Antilymphoproliferative activity of ethanolic extract of Boerhaavia diffusa roots

Extracts of plants have been widely evaluated for possible antiproliferative and anticarcinogenic properties. The antiproliferative activity of ethanolic extract of Boerhaavia diffusa, a plant used in traditional medicine, was evaluated in several cells. It inhibited T cell mitogen phytohemagglutinin and concanavalin A-stimulated proliferation of human peripheral blood mononuclear cells (PBMC). It also inhibited purified protein derivative antigen-stimulated PBMC proliferation and human mixed lymphocyte culture. In addition, B. diffusa extract inhibited the growth of several cell lines of mouse and human origin, such as mouse macrophage cells (RAW 264.7), human macrophage cells (U937), human monocytic cells (THP-1), mouse fibroblast cells (L929), human embryonic kidney cells (HEK293), mouse liver cells (BNLCL.2), African green monkey kidney cells (COS-1), mouse lymphoma cells (EL-4), human erythroleukemic cells (K562), and human T cells (Jurkat). The present study has demonstrated the antiproliferative potential of ethanolic extract of B. diffusa in vitro.     

细胞毒性T淋巴细胞相关抗原4融合蛋白抑制狼疮肾炎患者外周血单个核细胞B7分子表达及

目的探讨细胞毒性T淋巴细胞相关抗原4融合蛋白(CTLA-4Ig)对活动期狼疮肾炎(LN)患者外周血单个核细胞(PBMC)细胞表面B7-1(CD80)和B7-2(CD86)表达的影响及其对抗双链DNA(dsDNA)抗体和免疫球蛋白产生的影响。方法将18例活动期LN患者的抗凝血标本随机分为LN的CTLA-4Ig处理组(LN-T组9例)和普通培养组(LN-NC组9例)。另以14例正常人的抗凝血标本为对照,随机分为正常人的CTLA-4Ig处理组(NC-T组7例)和普通培养组(NC-NC组7例)。用密度梯度离心法分离PBMC。处理组加入CTLA-4Ig(10ng/L)、普通培养组加入等量普通培养基37℃孵育72h后,采用流式细胞仪技术检测PBMC细胞表面B7-1和B7-2分子的表达;ELISA法检测孵育液中抗dsDNA抗体、IgG及IgM水平。结果活动期LN患者CTLA-4Ig处理组与普通培养组比较,PB-MC表面B7-2分子表达明显下降(P<0.01);B7-1分子表达无显著变化(P>0.05);孵育液中抗dsD-NA抗体、IgG及IgM的生成均明显减少(P<0.01)。而正常人的两组间各指标差异均无统计学意义(P>0.05)。结论CTLA-4Ig可抑制活动期LN患者的PBMC表面B7-2分子的表达,并可减少其抗dsDNA抗体、IgG及IgM的分泌。     

HCV核心基因插入突变体编码蛋白对人肝癌细胞系(Huh-7)细胞基因表达的影响

目的用基因表达分析法鉴定丙型肝炎病毒(HCV)核心(Core，C)区基因插入突变体编码蛋白在人肝癌细胞系(Huh-7)表达，探讨该蛋白生物学功能及其基因表达改变与致病的关系。方法构建HCV-1bc基因插入突变体编码蛋白重组表达质粒，建立表达c基因插入突变体编码蛋白Huh-7细胞系，按Affymetrix公司实验程序制备探针、再与该公司H0u133A和Hg-u133b芯片杂交。对基因表达上调或下调≥3倍的基因，用NetAflk作进一步分析。并用半定量RT-PCR对其中3个上调基因进行鉴定。结果Microarray分析显示，HCV-1b C基因插入突变体编码蛋白比c蛋白引起更多的基因表达改变，主要集中在信号传导、蛋白酶活性、分子转运、免疫反应等，特别是免疫反应基因表达更加显著。C基因插入突变体编码蛋白表达可同时导致凋亡基因／抗凋亡基因表达上调或下调及致癌基因上调。半定量RT-PCR对有趣的致癌基因FHL2、抗凋亡基因PRKCZ和凋亡基因LGALSI的鉴定结果表明，FHL2、PRKCZ和LGALSI基因的表达比空载体转染对照组相同基因明显上调。结论Hcvc基因插入突变体编码蛋白在Huh-7细胞表达对其基因表达有很大影响，其中对免疫反应基因的影响更明显，这一结果对理解HCV C基因插入突变体编码蛋白在HCV致病过程中的作用及其研制抗HCV药物均有重大的参考价值。     

