-G gamma A gamma-Thalassemia and gamma-chain variants in Chinese newborn babies

The occurrence of gamma-chain abnormal hemoglobins and of gamma-thalassemia in Chinese newborns was evaluated through analyses of the Hb F of over 1,100 babies and of the DNA from one baby and his parents. Gene mapping data identified this baby as a homozygote for -G gamma A gamma-thalassemia, which is caused by a deletion of about 5 kb due to an unequal crossing-over between the -G gamma- and -A gamma- genes. This condition is the same as that observed in Indian and Japanese babies [2,3]. Its gene frequency among babies from the Shanghai area was 0.012. A previously unrecognized G gamma chain variant, Hb F-Shanghai or alpha 2 G gamma 266(E10)Lys----Arg, was observed in one newborn. This variant was not detected by conventional techniques but only by high performance liquid chromatography, as the G gamma 66 Lys and G gamma 66 Arg chains had slightly different chromatographic mobilities. Lys at position gamma 66 participates in contacts with the heme group, and its substitution by another amino acid residue might interfere with physiochemical and/or functional properties. No other gamma-chain variants have been detected except the well-known A gamma T or F-Sardinia chain (f.A gamma T = 0.076).     

Two arrangements of the human fetal globin genes may be responsible for high G gamma values in Chinese newborns: -G gamma-G gamma-delta-beta and -G gamma-A gamma G gamma-A gamma-delta-beta

High G gamma values (78% and higher) have been observed in about 3% of Chinese newborns from the Shanghai area. We describe here two arrangements different from the normal -G gamma-A gamma-delta-beta arrangement which have been characterized in the DNA from three of these babies. One baby was heterozygous for a chromosome with two linked G gamma globin genes (-G gamma-G gamma-delta-beta), which has also been observed in Black newborns [Powers et al, Nucleic Acids Res 12:7023, 1984], while the two other babies were heterozygous for a chromosome with triplicated gamma globin genes, presumably of the -G gamma-A gamma G gamma-A gamma-delta-beta arrangement. A similar triplication has been observed in natives of the New Hebrides [Trent et al, Nucleic Acids Res 9:6723, 1981].     

前列腺癌特异突变DNA聚合酶β表达载体转染NIH3T3的细胞生物学行为变化

目的观察前列腺癌(CPa)特异突变DNA聚合酶β(polβ)表达载体转染NIH3T3的细胞生物学行为变化。方法采用业已成功构建的2例特异突变polβ表达载体(pCDNA3.1-M1和pCDNA3.1-M2),采用脂质体包裹转染NIH3T3细胞,观察比较这些突变对细胞polβ基因mRNA和蛋白表达的影响以及引发的相应的细胞周期和细胞自发突变率等细胞生物学行为的变化。结果筛选得到高表达PCa特异突变polβ的NIH3T3细胞株(NIH3T3-M1,NIH3T3-M2)和高表达野生型polβ的NIH3T3细胞株(NIH3T3-W)。RT-PCR和Western印迹结果显示,外源DNApolβ在转化的NIH3T3细胞中呈现高表达。与转染空载体和未转染的NIH3T3细胞相比,转染PCa特异突变polβ的2个细胞株和转染野生型polβ的细胞株的细胞周期的S期比例及转染细胞的HGPRT(次黄嘌呤-鸟嘌呤磷酸核糖转移酶)基因的自发突变率均显著增加,且转染野生型polβ的细胞株亦显著高于转染PCa特异突变DNApolβ的细胞株。结论 DNApolβ高表达介导了肿瘤发生的分子机制。     

The Greek A gamma beta+-HPFH observed in a large black family

Several members of a Black family with a heterozygosity for an A gamma beta+-HPFH, shown in 1969 to have relatively low levels of Hb F and a low glycine to alanine ratio in the gamma chain of this Hb F, were reinvestigated. Thirteen of 30 available family members in two generations had the heterozygous form of this condition, which was characterized by a decreased level of Hb A2, an average Hb FAD value of 13.3%, an equal distribution of Hb F over the red cells, and normal hematological values. The gamma chain composition of isolated Hb F was determined by reversed phase high performance liquid chromatography for all 13 heterozygotes and showed an average A gamma value of 84.5%. Hybridization with synthetic oligonucleotides, specific for normal and mutant sequences at positions 111-129 5' to the A gamma globin gene, identified a G----A base substitution at position 117, similar to that seen in subjects with the Greek A gamma-HPFH. Our data support conclusions by others that this replacement is causative of the increased A gamma chain synthesis in this condition. Haplotype analysis supported the suggestion that the G----A substitution occurred as an independent event in this Black family.     

