Lipid-induced up-regulation of human acyl-CoA synthetase 5 promotes hepatocellular apoptosis

In the pathogenesis of nonalcoholic fatty liver disease, accumulation of lipids in hepatocytes and hepatocyte apoptosis are strongly implicated in disease progression from the potentially reversible condition of steatosis to severe acute and chronic liver injury. Acyl-CoA synthetase 5, a member of the ACSL gene family that catalyzes the activation of long-chain fatty acids for lipid biosynthesis, is the only ACSL isoform that is both, located on mitochondria and functionally involved in enterocyte apoptosis. In this study, the regulation of human ACSL5 in hepatocellular fatty acid degeneration and its involvement in hepatocyte apoptosis was investigated using models of in vitro and in vivo steatosis as well as plasmid-mediated stable gene transfer and RNAi-mediated gene silencing. ACSL5 mRNA and protein were strongly increased by uptake of dietary derived fatty acids in primary human hepatocytes, HepG2 cells and human steatotic liver. Over-expression of ACSL5 decreased HepG2 cell viability and increased susceptibility to TRAIL- and TNFα-, but not FAS- induced apoptosis, whereas knock down of ACSL5 reduced apoptosis susceptibility. High ACSL5 activity resulted in enhanced caspase-3/7 activity, but was not accompanied by up-regulation of death receptors, DR4, DR5 or TNF-R1. This study gives evidence that hepatocyte steatosis is associated with ACSL5 up-regulation resulting in increased susceptibility to hepatic cell death. We propose that ACSL5 could play a role in promoting fatty acid-induced lipoapoptosis in hepatocytes as important mechanism in fatty liver-related disorders.     

CMV promotor activity during ES cell differentiation: potential insight into embryonic stem cell differentiation

The activity of the P(CMV IE) promoter was studied during the differentiation of ES cells into neurons. In order to do this, stable embryonic stem (ES) cell lines that express enhanced green fluorescent protein (EGFP) under the control of P(CMV IE) were created and these ES cells were differentiated by aggregation of cells in the presence of retinoic acid (RA). Based on our observations that the activity of P(CMV IE) was highest in undifferentiated cells, and that cell-cell interaction and addition of RA that lead to enhanced cell proliferation also increased expression from P(CMV IE), we hypothesized that the activity of P(CMV IE) was positively regulated in cycling cells. However, when analysis was done at the single cell level it was found that BrdU label and EGFP expression were not correlated. EGFP expression was found to be down-regulated in many cells that were BrdU positive and conversely there were significant numbers of BrdU negative cells that were EGFP positive. Further, P(CMV IE) activity was not observed in cells that were nestin positive or in differentiated neurons, but P(CMV IE) was active in cells with a fibroblast-like morphology. Finally, several proteins present in undifferentiated ES cells were found to bind to regulatory regions of P(CMV IE). These were absent when cells were aggregated in the presence of RA. The above results have implications for expression of transgenes in ES cells as well as providing new insight into the mechanism of lineage restriction.     

Role of the cytomegalovirus (CMV)-antigenemia assay as a predictive and follow-up detection tool for CMV disease in AIDS patients

Forty-two patients were evaluated to determine the value of the CMV antigenemia (CMV-Ag) test as a follow-up marker as well as a prediction marker of CMV disease. Twenty patients were positive for at least one positive CMV-Ag assay and 9 of them developed CMV retinitis. With the threshold value (10 positive cells), sensitivity was 56% and specificity was 94%. The CMV-Ag assay, with the threshold value, produced high specificity, positive predictive value and negative predictive value but relatively poor sensitivity. Eight patients experienced CMV disease relapse a total of 16 times. At relapse, 8 of the 16 times showed negative for CMV-Ag assay; 7 underwent systemic maintenance while 1 underwent local maintenance. It is inferred that the CMV-Ag test is a poor follow-up marker to detect the relapse of CMV disease, particularly in patients undergoing systemic maintenance.     