Fusion and Matrix Protein Gene Sequence Analysis of Paramyxoviruses of Type 1(PMV-1) Isolated from Pigeons in Slovenia

Paramyxoviruses of type 1 (PMV-l) isolated from pigeons were genetically analyzed. A part of the fusion and the matrix protein genes were amplified and sequenced, Typical amino acid sequences associated with virulence were determined at the fusion protein cleavage site in all PMV-1 isolates. All Slovene pigeon PMV-1 strains share high amino acid sequence similarity with other pigeon strains. In the phylogenetic tree, they are clustered together with pigeon PMV-1 isolates with moderate pathogenicity. Phylogenetic analysis obtained from the fusion and the matrix protein gene alignments showed the same branching order. Viruses circulating among pigeons were found to form quite unique lineage of virulent NDV strains.     

Human testicular protein TPX1/CRISP-2: localization in spermatozoa, fate after capacitation and relevance for gamete interaction

Testicular protein Tpx-1, also known as CRISP-2, is a cysteine-rich secretory protein specifically expressed in the male reproductive tract. Since the information available on the human protein is limited to the identification and expression of its gene, in this work we have studied the presence and localization of human Tpx-1 (TPX1) in sperm, its fate after capacitation and acrosome reaction (AR), and its possible involvement in gamete interaction. Indirect immunofluorescence studies revealed the absence of significant staining in live or fixed non-permeabilized sperm, in contrast to a clear labelling in the acrosomal region of permeabilized sperm. These results, together with complementary evidence from protein extraction procedures strongly support that TPX1 would be mainly an intra-acrosomal protein in fresh sperm. After in vitro capacitation and ionophore-induced AR, TPX1 remained associated with the equatorial segment of the acrosome. The lack of differences in the electrophoretic mobility of TPX1 before and after capacitation and AR indicates that the protein would not undergo proteolytical modifications during these processes. The possible involvement of TPX1 in gamete interaction was evaluated by the hamster oocyte penetration test. The presence of anti-TPX1 during gamete co-incubation produced a significant and dose-dependent inhibition in the percentage of penetrated zona-free hamster oocytes without affecting sperm motility, the AR or sperm binding to the oolema. Together, these results indicate that human TPX1 would be a component of the sperm acrosome that remains associated with sperm after capacitation and AR, and is relevant for sperm-oocyte interaction.     

携带肝细胞生长因子基因重组质粒在大鼠体内的组织分布研究

目的 研究携带肝细胞生长因子基因的重组质粒（pUDKH）经肌肉注射给药后在大鼠体内的组织分布及其与大鼠基因组DNA的整合情况。 方法 二级Wistar大鼠雌雄各20只，按体重和取样时间的不同随机分为12h、24h、3d、7d、14d、21d的各实验组（给予pUDKH，2．0mg／kg）和注射后3d取样的对照组（给予PBS，200ul／只），依次将各组大鼠摘眼球取血后脱臼处死，按脑、心、肺、胸腺、肝、肠系膜淋巴结（MLN）、脾、肾、生殖腺、左腿肌肉、右腿肌肉的顺序取样。经典酚／氯仿抽提法提取各组织总DNA，应用紫外分光光度计测定其浓度和纯度，PCR方法检测其中的D肌动蛋白基因；巢式PCR检测各组织总DNA中pUDKH的分布情况；ApnL I酶切分离pUDKH分布阳性各组织中的pUDKHDNA和基因组DNA，巢式PCR检测pUDKHDNA与基因组DNA的整合情况。 结果 各组织总DNA浓度与纯度均符合实验需要，并适于采用PCR或巢式PCR方法进行体内组织分布及基因组DNA整合情况检测。巢式PCR检测显示，在各时间点的右腿肌肉与外周血、注射后3和7d的肝脏、3和14d的生殖腺、7d的胸腺、7和14d的MLN与左腿肌肉等组织中pUDKH分布均呈阳性，而在各时间点的脑、心、肺、脾、肾等组织中pUDKH分布均呈阴性。pUDKH分布阳性的各组织中均未检测到pUDKHDNA与基因组DNA的整合。 结论 pUDKH经肌肉注射给药后，在各时间点大鼠体内各组织中呈现不同的分布谱。在pUDKH分布阳性的各组织中，pUDKHDNA与基因组DNA发生随机整合的概率较低。     

