neonatal rat colon. In this study we demonstrate that human colon carcinoma CaCo-2 cells, which differentiate spontaneously into enterocytes when maintained confluent for several days, exhibit a very high expression of alpha 2,6 ST both in the cell-bound and soluble form. When the CaCo-2 cells were cultured on porous membranes the soluble alpha 2,6 ST was mainly detected in the medium collected from the chamber corresponding to the basolateral face of the monolayer. The soluble alpha 2,6 ST could be concentrated and purified from the alpha 2,3 sialyltransferase by affinity chromatography on Blue Sepharose.     

Long-term effectiveness of high-dosed ornithine-aspartate on urea synthesis rate and portal hypertension in human liver cirrhosis

Summary The effectiveness of ammonia reducing amino acids on hyperammonemia and hepatic encephalopathy is well known in patients suffering from liver cirrhosis. Data concerning long-term therapy on hepatic function and urea synthesis rate (UNSR) are still lacking. According to Vilstrup/Poulsen it is a good standard for functioning liver mass. Therefore, 25 patients with histologically proven liver cirrhosis and distinct portal hypertension were treated daily with 9 gr. ornithinasparte over 13 years (8–20 years). Shunt operations, esophageal varicosis sclerosis, or portal pressure reducing medication were not applied. Rigorous alcohol abstinence and 60 gr protein/day were prescribed. During the investigation, 3 laparoscopies and 4 liver biopsies were performed, on the average, on each individual. Significant improvements of clinical and biochemical results (Child-Pugh-Index; Composite Clinical and Laboratory Index) were obtained during the long-term therapy with ornithine-aspartate. Esophageal varicosis II–III was either reduced to 0-I or totally eliminated. Also significant was an increased urea synthesis rate and a decreased hyperammonemia.A plausible explanation for the long-term therapy effectiveness with ornithine-aspartate is the possible recovery of the functioning mass without hepatic size increase. Also important is the rigorous alcohol abstinence. It leads to a significant reduction of portal hypertension in patients suffering from alcohol induced liver cirrhosis (Reynolds, own observations).Additional favorable factors are intensive muscle training and absence of gastrointestinal bleeds.     

Amino acid composition of human liver mitochondrial membranes in normal and pathological conditions

The amino acid composition of proteins from liver mitochondrial membranes has been studied in patients with normal liver, with biliary diseases and fatty liver, with obstructive jaundice or liver cirrhosis. A characteristic pattern of the amino acid composition in patients with normal liver has been found. In the mitochondrial membranes of patients with fatty liver tryptophan and lysine were decreased while [aspartic acid plus asparagine] and [glutamic acid plus glutamine] were increased compared to their counterpart in the normal liver. In patients with obstructive jaundice of short duration (less than two months) only a slight decrease in methionine content was found, while in the case of liver cirrhosis amino acid composition was markedly changed.deceased.     

Relationship between the level of serum L-tryptophan and its hepatic uptake and metabolism in rats with carbon tetrachloride-induced liver cirrhosis

Summary In the serum of rats with liver cirrhosis induced by 12-week intermittent carbon tetrachloride (CCl₄) injection, free L-tryptophan (Trp) levels increased with decreases in total Trp, albumin-bound Trp, and albumin levels. In the serum of the cirrhotic rats, there were no changes in the ratio of albumin-bound Trp to albumin and the level of free fatty acids which are known to weaken the binding of Trp to albumin. In the liver of the cirrhotic rats, there were increases in protein and free Trp (i.e., non-protein Trp) contents and a decrease in total tryptophan 2,3-dioxygenase (TDO) activity. The decreased TDO activity was mainly due to the reduction of apo-TDO activity. When [³H]Trp was injected into the portal vein of the cirrhotic and control rats, radioactivity derived from the injected [³H]Trp in the liver was higher in the cirrhotic rats than in the control rats at 10min after the injection, while the radioactivity in the serum was lower in the former rats than in the latter rats. These results indicate that the increased Trp is easily taken up into the cirrhotic liver, and suggest that the Trp taken up into the cirrhotic liver could be utilized for the maintenance of synthesis of proteins in the tissue through the reduction of Trp metabolism due to reduced TDO activity in the tissue.     

Studies on the effect of 1-deoxynojirimycin on the release of albumin, sialyltransferase and alpha 1 acid glycoprotein from liver slices from normal and inflamed rats

Liver slices from control and 24hr inflamed rats were incubated for up to 20hr with 5mM 1-deoxynojirimycin (DN), an inhibitor of the processing glucosidases. The amounts of albumin and alpha 1-acid glycoprotein (AGP) and the activities of sialyltransferase were determined in liver and medium. The presence of DN significantly inhibited the release of AGP and sialyltransferase. The inhibitory effect of DN was most pronounced with slices from inflamed rats. Secretion of albumin was not inhibited. Incorporation studies with labelled leucine and mannose showed that the inhibitor did not significantly affect protein synthesis, but it did inhibit mannose incorporation into AGP and sialyltransferase. The results show that DN inhibits the secretion of acute phase AGP and sialyltransferase in liver slices and further suggests that sialyltransferase is a glycoprotein.     

