Molecular epidemiology of norovirus variants detected in children under five years of age in Hyderabad,India

PurposeNoroviruses are common viral agents in acute diarrhea in all age groups worldwide. Norovirus has been classified into 10 genogroups, GI to GX with over 48 genotypes among them the GII.4 genotype has evolved over time with a clear pattern of periodic variant replacement. Immunity is strain or genotype specific with little or no protection conferred across genogroups. The present study was aimed to determine the epidemiology, prevalent genotypes of norovirus in children below five years of age in the Hyderabad region, India.MethodsThe stool samples and clinical data were collected from 458 children below 5 years of age comprising of cases with acute gastroenteritis (n ?= ?366) and a control group (n ?= ?92) admitted to the pediatric ward. All the samples were tested for Norovirus by ELISA and RT-PCR. Sequencing was done for predominant strains.Results10.3% (n ?= ?38) of cases and 3.2% (n ?= ?3) of the control group were found to be Norovirus positive. Predominant genotypes were GII-82.5% followed by GI-12.5%.ConclusionSequencing and Phylogenetic analyses of 20 GII.4 strains was done. All of the isolates are clustered away from published the GII.4 variants thus suggesting the appearance of a new variant.     

PD98059对人骨髓间质干细胞分化为成骨细胞的影响

目的:探讨胞外信号调节激酶(ERK)信号传导途径对骨髓间质干细胞(MSC)分化为成骨细胞的影响。方法:采用Ficoll-Paque淋巴细胞分离液分离成人MSC,体外扩增,应用地塞米松、β-甘油磷酸钠、vitaminC定向诱导MSC分化为成骨细胞。在成骨诱导液中加入不同剂量的PD98059,观察其对成骨细胞形成的影响。结果:MSC体外扩增15代可获得(3-4)×10¹²个细胞。在成骨诱导液作用下,MSC可在体外定向分化为成骨细胞。不同剂量的PD98059均可抑制MSC分化为成骨细胞,并有剂量依赖关系;同时促使部分细胞转化为脂肪细胞。结论:ERK信号传导途径可能在MSC分化为成骨细胞和脂肪细胞过程中起关键作用。     

HIV核心抗原p24原核系统的高效表达、纯化及活性鉴定

目的　在原核表达系统中对HIVp2 4抗原基因进行克隆和高效表达、纯化并鉴定其活性。方法　利用PCR技术从HIV-1全基因质粒 (B2N)中扩增p2 4抗原基因 ,并克隆入T载体中。通过酶切消化后连接到表达载体pRSET上 ,用此连接产物转化大肠埃希菌BL2 1,经IPTG诱导 ,表达p2 4抗原。利用固定化金属离子 (Ni2 )配体亲和层析技术从表达蛋白中纯化目的蛋白。并运用双酶切技术、SDS-PAGE电泳、WesternBlot(WB)及ELISA法分别对插入基因片段的正确性、表达产物的活性及特异性进行检测。结果　PCR产物约为 6 90bp ,与预期p2 4抗原全基因片段大小一致。重组质粒T-p2 4和pRSET-p2 4经BamHⅠ和HindⅢ双酶切 ,其插入的外源基因片段均为 6 90bp。将纯化前与纯化后的蛋白作SDS PAGE电泳 ,均可见一条约 2 4× 10 3的外源基因表达带 ,与计算的相对分子质量相符。经WB和ELISA试验 ,证明基因工程表达的p2 4抗原具有较高的特异性及活性。结论　成功构建了HIVp2 4表达载体pRSET-p2 4 ,并在原核细胞中高效表达 ,其表达产物具有良好的特异性及活性     