人胎盘CD133+细胞具有高增殖潜能集落形成细胞特性

目的 通过对人胎盘CD133⁺细胞群中高增殖潜能集落形成细胞(HPP-CFC)检测与生物学特性的分析，证明人胎盘存在早期造血干/祖细胞（HSPC）。 方法 采用机械法制备人胎盘组织（PT）单细胞悬液，用Histopaque-1007分离出单个核细胞(MNC)，经磁式分选（MACS）富集CD133⁺细胞，培养28 d后观察HPP-CFC集落形成能力，用流式细胞仪（FCM）对分选的细胞组份和HPP-CFC进行表型分析，实验全程用脐带血（UCB）作平行比较分析。 结果 培养28 d后，PT-CD133⁺与UCB-CD133⁺细胞组份分别扩增了266和362倍，前者低于后者（P<0.01）；PT-CD133⁺与UCB-CD133⁺细胞中HPP-CFC分别为(32.4±11.2)/5×10³、(17. 7±5.7)/5×10³，前者形成的HPP-CFC数量明显高于后者（P<0.01）；PT-CD133⁺、UCB-CD133⁺细胞培养至28 d时，除UCB-CD133⁺组的CD133⁺CD34^-亚群比例无明显改变外，CD133⁺CD34⁺、CD133^-CD34⁺和CD133⁺CD34^-（PT-CD133⁺组）亚型均比培养前减少。 结论 人胎盘组织CD133⁺细胞中存在HPP-CFC，说明胎盘CD133⁺细胞群中存在早期HSPC。     

Isolation and radiation hybrid mapping of dinucleotide repeat polymorphism at the human matrix Gla protein (MGP) locus

Matrix Gla protein (MGP) is an 84-residue, vitamin K-dependent protein expressed by chondrocytes and vascular smooth muscle cells, and is a potent regulator of calcium deposition in cartilage and arterial wall. We isolated a polymorphic dinucleotide CA repeat marker from a genomic clone containing the human MGP gene. This polymorphism will be useful in genetic studies of arteriosclerosis and osteoporosis. Received: November 5, 1997 / Accepted November 27, 1997     

Localization of hepatitis B surface antigen epitopes present on variants and specifically recognised by anti-hepatitis B surface antigen monoclonal antibodies

Small hepatitis B surface antigen (HBsAg) is considered to be the best marker for the diagnosis of Hepatitis B virus infection. However, HBsAg variants with mutations within the "a" determinant may be poorly or not detected by diagnostic assays. Three anti-HBsAg monoclonal antibodies (6H6B6, 27E7F10, and 2G2G10), directed against conformational epitopes, were tested for their ability to detect the wild-type HBsAg as well as variant forms and their respective epitopes were localised on the HBsAg sequence by using the phage-displayed peptide library technology. Whereas 6H6B6 did not detect mutations T123N, S143L, D144A and G145R, 27E7F10 binding was affected by mutations P120T and G145R. In contrast, 2G2G10 reacted strongly with all tested variants including variant with the G145R mutation. Part of the 6H6B6 epitope was located in the major hydrophilic region (MHR) at residues 101-105, the 27E7F10 epitope (residues 214-219) was located near the C-terminal end of the antigen and the 2G2G10 epitope at residues 199-208, within the theoretical fourth transmembrane helix. The 2G2G10 epitope localisation brings information about the HBsAg structure and the validity of established topological models. Finally, 2G2G10 is a valuable tool for HBsAg variant detection that is used as capture phase in a new bioMérieux diagnostic assay, which is currently in development.     

Rezidivierende Meningitis bei familiärem Mangel der achten Komponente des Komplementsystems

Summary An 18-year-old man suffered from recurrent bacterial meningitis. Investigation of the complement system revealed deficiency of the 8th complement component (C8) in the patient and his sister. Genetic defects of the terminal complement components C5 to C8 predispose toNeisseria infections, probably due to a lack in bacteriolytic activity. It is to be noted that 1 year ago the patient had been hospitalized for a culture-proved pneumococcal meningitis.