基因毒物血清对胎鼠肺细胞SCE效应的研究

本文以胎鼠肺细胞ＳＣＥ作为观察指标，以环磷酰胺（ＣＰＡ）接种大鼠血清作为阳性血清，应用抗癌药物妇女血清为阳性对照，正常生育史妇女血清为阴性对照，有异常生育史妇女血清为观察血清。结果显示：异常生育史妇女血清组、有极显著差异（Ｐ＜０．０１），并且ＳＣＥ频率＞５０％，有生物学意义。ＣＰＡ血清组，异常生育史妇女血清组，应用抗癌药物妇女血清组之间ＳＣＥ频率也有极显著差异，但ＳＣＥ＜５０％，提示异常生育史妇女血清中含有某种致畸物质。

肝癌相关基因突变与肝癌易感性的研究

全文就近几年肝癌相关基因的研究进展进行分类归纳,对原癌基因、抑癌基因、毒物代谢酶基因和DNA修复基因中的相关基因突变与肝癌易感性的关联作一综述。

体外硫代乙酰胺处理后鼠肝脏上皮细胞中早期应答相关基因的表达分析

硫代乙酰胺(TA)是一种影响肝脏代谢,抑制mRNA运输,诱导免疫抑制的强效肝毒物。应用微阵列技术能很好地阐释该生物毒性复合物的遗传机制。因此,本试验应用高通量鼠基因组寡核苷酸微阵列(大约包含22000个基因)来研究WB-F344鼠肝脏上皮细胞中TA相关细胞毒性遗传组分。试验用TA处理过的细胞分别于TA处理后1、3、6、12、24 h提取总RNA,并使RNA杂交呈微阵列。聚类分析区分2组基因,分别为早期基因组(TA处理后1、3h)和晚期基因组(TA处理后6、122、4 h)。低浓度和高浓度TA处理后分别共鉴定出2129和2348个差异表达基因。用低浓度(1000 mol/L)和高浓度(10000 mol/L)TA处理后出现差异表达的1229个基因具有相似的表达模式。低浓度TA处理过的1410个基因和高浓度TA处理过的1858个基因早期存在差异表达,该结果表明,这些与TA早期应答相关的基因也许可以作为肝中毒的早期标志物。

CTD数据库架构及数据获取查询与提取方法

CTD即比较毒物基因组学数据库,CTD是一个强大的、公开的研究资源,是用于描述化学物质、基因和人类疾病之间关系的科学数据。这些数据与功能和通路数据相结合,以帮助建立关于环境影响疾病的机制的假说,其主要目标是促进对环境化学品对人类健康影响的理解。CTD收录了许多描述跨物种化学基因/蛋白质相互作用和化学-疾病关系以及基因-疾病关系的精确数据,这些研究结果有助于了解潜在、可变的易感性和环境影响疾病的分子机理,还有助于了解化学基因和蛋白质之间复杂的相互作用网络。感兴趣的研究者可以申请其中的数据进行相关研究,本文旨在对CTD数据库的结构以及毒性与基因数据查询和获取进行介绍。

Designing Primers for H5 and H7 Subtypes of Avian Influenza Virus and Multiplex RT-PCR Amplification

[Objective] The research aimed to design primers that are suitable for detecting H5 and H7 subtypes of avian influenza virus （AIV） ; [Method] DNAStar was used to analyze the homology of the sequences of H5 and H7 subtypes of AIV accessed in GenBank, and design primers（ by Primer Premier 5.0） on high homologous region of these sequences, and then amplified by RT-PCR. [Result] The multiplex RT-PCR amplification, agarose gel electrophoresis and sequencing results showed that the self-designed primers are successful for detecting AIV. [Conclusion] It is feasible to rapidly diagnose AIV through this method.

Virus Movement Protein Gene Mediated Resistance Against Cucumber Mosaic Virus Infection

Tobacco ( Nicotiana tabacum L.) “NC89” plants were transformed with deletion mutant of cucumber mosaic virus (CMV) movement protein (MP) gene and full_length CMV MP gene, respectively. The transformed plants were analyzed with polymerase chain reaction (PCR), PCR_Southern, Southern and Western blots. R 0 generation of the transgenic plants were inoculated with CMV. Five out of 10 lines of tobacco plants (BMPK) transformed with CMV MP deletion mutant gene showed high resistance to CMV infection and remained symptomless for up to 50 days post_inoculation. In contrast, tobacco plants (BMPR) transformed with full_length CMV MP gene did not show resistance to CMV infection. However, most of the infected full_length CMV MP gene transgenic plants recovered by showing none or very mild mosaic symptoms in 40 days post_inoculation. The results of R 1 generation of the BMPK transgenic plants tested under field conditions showed that all 5 lines of transgenic plants could delay the virus disease development.