叶酸拮抗同型半胱氨酸诱导的内皮细胞凋亡的作用机制

目的： 明确同型半胱氨酸（Hcy）对内皮细胞凋亡的影响以及叶酸的拮抗作用，阐明Bax和Bcl-2在同型半胱氨酸诱导内皮细胞凋亡及叶酸拮抗中的作用。方法: 用不同浓度的Hcy处理内皮细胞后，应用末端转移标记技术（TUNEL）以及Annexin V/PI染色加流式细胞术了解细胞凋亡状态，免疫组化方法检测Bax、Bcl-2的表达。结果: Hcy能促进细胞凋亡，叶酸具有拮抗作用。Hcy能促进细胞Bax、Bcl-2的表达，上调Bax/Bcl-2比值，叶酸能减少细胞表达Bax及Bcl-2，下调Bax/Bcl-2比值。结论: Bax、Bcl-2参与了Hcy诱导内皮细胞凋亡以及叶酸拮抗作用的过程。     

14-3-3σ调控p27抑制Rat1-Akt细胞增殖

目的： 研究腺病毒介导14-3-3·σ（Ad-14-3-3·σ）对Akt过表达Rat1-Akt细胞增殖的影响，并探讨其作用是否通过调控p27而实现。 方法： 通过5-溴-2′脱氧尿嘧啶（BrdU）实验检测Ad-14-3-3·σ对Rat1-Akt细胞增殖的影响，并通过激酶分析法和免疫荧光实验探讨Ad-14-3-3·σ对p27磷酸化水平及其在细胞内定位的影响。 结果： Ad-14-3-3σ转染的细胞BrdU阳性率（45%）低于PBS处理组（100%）或Ad-β-gal转染的对照组细胞（98%）。14-3-3σ可降低磷酸化p27的水平和减少Akt介导的p27在胞浆中的定位。 结论： 转染14-3-3σ基因能抑制Akt过表达细胞株Rat1-Akt增殖，14-3-3σ通过降低Akt激酶磷酸化p27的活性，阻断Akt介导的p27胞浆错位，从而发挥其抑制Rat1-Akt细胞增殖。     

预测蛋白质相互作用位点的计算方法研究(一)

许多重要的细胞过程如信号转导、转运、细胞运动以及多数调节机制均由蛋白-蛋白之间的相可作用介导，蛋白质之间的相互作用在物理上是通过在两个相互作用蛋白之间形成接触面的短残基序列来实现。识别蛋白-蛋白相互作用位点，以及检测相互作用氨基酸残基之间的特异性与强度特异性，是一个具有重要应用前景的课题，它的应用范围从理性的药物设计到代谢和信号转导网络的分析。虽然有不少准确度不断提高的实验技术和计算方法来检测蛋白质之间的相互作用，但很少有方法能够精确地指出参与蛋白质相互作用的特定残基及其位置，而这些信息是将相互作用数据直接应用于药物开发所必需的。随着生物信息学和计算生物学的发展，通过研究已知蛋白-蛋白相互作用位点的这些不同特征．出现了一些利用序列与结构信息顶测蛋白-蛋白相互作用位点的计算方法。本文简要介绍了近年来在顶测蛋白-蛋白的相互作用位点方面取得一定进展的计算方法，包括基于基因组信息的计算方法、基于蛋白质初级序列的计算方法以及基于蛋白复合物结构信息的计算方法。虽然这些方法在过去儿年里取得了显著的进展，但是大多数在这方面的研究仍处于起步阶段．而现在数据库的不足和实验技术的缺陷对计算预测方法的进一步发展和公平性评价也存在着较大的影响，要提高蛋白-蛋白相巨作用位点预测的鲁棒性与可靠性，仍要有很多的工作要做。（发表在这里的是第一部分）     

Are thrombocyte membranes altered in Alzheimer''s disease? A morphometric and biochemical study

Morphometric analysis of thrombocytes from patients with Alzheimer's disease, from patients with multi-infarct dementia, and from young and agematched healthy control donors, did not reveal any Alzheimer-related increase in internal membranes. Biochemical analysis showed a reduced cholesterol content of thrombocyte membrane preparations from Alzheimer patients relative to age-matched controls, but not relative to multi-infarct dementia patients. Overall distribution of protein kinase C activity (PKC) between cytosol and membrane, in resting as well as in activated thrombocytes from Alzheimer patients, was similar to that in the control groups. However, both Alzheimer and multi-infarct dementia patients had lower cytosolic levels of basal kinase and PKC activities than age-matched controls, while only Alzheimer patients had lower cytoskeletal PKC activity than controls.     