Ischemic preconditioning induces autophagy and limits necrosis in human recipients of fatty liver grafts, decreasing the incidence of rejection episodes

Whether ischemic preconditioning (IP) reduces ischemia/reperfusion (I/R) injury in human normal and fatty livers remains controversial. We compared two independent groups of liver donor transplants with versus without steatosis to evaluate IP consequences. Liver donors with (n=22) or without (n=28) steatosis either did or did not undergo IP before graft retrieval. Clinical data from the recipients, as well as histological and immunohistological characteristics of post-reperfusion biopsies were analyzed. Incidence of post-reperfusion necrosis was increased (10/10 versus 9/14, respectively; P<0.05) and the clinical outcome of recipients was worse for non-IP steatotic liver grafts compared with non-IP non-steatotic grafts. IP significantly lowered the transaminase values only in patients receiving a non-steatotic liver. An increased expression of beclin-1 and LC3, two pro-autophagic proteins, tended to decrease the incidence of necrosis (P=0.067) in IP steatotic livers compared with non-IP steatotic group. IP decreased the incidence of acute and chronic rejection episodes in steatotic livers (2/12 versus 6/10; P=0.07 and 2/12 versus 7/10; P<0.05, respectively), but not in non-steatotic livers. Thus, IP may induce autophagy in human steatotic liver grafts and reduce rejection in their recipients.     

Levels and values of circulating endothelial progenitor cells,soluble angiogenic factors,and mononuclear cell apoptosis in liver cirrhosis patients

Background

The roles of circulating endothelial progenitor cell (EPC) and mononuclear cell apoptosis (MCA) in liver cirrhosis (LC) patients are unknown. Moreover, vascular endothelial growth factor (VEGF) and stromal cell-derived factor (SDF)-1α are powerful endogenous substances enhancing EPC migration into circulation. We assessed the level and function of EPCs [CD31/CD34 (E₁), KDR/CD34 (E₂), CXCR4/CD34 (E₃)], levels of MCA, VEGF and SDF-1α in circulation of LC patients.

Methods

Blood sample was prospectively collected once for assessing EPC level and function, MCA, and plasma levels of VEGF and SDF-1α using flow cytometry and enzyme-linked immunosorbent assay (ELISA), respectively, in 78 LC patients and 25 age- and gender-matched healthy controls.

Results

Number of EPCs (E₁, E₂, E₃) was lower (all p < 0.0001), whereas SDF-1α level and MCA were higher (p < 0.001) in study patients compared with healthy controls. Number of EPCs (E₂, E₃) was higher but MCA was lower (all p < 0.05) in Child''s class A compared with Child''s class B and C patients, although no difference in VEGF and SDF-1α levels were noted among these patients. Chronic hepatitis B and esophageal varices bleeding were independently, whereas chronic hepatitis C, elevated aspartate aminotransferase (AST), and decompensated LC were inversely and independently correlated with circulating EPC level (all p < 0.03). Additionally, angiogenesis and transwell migratory ability of EPCs were reduced in LC patients than in controls (all p < 0.001).

Conclusion

The results of this study demonstrated that level, angiogenic capacity, and function of circulating EPCs were significantly reduced, whereas plasma levels of SDF-1α and circulating MCA were substantially enhanced in cirrhotic patients.     

Determination of copper, manganese and zinc in human liver

Autopsied liver tissue samples collected from 42 males and 31 females were analyzed for copper, manganese and zinc using atomic absorption spectrometry (AAS). With the exception of two liver samples for which the copper levels were determined to be 74.8 and 104.0 g/g (dry weight), hepatic copper concentrations were found to range from 1.7 to 32.4 g/g with a mean concentration of 14.2 g/g and standard deviation of 7.0 g/g. Manganese concentrations (with the exception of one sample having 12.9 g/g) ranged from 0.22 to 4.6 g/g with a mean of 2.26 ± 1.00 g/g. Hepatic zinc levels averaged 118.3 ± 44.4 g/g and ranged from 38.5 to 231.3 g/g. There were no apparent trends for the levels of any metals versus age nor were there any differences in average hepatic metal concentrations for males and females. © Rapid Science 1998.     

Urokinase-type plasminogen activator gene therapy in liver cirrhosis is mediated by collagens gene expression down-regulation and up-regulation of MMPs, HGF and VEGF

Human urokinase-type plasminogen activator (uPA) gene administration via an adenoviral (Ad)-vector induced cirrhosis regression and ameliorated hepatic dysfunction in a model of experimental liver cirrhosis. The administration of a single dose of 6 x 10(11) viral particles per kilogram of a clinical-grade Ad-vector was evaluated after the onset of rat liver cirrhosis via degradation of deposited collagen and a substantial decrease of alpha-sma-positive cells. Also, gene expression for pro-fibrogenic molecules (Col I, III, IV, TIMP-1 and PAI-1) was clearly down-regulated. In contrast, gene expression for collagen-degrading enzymes such as MMP-13 and MMP-2 was up-regulated. These events correlated with increased amounts of proteic free-TIMP-1, i.e. non-complexed with metalloproteinases (MMPs), indicating the presence of higher amounts of active MMPs inside the liver of cirrhotic animals treated with Ad-huPA. The harmonized and concerted expression of HGF and c-met resulted in exacerbated hepatocyte proliferation, although these events did not induce an abnormal liver growth. Angiogenesis, i.e. formation of new blood vessels, was evaluated by vascular endothelial growth factor (VEGF) expression which was notably detected to be 10 times higher during the first 6 days after Ad-huPA-treatment in cirrhotic animals as compared with controls. These events provide a clearer rationale as to how Ad-huPA-induced liver regeneration on CCl(4)-induced liver fibrosis takes place.