Agroinoculation as a Simple Way to Deliver a Tobacco Mosaic Virus- Based Expression Vector

烟草花叶病毒(TMV)表达载体30B是一个目前广泛应用的植物病毒表达载体,但用其生产外源蛋白时,必须先将它体外转录成RNA,才能被用来接种宿主植物。由于RNA体外转录费用昂贵、操作复杂,因此限制了30B表达载体的进一步应用。针对这一不足,我们用农杆菌接种法(agroinoculation)接种该病毒载体,即将30B cDNA置于花椰菜花叶病毒(CaMV)的35S启动子和终止子之间,再将整个表达框架插入到农杆菌T-DNA的左边界和右边界之内,构建成质粒p35S-30B,将转入该质粒的农杆菌注射到植物的叶片中,30B cDNA随T-DNA进入植物细胞后,被转录成可自我复制的RNA形式,进而发生系统侵染。为了检测此接种方式的可行性,绿色荧光蛋白(GFP)报告基因被克隆到p35S-30B中,构建成p35S-30B∶∶GFP,用含有该质粒的农杆菌进行注射操作。证实该病毒载体可通过简便的农杆菌接种法侵染Nicotiana benthamiana,在被接种植物的系统叶中,GFP的表达量可占植物总可溶蛋白的5.2％。

Cloning and Sequence Analysis of Sheeppox Virus RPO30 Gene

[Objective] The study aimed to clone RPO30 gene from Sheeppox virus （SPPV） and predict the structure and function of the sequence. [Method] RPO30 gene of SPPV was cloned with PCR, linked into pMD18-T simple vector and then transformed into E. coli DH5a. In blue-white screen, the white colonies were selected to prepare plasmids. The positive plasmids were selected by double digestion and PCR, and then sequenced. Finally, the structure and function of the sequence obtained were predicted by bioinformatics methods. [Results] The RPO30 gene was successfully obtained; its ORF was 585 bp, encoding 193 amino acids and containing a recognition site for Hind III. Moreover, the SPPV RPO30 gene shared different homologies with the RPO30 gene sequences of other pox virus strains from GenBank database. Further analysis by biological software showed that in RPO30 protein, amino acids 4-12, 18-26, 50- 61, 68- 92 and 176-190 had a high possibility to form the active center, and acting to these regions was likely to inactivate the enzyme encoded by the sequence, thus to inhibit viral replication efficiently. [Conclusion] This study will lay foundation for further study on the structure and function of RPO30.

Toll-like receptor 4 plays an anti-HBV role in a murine model of acute hepatitis B virus expression

AIM： Toll-like receptor 4 （TLR4） has been shown to be important for bacterial infection, especially to lipopolysaccharide signaling. Its possible role in HBV infection is studied in the present study. MATERIALS AND METHODS： pHBV3.6 plasmid, containing full-length HBV genome was used in the murine model of acute HBV expression by hydrodynamics in vivo transfection. TLR4 normal or mutant mouse strain was compared to investigate the possible role of TLR4 in acute HBV expression. RESULTS： After pHBV3.6 injection, the infiltrating leukocytes expressed TLR4 were observed nearby the HBsAg-expressing hepatocytes. The HBV antigenemia as well as the replication and transcription were higher in TLR4-mutant C3H/HeJ mice than in normal C3H/ HeN mice. The HBV-specific immune responses were impaired in the liver or spleen of the C3H/HeJ mice. Their inducible nitric oxide synthase （iNOS） expression on the hepatic infiltrating cells was also impaired. When adoptively transferring splenocytes from C3H/HeN mice to C3H/HeJ mice, the HBV replication was inhibited to the level as that of C3H/HeN. CONCLUSION： These results suggest that TLR4 plays an anti-HBV role in vivo through the induction of iNOSexpression and HBV-specific immune responses after HBV expression.

