Phage phi29 protein p56 prevents viral DNA replication impairment caused by uracil excision activity of uracil-DNA glycosylase

Protein p56 encoded by the Bacillus subtilis phage 29 inhibits host uracil-DNA glycosylase (UDG) activity. In previous studies, we suggested that this inhibition is likely a defense mechanism developed by phage 29 to prevent the action of UDG if uracilation occurs in DNA either from deamination of cytosine or the incorporation of dUMP during viral DNA replication. In this work, we analyzed the ability of 29 DNA polymerase to insert dUMP into DNA. Primer extension analysis showed that viral DNA polymerase incorporates dU opposite dA with a catalytic efficiency only 2-fold lower than that for dT. Using the 29 DNA amplification system, we found that 29 DNA polymerase is also able to carry out the extension of the dA:dUMP pair and replicate past uracil. Additionally, UDG and apurinic-apyrimidinic endonuclease treatment of viral DNA isolated from 29-infected cells revealed that uracil residues arise in 29 DNA during replication, probably as a result of misincorporation of dUMP by the 29 DNA polymerase. On the other hand, the action of UDG on uracil-containing 29 DNA impaired in vitro viral DNA replication, which was prevented by the presence of protein p56. Furthermore, transfection activity of uracil-containing 29 DNA was significantly higher in cells that constitutively synthesized p56 than in cells lacking this protein. Thus, our data support a model in which protein p56 ensures an efficient viral DNA replication, preventing the deleterious effect caused by UDG when it eliminates uracil residues present in the 29 genome.     

乙型肝炎病毒基因型、YMDD变异与拉米夫定治疗后HBV DNA反弹关系的研究

目的探讨HBV基因型、YMDD变异与拉米夫定抗病毒治疗后HBV DNA反弹的关系。方法应用多引物对巢式PCR法、PCR-序列分析法检测拉米夫定治疗的27例乙型肝炎患者和19例从未用过抗病毒治疗的患者HBV基因型和P区（YMDD）的突变位点。结果在27例HBV DNA反弹的患者中，13例（48．15％）检出YMDD变异，而对照人群无YMDD变异（P〈0．05）。YMDD变异的位点为rtM204V／I（C区）±rtL180M（B区）；在治疗组YMDD变异的患者中，B、C基因型构成比（46．15％和59．26％）与对照组（53．85％和68．42％）比较无显著性差异（P〉0．05）。结论YMDD变异是拉米夫定治疗后出现耐药导致HBV DNA反弹的主要原因；YMDD变异的常见位点依然为rtM204V／I（C区）±rtL180M（B区）；YMDD变异在B、C基因型病人中无差别。     

我国4省、自治区细粒棘球绦虫DNA限制性片段长度多态性分析

运用 DNA探针pHD5、pEG18、pSM889 结合 Southern blot 杂交技术对采自我国新疆、青海、甘肃和宁夏等地的羊源细粒棘球蚴与采自青海、甘肃的牦牛源细粒棘球蚴进行了限制性片段长度多态性(RFLP)分析。结果表明我国这些省、自治区采集的羊源细粒棘球蚴彼此之间未见有明显而稳定的杂交条带差异,属于同一虫株。青海和甘肃两省牦牛源细粒棘球蚴彼此之间也未见明显而稳定杂交条带差异,据杂交图谱判断似与羊源细粒棘球蚴属同一虫株。     

Worldwide Distribution of Four SNPs in X‐Ray and Repair and Cross‐Complementing Group 1 (XRCC1)

Purpose

X‐ray repair cross‐complementing group 1 (XRCC1) repairs single‐strand breaks in DNA. Several reports have shown the association of single nucleotide polymorphisms (SNPs) (Arg194Trp, Pro206Pro, Arg280His, Arg399Gln) in XRCC1 to diseases. Limited population data are available regarding SNPs in XRCC1, especially in African populations. In this study, genotype distributions of four SNPs in worldwide populations were examined and compared with those reported previously.

Materials and Methods

Four SNPs (Arg194Trp, Pro206Pro, Arg280His, Arg399Gln) in XRCC1 from genomic DNA samples of 10 populations were evaluated by using polymerase chain reaction followed by restriction fragment length polymorphism analysis.

Results

The frequency of the minor allele corresponding to the Trp allele of XRCC1Arg194Trp was higher in Asian populations than in African and Caucasian populations. As for XRCC1Pro206Pro, Africans showed higher minor allele frequencies than did Asian populations, except for Tamils and Sinhalese. XRCC1 Arg280His frequencies were similar among Africans and Caucasians but differed among Asian populations. Similarly, lower mutant XRCC1 Arg399Gln frequencies were observed in Africans.

Conclusions

This study is the first to show the existence of a certain genetic heterogeneity in the worldwide distribution of four SNPs in XRCC1.     