Reappearance of Cucumber mosaic virus Isolates Belonging to Subgroup IB in Tomato Plants in North-eastern Spain

A disease characterized by symptomless leaves and fruit decolouration, loss of consistency and mild deformation on ripening was detected in tomato fields in north‐eastern Spain during 2003 and 2004. DAS‐ELISA analysis revealed the presence of the Cucumber mosaic virus (CMV) in all diseased plants. CMV isolates were characterized by polyacrylamide gel electrophoresis (PAGE) analysis of double‐stranded RNAs (dsRNAs) and nucleotide sequence analysis, and compared with some CMV isolates belonging to different subgroups used as controls. A total of 12 isolates obtained from the infected tomato plants selected for this study gave the same electrophoresis pattern for the three genomic dsRNAs, which was different to the patterns showed by the CMV isolates collected in the same region a few years ago. The identity of the complete nucleotide sequence of one of these CMV isolates and the partial sequence of other five isolates compared to the Tfn strain from Italy and the BAR92/1 isolate from tomato collected in Barcelona in 1992 was higher than 99%, both belonging to subgroup IB of CMV. The CMV isolates of this subgroup found in eastern Spain in previous studies were not detected after 1996. The nucleotide sequences of two isolates that were chosen as representatives of the CMV isolates more frequently detected in previous years revealed that they belonged to the CMV subgroups IA. The origin and the possible causes of reappearance of CMV IB isolates in north‐eastern Spain are discussed.     

Detection of Single Nucleotide Variations in Viral RNA Populations by Primer Extension

A method for identifying and differentiating between two highly similar sequence variants in the cucumber mosaic virus RNA 5 population is described. The technique is based on the use of different primers with 3 terminal mismatches for primer extension analysis. Primers varied in their length, number of 3 mismatches, and sequence context of the mismatch. The results indicate that the size of the primer is critically important to the ability of this method to differentiate between highly similar RNAs. This technique should prove very useful for the genetic analysis of characteristics that are determined by single nucleotide variations in viral RNA genomes.Abbreviations: nt, nucleotide; CMV, cucumber mosaic virus.     

矮牵牛病毒病的初步研究

矮牵牛是一种草本花卉,主要用来布置花坛,但病毒侵染却严重地影响其观赏价值。国内外有关危害矮牵牛的病毒报道较多［１,２］,但较系统研究矮牵牛病毒病的报道较少,本文从矮牵牛病毒鉴定、优势病毒种类确定及防治矮牵牛病毒病进行了初步研究,现报道如下：材料与方法...     

豆科植物上分离的黄瓜花叶病毒（CMV）五个分离物的比较研究

从豌豆、扁豆、菜豆和赤豆分离到五个ＣＭＶ分离物,除寄主反应及核酸组分外,５个分离物在体外抗性、蚜虫传毒、提纯病毒粒体形态和稳定性、衣壳蛋白分子过和病毒粒体迁移率等方面无明显差异．在琼脂糖双扩散试验中五个分离物与ＣＭＶ抗血清形成的沉淀线相互融合,在Ａ蛋白双抗体夹心ＥＬＢＡ测定中,也无明显差异。根据在四种豆科植物上的症状反应,五个分离物可归为两个型：Ⅰ型在豌豆、蚕豆、菜豆和豇豆上产生局部枯斑症状;Ⅱ型在这四种植物上产生系统症状。五个分离物中,ＣＭＶＰ＿２含有五个枝酸组分,其它分离物为四个组分,即ＣＭＶＰ＿２可能含有卫星ＲＮＡ．     

Cloning of the complete human cytomegalovirus genome in cosmids

Purified virion DNA (155 X 10(6) Mr) of human cytomegalovirus (CMV) strain Ad169 was partially cleaved with restriction endonucleases HindIII and EcoRI and cloned in the respective cleavage sites of cosmid pHC79. A complete gene library was established in a set of clones containing the viral DNA in long overlapping segments. Restriction maps for HindIII (29 fragments) and EcoRI (36 fragments) were constructed from the linkage of cosmid-cloned fragments, from double digestions of cloned DNA, and from blot hybridization of labeled cloned viral DNA with restriction fragments of virion DNA and singly or doubly cleaved cosmid clones.