Ribosome Inactivating Proteins from Plants Inhibiting Viruses

Many plants contain ribosome inactivating proteins (RIPs) with N-glycosidase activity, which depurinate large ribosomal RNA and arrest protein synthesis. RIPs so far tested inhibit replication of mRNA as well as DNA viruses and these proteins, isolated from plants, are found to be effective against a broad range of viruses such as human immunodeficiency virus (HIV), hepatitis B virus (HBV) and herpes simplex virus (HSV). Most of the research work related to RIPs has been focused on antiviral activity against HIV; however, the exact mechanism of antiviral activity is still not clear. The mechanism of antiviral activity was thought to follow inactivation of the host cell ribosome, leading to inhibition of viral protein translation and host cell death. Enzymatic activity of RIPs is not limited to depurination of the large rRNA, in addition they can depurinate viral DNA as well as RNA. Recently, Phase I/II clinical trials have demonstrated the potential use of RIPs for treating patients with HIV disease. The aim of this review is to focus on various RIPs from plants associated with anti-HIV activity.

Apolipoprotein B100 is required for hepatitis C infectivity and Mipomersen inhibits hepatitis C

AIM To characterize the role of apolipoprotein B100(apoB 100) in hepatitis C viral(HCV) infection. METHODS In this study, we utilize a gene editing tool, transcription activator-like effector nucleases(TALENs), to generate human hepatoma cells with a stable genetic deletion of APOB to assess of apoB in HCV. Using infectious cell culture-competent HCV, viral pseudoparticles, replicon models, and lipidomic analysis we determined the contribution of apoB to each step of the viral lifecycle. We further studied the effect of mipomersen, an FDAapproved antisense inhibitor of apoB 100, on HCV using in vitro cell-culture competent HCV and determined itsimpact on viral infectivity with the TCID50 method. RESULTS We found that apo B100 is indispensable for HCV infection. Using the JFH-1 fully infectious cell-culture competent virus in Huh 7 hepatoma cells with TALENmediated gene deletion of apoB(APOB KO), we found a significant reduction in HCV RNA and protein levels following infection. Pseudoparticle and replicon models demonstrated that apo B did not play a role in HCV entry or replication. However, the virus produced by APOB KO cells had significantly diminished infectivity as measured by the TCID-50 method compared to wildtype virus. Lipidomic analysis demonstrated that these virions have a fundamentally altered lipidome, with complete depletion of cholesterol esters. We further demonstrate that inhibition of apoB using mipomersen, an FDA-approved anti-sense oligonucleotide, results in a potent anti-HCV effect and significantly reduces the infectivity of the virus. CONCLUSION Apo B is required for the generation of fully infectious HCV virions, and inhibition of apo B with mipomersen blocks HCV. Targeting lipid metabolic pathways to impair viral infectivity represents a novel host targeted strategy to inhibit HCV.

Genetic Modification of Baculovirus Expression Vectors

As a protein expression vector, the baculovirus demonstrates many advantages over other vectors. With the development of biotechnology, baculoviral vectors have been genetically modified to facilitate high level expression of heterologous proteins in both insect and mammalian cells. These modifications include utilization of different promoters and signal peptides, deletion or replacement of viral genes for increasing protein secretion, integration of polycistronic expression cassette for producing protein complexes, and baculovirus pseudotyping, promoter accommodation or surface display for enhancing mammalian cell targeting gene delivery. This review summarizes the development and the current state of art of the baculovirus expression system. Further development of baculovirus expression systems will make them even more feasible and accessible for advanced applications.

Hepatitis C virus infection down-regulates the expression of peroxisome proliferator-activated receptor a and carnitine palmitoyl acyl-CoA transferase 1A

