Stable replication of the EBNA1/OriP-mediated baculovirus vector and its application to anti-HCV gene therapy

<p>Abstract</p> <p>Background</p> <p>Hepatitis C virus (HCV) is one of the main causes of liver-related morbidity and mortality. Although combined interferon-α-ribavirin therapy is effective for about 50% of the patients with HCV, better therapies are needed and preventative vaccines have yet to be developed. Short-hairpin RNAs (shRNAs) inhibit gene expression by RNA interference. The application of transient shRNA expression is limited, however, due to the inability of the shRNA to replicate in mammalian cells and its inefficient transduction. The duration of transgene (shRNA) expression in mammalian cells can be significantly extended using baculovirus-based shRNA-expressing vectors that contain the latent viral protein Epstein-Barr nuclear antigen 1 (EBNA1) and the origin of latent viral DNA replication (OriP) sequences. These recombinant vectors contain compatible promoters and are highly effective for infecting primary hepatocyte and hepatoma cell lines, making them very useful tools for studies of hepatitis B and hepatitis C viruses. Here, we report the use of these baculovirus-based vector-derived shRNAs to inhibit core-protein expression in full-length hepatitis C virus (HCV) replicon cells.</p> <p>Results</p> <p>We constructed a long-term transgene shRNA expression vector that contains the EBV <it>EBNA1 </it>and <it>OriP </it>sequences. We also designed baculovirus vector-mediated shRNAs against the highly conserved core-protein region of HCV. HCV core protein expression was inhibited by the EBNA1/OriP baculovirus vector for at least 14 days, which was considerably longer than the 3 days of inhibition produced by the wild-type baculovirus vector.</p> <p>Conclusion</p> <p>These findings indicate that we successfully constructed a long-term transgene (shRNA) expression vector (Ac-EP-shRNA452) using the EBNA1/OriP system, which was propagated in <it>Escherichia coli </it>and converted into mammalian cells. The potential anti-HCV activity of the long-term transgene (shRNA) expression vector was evaluated with the view of establishing highly effective therapeutic agents that can be further developed for HCV gene therapy applications.</p

Caspase-8-and JNK-dependent AP-1 activation is required Fas Ligand-induced IL-8 production

Division of Immunology and Molecular Biolog

Caspase-8- and JNK-dependent AP-1 activation is required for Fas ligand-induced IL-8 production

Despite a dogma that apoptosis does not induce inflammation, Fas ligand (FasL), a well-known death factor, possesses pro-inflammatory activity. For example, FasL induces nuclear factor κB (NF-κB) activity and interleukin 8 (IL-8) production by engagement of Fas in human cells. Here, we found that a dominant negative mutant of c-Jun, a component of the activator protein-1 (AP-1) transcription factor, inhibits FasL-induced AP-1 activity and IL-8 production in HEK293 cells. Selective inhibition of AP-1 did not affect NF-κB activation and vice versa, indicating that their activations were not sequential events. The FasL-induced AP-1 activation could be inhibited by deleting or introducing the lymphoproliferation (lpr)-type point mutation into the Fas death domain (DD), knocking down the Fas-associated DD protein (FADD), abrogating caspase-8 expression with small interfering RNAs, or using inhibitors for pan-caspase and caspase-8 but not caspase-1 or caspase-3. Furthermore, wildtype, but not a catalytically inactive mutant, of caspase-8 reconstituted the FasL-induced AP-1 activation in caspase-8-deficient cells. Fas ligand induced the phosphorylation of two of the three major mitogen-activated protein kinases (MAPKs): extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) but not p38 MAPK. Unexpectedly, an inhibitor for JNK but not for MAPK/ERK kinase inhibited the FasL-induced AP-1 activation and IL-8 production. These results demonstrate that FasL-induced AP-1 activation is required for optimal IL-8 production, and this process is mediated by FADD, caspase-8, and JNK. © 2007 The Authors

Caspase-8-and JNK-dependent AP-1 activation is required for Fas ligand-induced IL-8 production

取得学位 : 博士（医学）, 学位授与番号 : 医博甲第1877号 , 学位授与年月日 : 平成19年6月30日, 学位授与大学 : 金沢大学, 主査教授 : 佐藤 博, 副査教授 : 向田 直史 , 山本

Estimation of diameter growth parameters for Cryptomeria Plantations in� Taiwan Using the Local Yield Table Construction System