羟丁酸钠对缺氧缺血后新生大鼠海马CA1区Bcl-2、Bax蛋白表达的影响

目的：观察羟丁酸钠(GHB)对新生大鼠缺氧缺血性脑损伤（HIBD）后海马CA1区神经元Bcl-2、Bax蛋白表达的影响。方法：生后 7 d SD大鼠采用Rice等法，制成HIBD动物模型。新生大鼠随机分成假手术（sham）组、缺氧缺血（HI）组、GHB组。其中GHB组包括GHB50（50 mg/kg）、GHB100（100 mg/kg）、GHB200（200 mg/kg）亚组。各组在缺氧完成后 1 h、3 h、24 h、72 h 和 168 h 时点取脑切片作HE染色，用免疫组化染色观察Bcl-2、Bax蛋白的表达。结果：①光镜下HE染色结果：HI组海马CA1区锥体细胞排列紊乱，锥体细胞减少，海马带宽窄不一，可见细胞肿胀和核碎裂。GHB50组和GHB100组可减轻锥体细胞层病理改变。②免疫组化染色结果：HI组缺血缺氧后1h海马CA1区 Bcl-2、Bax表达开始增强，24 h 时达到高峰，其后逐渐减弱。在GHB50组和GHB100组可使Bcl-2表达明显高于HI组（P<0.05，P<0.01），Bax表达明显低于HI组（P<0.05，P<0.05）。结论：GHB可通过对Bcl-2、Bax表达的调控抑制新生大鼠HIBD后海马CA1区神经元损伤。     

钙激活的氯通道gob-5和粘蛋白基因Muc5ac在小鼠哮喘模型肺部的表达

目的：研究“钙激活的氯通道”成员之一gob-5和粘蛋白基因Muc5ac在小鼠哮喘模型肺部的表达。 方法： 22只清洁级BALB/c小鼠随机分成哮喘组和对照组，用逆转录聚合酶链反应法（RT-PCR）和免疫组化法分别测定肺组织gob-5 mRNA和粘蛋白基因Muc5ac蛋白的表达。 结果： 小鼠哮喘组肺组织gob-5 mRNA表达阳性（0.2297±0.0186，A），而对照组未出现gob-5 mRNA的表达(P<0.01)；哮喘组Muc5ac蛋白表达（0.6637±0.0127，A）明显高于对照组（0.2060±0.0179，A）（P<0.01）；哮喘组肺组织gob-5 mRNA表达与Muc5ac蛋白表达呈直线正相关（r=0.864, P<0.05）。 结论： Gob-5 mRNA表达的上调可能在哮喘小鼠气道粘液过度分泌中起着关键作用；抑制gob-5的表达将为哮喘的治疗提供新的策略。     

Serous papillary carcinoma of the endometrium arising from endometrial polyps: a clinical, histological, and immunohistochemical study of 13 cases

Serous papillary carcinoma is an aggressive tumor. Point mutations in the p53 suppressor gene might explain in part the rapid growth of this malignant tumor and its unfavorable outcome. The aims of this study were to evaluate the behavior of serous papillary carcinoma developing in endometrial polyps and to assess the p53 protein overexpression. Patients included in this study were treated in our institution between 1982 and 2003. All clinical and pathological materials were examined. A p53 protein immunohistochemical analysis was performed on paraffin-embedded tissues. Thirteen serous papillary carcinomas arising from benign polyps of the endometrium were identified. The patients' age averaged 73 years. All patients were treated surgically. After an average follow-up of 22 months, 54% of the patients were dead or alive with disease. Of 10 serous papillary carcinomas, 8 (80%) for which paraffin blocks were available overexpressed the p53 protein. A serous papillary carcinoma arising from benign polyps of the endometrium remains a malignant neoplasia with an unfavorable outcome even if the primary tumor is limited to the polyp. The high rate of protein p53 overexpression suggests that a p53 gene mutation occurs early in the disease and might explain the rapid growth of the tumor.