AIM： To elucidate the role of the peroxisome proliferator-activated receptor α （PPARα） and its target gene camitine palmitoyl acyl-CoA transferase 2A （CPT1A） in the pathogenesis of hepatitis C virus （HCV） infection.METHODS： Liver samples were collected from the patients with chronic HCV infection and controls. HepG2 cells were transfected with vector pEF352neo carrying. Two independent clones （clone N3 and N4） stably expressing HCV core protein were analyzed. Total RNA was extracted from cells and liver tissues. PPARα and CPT1A mRNAs were quantified by real-time polymerase chain reaction （PCR） using SYBR Green Master. Total extracted proteins were separated by polyacrylamide gel electrophoresis, and electroblotted. Membranes were incubated with the anti-PPARα antibody, then with a swine anti-rabbit IgG conjugated to horseradish peroxidase for PPARα. Protein bands were revealed by an enhanced chemiluminescence reaction for PPARα. For immunohistochemical staining of PPARα, sections were incubated with the primary goat polyclonal antibody directed against PPARα at room temperature.RESULTS： Real-time PCR indicated that the PPARα level and expression level of CPT1A gene in hepatitis C patients lowered significantly as compared with the controls （1.8±2.8 vs 13±3.4, P = 0.0002; 1.1±1.5 vs 7.4±1, P = 0.004）. Western blot results showed that the level of PPARα protein in the livers of hepatitis C patients was lower than that in controls （2.3±0.3 vs 3.6±0.2, P = 0.009）. The immunohistochemical staining results in chronic hepatitis C patients indicated a decrease in PPARα staining in hepatocytes compared with those in the control livers. The in vitro studies found that in the N3 and N4 colon stably expressing HCV core protein, the PPARα mRNA levels were significantly lower than that in the controls.CONCLUSION： The impaired intrahepatic PPARα expression is associated with the pathogenic mechanism in hepatic injury during chronic HCV infection. HCV infection reduced the expression of PPARα and CPT1A at the level of not only mRNAs but also proteins. PPARα plays an important role in the pathogenesis of chronic HCV infection, but the impaired function of this nuclear receptor in HCV infection needs further studies.

New combination test for hepatitis C virus genotype and viral load determination using Amplicor GT HCV MONITOR test v2.0

AIM: To develop a new sensitive and inexpensive hepatitis C virus (HCV) combination test (HCV Guideline test) that enables the determination of HCV genotypes 1, 2 and 3, and simultaneous determination of HCV viral load using commercial Amplicor GT HOV MONITOR test v2.0 (microwell version). METHODS: The HCV Guideline test used the PCR product generated in commercial Amplicor GT HCV Monitor test v2.0 for viral load measurement using microwell plate version of Amplicor HCV Monitor and also captured on separate plates containing capture probes and competitive oligonucleotide probes specific for HCV genotypes 1, 2 and 3, The HCV genotype was subsequently determined using the biotin-labeled PCR product and five biotin-labeled HCV-specific probes. RESULTS: The sensitivity of the HCV Guideline test was 0.5 KIU/mL. Specificity of the HCV Guideline test was confirmed by direct sequencing of HCV core region and molecular evolutionary analyses based on a panel of 31 samples. The comparison of the HCV Guideline test and an in-house HCV core genotyping assay using 252 samples from chronic hepatitis C patients indicated concordant results for 97.2% of samples (59.5% genotype 1, 33.7% genotype 2, 6.0% genotype 3, and 0.8% mixed genotypes). Similarly, the HCV Guideline test showed concordance with a serological test, and the serological test failed to assign any serotype in 12.7% of the samples, indicating a better sensitivity of the HCV Guideline test. CONCLUSION: Clinically, both viral load and genotypes (1, 2 and 3) have been found to be major predictors of antiviral therapy outcome regarding chronic hepatitis C based on guidelines and they are, in normal circumstances, performed as separate stand-alone assays. The HCV Guideline test is a useful method for screening large cohorts in a routine clinical setting for determining the treatment regimen and for predicting the outcome of antiviral therapy of chronic hepatitis C.

Novel approaches towards conquering hepatitis B virus infection

Currently approved treatments for hepatitis B virus （HBV） infection include the immunomodulatory agent, IFN-α, and nucleos（t）ide analogues. Their efficacy is limited by their side effects, as well as the induction of viral mutations that render them less potent. It is thus necessary to develop drugs that target additional viral antigens. Chemicals and biomaterials by unique methods of preventing HBV replication are currently being developed, including novel nucleosides and newly synthesized compounds such as capsid assembling and mRNA transcription inhibitors. Molecular therapies that target different stages of the HBV life cycle will aid current methods to manage chronic hepatitis B （CriB） infection. The use of immunomodulators and gene therapy are also under consideration. This report summarizes the most recent treatment possibilities for CHB infection. Emerging therapies and their potential mechanisms, efficacy, and pitfalls are discussed.