Epigenetics in the Pathogenesis of Esophageal Adenocarcinoma

Epigenetic influences, such as DNA methylation, histone acetylation, and up‐regulation/down‐regulation of genes by microRNAs, change the genetic makeup of an individual without affecting DNA base‐pair sequences. Indeed, epigenetic changes play an integral role in the progression from normal esophageal mucosa to Barrett''s esophagus to esophageal adenocarcinoma via dysplasia–metaplasia–neoplasia sequence. Many genes involved in esophageal adenocarcinoma display hypermethylation, leading to their down‐regulation. The classes of these genes include cell cycle control, DNA and growth factor repair, tumor suppressors, antimetastasis, Wnt‐related genes, and proapoptotic genes. Histone acetylation in the pathophysiology of esophageal diseases has not been thoroughly investigated, and its critical role in the development of esophageal adenocarcinoma is less defined. Many microRNAs have been associated with the development of Barrett''s esophagus and esophageal adenocarcinoma. Here, we critically addressed the specific steps most closely influenced by microRNAs in the progression from Barrett''s esophagus to esophageal adenocarcinoma. However, microRNAs can target up to hundreds of genes, making it difficult to correlate directly with a given phenotype of the disease. Esophageal adenocarcinoma progressing from premalignant condition of Barrett''s esophagus carries an extremely poor prognosis. Risk stratification for patients based on their epigenetic profiles may be useful in providing more targeted and directed treatment to patients.     

氢醌对K562细胞着色性干皮病基因D甲基化的影响

目的：了解不同剂量苯代谢物氢醌（ HQ）对人白血病细胞株K562细胞着色性干皮病基因D（ XPD）甲基化水平的影响。方法：分别以终浓度为0、15、30和60μmol/L HQ溶液重复处理K562细胞48 h,采用MTT比色法检测K562细胞增殖能力,采用亚硫酸氢盐处理后测序法检测XPD甲基化水平;观察各组细胞存活率及甲基化率。结果：HQ 0、15、30、60μmol/L 处理后,K562细胞存活率分别为（100.00±0.00）％、（85.46±0.60）％、（63.46±7.02）％和（51.20±6.49）％,15μmol/L 组与0μmol/L 组比较,差异无统计学意义（ P ＞0.05）,30μmol/L和60μmol/L组与0μmol/L组比较,差异有统计学意义（P＜0.05）;XPD基因甲基化水平分别依次为1.03％（3/290）、0.34％（1/290）、0.34％（1/290）和0.70％（2/290）,15、30、60μmol/L组与0μmol/L组比较,差异无统计学意义（χ2=1.531,P＞0.05）。结论：HQ对K562细胞生长有明显的抑制作用,但对细胞中XPD基因甲基化水平无影响。     

用分子生物学技术分析国内六株旋毛虫分离株

本文首次对来自我国不同地区、不同宿主来源有代表性的6个旋毛虫分离株从基因组DNA的限制性酸切长度多态性、同工酶及可溶性蛋白进行综合研究。核酸结果显示：长春株与其余各地域株的限制性酶切困话间存在着差异，用长春株特异性DNA片段（1．12kb）制成探针，与各株DNA进行Southern杂交，发现各虫株的杂交带型不一，且仅长春株出现1．12kb杂交带。同工酶结果显示：长春株与其余各珠在5种同工酶（GPI、G6PD、HK、6PGDH及AK）酶谱中有显著不同。等电聚焦电泳结果显示：长春株具有一条约4.1PI的特异带。上述结果提示：我国6株旋毛虫分离株间存在着差异，长春株与其余各株差异显著，从而推断我国旅毛虫虫株至少存在2种不同的生物学类型，其间的差异主要与不同的地理分布和／或不同宿主来源有关。     

表达Ag85B与ESAT6融合蛋白的基因疫苗的免疫学特性

目的测定表达Ag85B与ESAT6融合蛋白的两种重组质粒AZ-pcDNA3-EF和EZ-pcDNA3-AF在小鼠体内诱导的免疫应答及保护力。方法50只BALB/c小鼠随机分为5组(每组10只),将表达Ag85B与ESAT6融合蛋白的两种重组质粒AZ-pcDNA3-EF和EZ-pcDNA3-AF分别免疫小鼠3次,每次间隔2周,同时设卡介苗(BCG)免疫组、空载体质粒免疫组和生理盐水对照组。最后一次免疫结束后,每组取5只小鼠血清,酶联免疫吸附测定(ELISA)法检测特异性抗体的滴度,并分离小鼠的脾淋巴细胞,并在体外用结核分枝杆菌(MTB)培养滤液蛋白(cu lture filtrate prote in,CFP)刺激,测定脾淋巴细胞增殖指数和γ干扰素(IFN-γ)水平。用1 m l含1×105克隆形成单位(CFU)的MTB毒株H37Rv经尾静脉感染其余每组5只BALB/c小鼠,4周后计数脾脏细菌负荷数。结果质粒AZ-pcDNA3-EF和EZ-pcDNA3-AF免疫小鼠血清的特异性抗体滴度分别为1∶1 000和1∶1 500。其脾淋巴细胞刺激指数分别为2.2和2.4,而生理盐水对照组和空载体质粒免疫组的刺激指数只有0.9和1.1;脾淋巴细胞悬液中诱发的IFN-γ分别为(5.48±0.38)ng/m l和(5.76±0.51)ng/m l,显著高于生理盐水对照组和空载体质粒免疫组(P<0.05),但与BCG免疫组的(5.55±0.31)ng/m l比较差异无统计学意义。结核毒株攻击后,与空载体质粒免疫组相比,质粒AZ-pcDNA3-EF和EZ-pcDNA3-AF免疫的BALB/c小鼠其抗MTB在脾脏中增殖有显著作用,3组脾脏细菌负荷对数值(lg,CFU/g)分别为6.08±0.25、4.63±0.11、4.50±0.32,但不及BCG免疫组的4.09±0.27。结论表达Ag85B与ESAT6融合蛋白的基因疫苗在小鼠体内诱导的IFN-γ水平与BCG相当,其保护力有待进一步提高。