补体C3及C3 c和C3 d在IgA肾病肾组织中的表达

目的 阐明补体C3,C3c和C3 d分子在IgA肾病肾组织中的表达,探讨C3、C3c和C3d在IgA肾病发病中的作用.方法 对36例IgA肾病和43例无IgA沉积的系膜增生性肾炎患者进行回顾性分析,对其肾组织进行抗人C3,C3c和C3 d抗体检查,在免疫荧光显微镜下观察C3、C3c和C3 d在肾组织上各自的表达.结果 补体C3在IgA肾病组肾小球上的表达高于系膜增生性肾炎组患者.C3c和C3 d仅在IgA肾病上发现表达;它们的共同表达与肾脏病理损害有关(r=0.99176,P=0.0009).结论 补体C3、C3c和C3 d在IgA肾病肾组织上高表达,而且C3c和C3 d共同表达与肾脏损害有关,可能是向终末性肾衰竭转化的标志.     

HB F-Yamaguchi (γ75THR, γ80ASN, γ136ALA) is associated with Gγ-thalassemia

Restriction endonuclease analyses of DNA from a known Hb F-Yamaguchi heterozygote and three of his relatives have shown a deletion of about 5 kb, which includes one of the γ genes. This abnormality is similar to the ^Gγ-thalassemia described recently [4] and is probably caused by an unequal crossing over between -^Gγ- and -^Aγ^T-genes. The abnormal -^Gγ^Aγ^T-X- (X = Asp→Asn at γ80) hybrid gene produces the γ-Yamaguchi chain at a level usually seen for ^Gγ chains only.     

Different molecular defects of G gamma (A gamma delta beta)o-thalassaemia in Thailand

DNA from members of 2 Thai families with conditions considered to be delta beta-thalassaemia were studied by using restriction endonuclease DNA mapping. The propositus in family A is a double heterozygote for beta-thalassaemia and delta beta-thalassaemia. DNA analysis reveals a deletion of the beta-globin gene cluster starting at the area between the Sac I and Eco RI sites near the 3' end of the G gamma-gene and extending through the A gamma-, delta- and beta-genes to an unknown extent downstream. In family B, the propositus is delta beta-thalassaemia/Hb E. Deletion of the beta-globin gene cluster begins in the large intervening sequence of the A gamma-gene and removes both delta- and beta-genes downstream.     

人垂体瘤转化基因1对NIH3T3细胞无直接肿瘤转化作用

目的 克隆人垂体瘤转化基因1（hpTTG1)cDNA并确定其在不同细胞内的分布和其在体外是否具有直接的肿瘤转化作用。方法 （1）用快速扩增（RACE）克隆hPTTG1 cDNA；（2）用Northern印迹检测hPTTG1 mRNA在肿瘤细胞的表达水平；（3）构建pCMX-GFP-hPTTG1表达质粒并转染HeLa等6种细胞，用荧光共聚焦显微镜观察GFP－hPTTG1的细胞内分布；（4）构建pIRESneo-hPTTG1表达质粒并转染NIH3T3细胞，经G418选择后检测hPTTG1是否有直接的致肿瘤作用。结果 （1）hPTTG1与大鼠PTTG的同源性为79％。开放可读框架由609bp组成，编码202个氨基酸；（2）hPTTG1在血液系统肿瘤细胞表达水平较其它肿瘤细胞为高，其表达水平由强到弱依次为THP－1，Raji,CEM,AR230,DLD-1,H1299,HeLa,HepG2和A549肿瘤细胞；（3）hPTTG1的细胞内分布在HeLa、Cos-7和DU145细胞主要位于细胞核，在A549、DLD－1和NIH3T3细胞则呈胞浆和胞核的弥漫性分布；（4）hPTTG1明显抑制NIH3T3细胞的生长，降低[^3H]胸腺嘧啶脱氧核苷的掺入率。hPTTG1单独不具有细胞肿瘤转化作用。结论 （1）hPTTG1在血液系统肿瘤细胞表达水平较其它肿瘤细胞为高；（2）hPTTG1的细胞内分布与细胞类型有关；（3）hPTTG1单独不具有细胞肿瘤转化作用。     

mdig基因的研究进展

mdig意为"粉尘诱导的基因",是2005年在煤矿工人肺泡巨噬细胞中发现的,并证实是一种新的肺癌基因.mdig基因抑制H3K9me3形成,并且可以使H3K9me3脱甲基化变成H3K9me2、H3K9me1,促使肺癌发生发展.目前已经发现mdig在肺癌等几种恶性肿瘤中过表达.本文就mdig的最新研究进展作一综述.     