Adeno-associated virus mediated endostatin gene therapy in combination with topoisomerase inhibitor effectively controls liver tumor in mouse model

AIM:rAAV mediated endostatin gene therapy has been examined as a new method for treating cancer.However, a sustained and high protein delivery is required to achieve the desired therapeutic effects.We evaluated the impact of topoisomerase inhibitors in rAAV delivered endostatin gene therapy in a liver tumor model. METHODS:rAAV containing endostatin expression cassettes were transduced into hepatoma cell lines.To test whether the topoisomerase inhibitor pretreatment increased the expression of endostatin,Western blotting and ELISA were performed.The biologic activity of endostatin was confirmed by endothelial cell proliferation and tube formation assays. The anti-tumor effects of the rAAV-endostatin vector combined with a topoisomerase inhibitor,etoposide,were evaluated in a mouse liver tumor model. RESULTS:Topoisomerase inhibitors,including camptothecin and etoposide,were found to increase the endostatin exPression level in vitro.The over-expressed endostatin, as a result of pretreatment with a topoisomerase inhibitor, was also biologically active.In animal experiments,the combined therapy of topoisomerase inhibitor,etoposide with the rAAV-endostatin vector had the best tumor- suppressive effect and tumor foci were barely observed in livers of the treated mice.Pretreatment with an etoposide increased the level of endostatin in the liver and serum of rAAV-endostatin treated mice.Finally,the mice treated With rAAV-endostatin in combination with etoposide showed the longest survival among the experimental models. CONCLUSION:rAAV delivered endostatin gene therapy in combination with a topoisomerase inhibitor pretreatment is an effective modality for anticancer gene therapy.

The Flavivirus Protease As a Target for Drug Discovery

Many flaviviruses are significant human pathogens causing considerable disease burdens,including encephalitis and hemorrhagic fever,in the regions in which they are endemic.A paucity of treatments for flaviviral infections has driven interest in drug development targeting proteins essential to flavivirus replication,such as the viral protease.During viral replication,the flavivirus genome is translated as a single polyprotein precursor,which must be cleaved into individual proteins by a complex of the viral protease,NS3,and its cofactor,NS2B.Because this cleavage is an obligate step of the viral life-cycle,the flavivirus protease is an attractive target for antiviral drug development.In this review,we will survey recent drug development studies targeting the NS3 active site,as well as studies targeting an NS2B/NS3interaction site determined from flavivirus protease crystal structures.

Joannesia princeps： Evaluation of Aqueous Extracts Genotoxicity Utilizing Allium cepa Assay and Micronucleus Test

Evaluation of the genotoxic and cytotoxic potentials is a part of the analysis process where plant products with therapeutic properties must be submitted for being safely employed, mainly through long time points, doannesia princeps, also known as ＂cotieira＂ has been used on popular medicine as laxative and several diseases treatment. Aiming to analyze genotoxic potential ofd. princeps leaves extract, Allium cepa assay and micronucleus test were employed. No genotoxic activity presented J. princeps leaves extract; however, extracts cytotoxic activity over A. cepa meristematic cells and mice marrow cells was observed. J. princeps leaves extract presented antiproliferative activity at concentrations and systems employed indicating their therapeutic potential for cellular cycle inhibition. Moreover, results showed that in regard to its mutagenic effect, usage of J. princeps leaves tea on popular medicine has been a safe procedure, as well.

The Role of the Charged Residues of the GP2 Helical Regions in Ebola Entry

The glycoprotein （GP） of Ebola is the sole structural protein that forms the spikes on the viral envelope. The GP contains two subunits, GPI and GP2, linked by a disulfide bond, which are responsible for receptor binding and membrane fusion, respectively. In this study, the full length of GP gene of Ebola Zaire species, 2028 base pairs in length, was synthesized using 38 overlapping oligonucleotides by multiple rounds of polymerase chain reaction （PCR）. The synthesized GP gene was shown to be efficiently expressed in mammalian cells. Furthermore, an efficient HIV-based pseudotyping system was developed using the synthetic GP gene, providing a safe approach to dissecting the entry mechanism of Ebola viruses. Using this pseudotyping system and mutational analysis, the role of the charged residues in the GP2 helical regions was examined. It was found that substitutions of the most charged residues in the regions did not adversely affect GP expression, processing, or viral incorporation, however, most of the mutations greatly impaired the ability of GP to mediate efficient viral infection. These results demonstrate that these charged residues of GP2 play an important role in GP-mediated Ebola entry into its host cells. We propose that these charged residues are involved in forming the intermediate conformation（s） of GP in membrane fusion and Ebola entry.