We applied the Local Yield Table Construction System (LYCS), a computer program that estimates stand growth as a function of various density control strategies, to Cryptomeria plantations in Taiwan. Parameters of the growth model were estimated from permanent plot data on Cryptomeria stands stored in a database at the Experimental Forest of National Taiwan University. The diameter at breast height (DBH) and the number of trees measured in permanent plots were used as parameters to estimate the curve of the DBH growth rate, the effects of stand density on diameter growth, growth in terms of DBH, and diameter distribution. The estimated stand growth could be adapted to the observed values in the permanent plots. Based on these results, yield tables for various stand density control strategies can now be constructed for Cryptomeria stands in Taiwan

Fission Yeast CENP-C (Cnp3) Plays a Role in Restricting the Site of CENP-A Accumulation

The centromere is a chromosomal locus where a microtubule attachment site, termed kinetochore, is assembled in mitosis. In most eukaryotes, with the exception of holocentric species, each chromosome contains a single distinct centromere. A chromosome with an additional centromere undergoes successive rounds of anaphase bridge formation and breakage, or triggers a cell cycle arrest imposed by DNA damage and replication checkpoints. We report here a study in Schizosaccharomyces pombe to characterize a mutant (cnp3-1) in a gene encoding a homolog of mammalian centromere-specific protein, CENP-C. At the restrictive temperature 36 degrees , the Cnp3-1 mutant protein loses its localization at the centromere. In the cnp3-1 mutant, the level of the Cnp1 (a homolog of a centromere-specific histone CENP-A) also decreases at the centromere. Interestingly, the cnp3-1 mutant is prone to promiscuous accumulation of Cnp1 at non-centromeric regions, when Cnp1 is present in excess. Unlike the wild type protein, Cnp3-1 mutant protein is found at the sites of promiscuous accumulation of Cnp1, suggesting that Cnp3-1 may stabilize or promote accumulation of Cnp1 at non-centromeric regions. From these results, we infer the role of Cnp3 in restricting the site of accumulation of Cnp1 and thus to prevent formation of de novo centromeres

Analysis of Acute Transfusion Reactions and Their Occurrence Times

Acute transfusion reactions (ATRs) are significantly relevant to the morbidity and mortality of patients. ATRs are mostly not severe and rarely cause severe conditions, including anaphylactic shock. The aim of this study was to clarify the frequency of ATRs and the time of event occurrence. A total of 18,745 transfusions were administered to 11,718 patients during a 3-year period. Adverse reactions including at least one sign or symptom were collected through a report system in 143 of 2,478 (5.7%) platelet concentrate transfusions, 105 of 6,629 (1.6%) red blood cell component transfusions and 51 of 2,307 (2.2%) fresh frozen plasma transfusions. Allergic signs and symptoms accounted for 70% of all adverse events. Severe signs and symptoms were observed in 7.1% of patients. These events appeared significantly earlier than those of non-severe signs and symptoms (median time 20 min vs 100 min, P < 0.05). For patients who have had repetitive transfusion-associated adverse events, preventive treatments for adverse events should be proactively promoted

Mechanism and repertoire of ASC-mediated gene expression

金沢大学がん研究所Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is an adaptor molecule that mediates inflammatory and apoptotic signals. Although the role of ASC in caspase-1-mediated IL-1beta and IL-18 maturation is well known, ASC also induces NF-kappaB activation and cytokine gene expression in human cells. In this study, we investigated the molecular mechanism and repertoire of ASC-induced gene expression in human cells. We found that the specific activation of ASC induced AP-1 activity, which was required for optimal IL8 promoter activity. ASC activation also induced STAT3-, but not STAT1-, IFN-stimulated gene factor 3- or NF-AT-dependent reporter gene expression. The ASC-mediated AP-1 activation was NF-kappaB-independent and primarily cell-autonomous response, whereas the STAT3 activation required NF-kappaB activation and was mediated by a factor that can act in a paracrine manner. ASC-mediated AP-1 activation was inhibited by chemical or protein inhibitors for caspase-8, caspase-8-targeting small-interfering RNA, and p38 and JNK inhibitors, but not by a caspase-1 inhibitor, caspase-9 or Fas-associated death domain protein (FADD) dominant-negative mutants, FADD- or RICK-targeting small-interfering RNAs, or a MEK inhibitor, indicating that the ASC-induced AP-1 activation is mediated by caspase-8, p38, and JNK, but does not require caspase-1, caspase-9, FADD, RICK, or ERK. DNA microarray analyses identified 75 genes that were induced by ASC activation. A large proportion of them was related to transcription (23%), inflammation (21%), or cell death (16%), indicating that ASC is a potent inducer of inflammatory and cell death-related genes. This is the first report of ASC-mediated AP-1 activation and the repertoire of genes induced downstream of ASC activation