日本血吸虫重组信号蛋白14-3-3疫苗免疫保护性的观察

目的　研究日本血吸虫(中国大陆株)重组信号蛋白1433(rSj1433)作为血吸虫病疫苗分子的潜能,并探讨rSj1433、rSjGST两种重组蛋白作为疫苗的协同作用及结核杆菌低分子量耐热多肽(Mtb)激活的γδT细胞在抗血吸虫病中的作用。　方法　用SDSPAGE、电洗脱和透析的方法制备rSj1433和rSjGST抗原,将两种抗原(分别用福氏佐剂和Mtb为佐剂)分别免疫BALB/c小鼠后,进行尾蚴攻击感染实验。在攻击感染6wk后,剖杀小鼠计算各组的减虫率。　结果　各组的减虫率为rSj1433+福氏佐剂组32.20%,rSj1433+rSjGST+福氏佐剂组31.10%,rSj1433+Mtb佐剂组27.96%,rSj1433+rSjGST+Mtb佐剂组26.00%,rSjGST+Mtb佐剂组27.10%;各组的减卵率分别为(按以上组序)50.40%、53.30%、51.10%、58.60%和51.30%。　结论　rSj1433具有一定的抗血吸虫潜能,有可能成为抗日本血吸虫疫苗,但未见rSj1433和rSjGST的协同作用;Mtb激活扩增的γδT细胞在抗血吸虫免疫中的效果与福氏佐剂产生的免疫作用类似。     

高糖对成骨细胞MC3T3-E1活性影响的研究

目的探讨高糖对体外培养小鼠前体成骨细胞MC3T3-E1活性的影响。方法将培养的MC3T3-E1细胞分为5．5mmol／L正常糖对照组、12．5mmol／L高糖组和25mmol／L高糖组,观察不同浓度糖对MC3T3-E1细胞增殖、碱性磷酸酶（ALP）、矿化结节、骨钙素（OC）及Caspase-3的影响。结果与5．5mmol／L正常糖对照组相比,12．5mmol／L高糖组培养5d细胞增殖和ALP分泌不受影响,在培养7d和14d时明显促进矿化结节形成,在培养5d时促进细胞内0（2表达,在培养3d时促进Caspase-3的表达;而25mmol／L高糖组在培养5d则可明显抑制细胞增殖及ALP分泌,在培养14d减少矿化结节的形成,在培养第5d时减少0（2表达,在培养3d时促进Caspase-3的表达。结论高糖可影响MC3T3-E1细胞活性,12．5mmol／L糖在培养时间内不影响细胞增殖与分化,可促进矿化;25mmol／L糖则对细胞产生明显的抑制效应。     

过敏性哮喘患者外周血T-bet、GATA-3、RORγt和FoxP3 mRNA的表达变化

目的：探讨特异性转录因子T-bet、GATA-3、RORγt和FoxP3 mRNA在过敏性哮喘患者外周血中的表达变化。方法：选择30例慢性持续期的屋尘螨过敏性哮喘患者,其中轻度哮喘17例、中重度哮喘13例,14名正常人作为对照。通过荧光实时定量PCR方法检测3组患者外周血单个核细胞（PBMC）中T-bet、GATA-3、RORγt和FoxP3mRNA的表达。结果：轻度和中重度哮喘患者的GATA-3 mRNA表达均显著高于正常人（均P〈0.01）。中重度哮喘患者的RORγt mRNA表达明显高于正常人和轻度哮喘患者（均P〈0.01）。T-bet和FoxP3 mRNA表达在3组间差异无统计学意义（均P〉0.05）。结论：过敏性哮喘中GATA-3表达增加,中重度哮喘中RORγt表达增加,GATA-3和RORγt在哮喘发病中起着重要的作用。     

Level and composition of fetal hemoglobin expression in normal newborn babies are not dependent on beta cluster DNA haplotype.

Interindividual variations in the level and composition of fetal hemoglobin observed in 604 cord blood samples from normal white and nonwhite newborns, unlike those in adults, are not dependent on beta gene cluster DNA haplotype. The data suggest that the mechanism(s) involved in the neonatal expression of Hb F is distinct from that at the adult stage.     

Identification of an epithelial cell receptor responsible for Clostridium difficile TcdB-induced cytotoxicity

Clostridium difficile is the leading cause of hospital-acquired diarrhea in the United States. The two main virulence factors of C. difficile are the large toxins, TcdA and TcdB, which enter colonic epithelial cells and cause fluid secretion, inflammation, and cell death. Using a gene-trap insertional mutagenesis screen, we identified poliovirus receptor-like 3 (PVRL3) as a cellular factor necessary for TcdB-mediated cytotoxicity. Disruption of PVRL3 expression by gene-trap mutagenesis, shRNA, or CRISPR/Cas9 mutagenesis resulted in resistance of cells to TcdB. Complementation of the gene-trap or CRISPR mutants with PVRL3 resulted in restoration of TcdB-mediated cell death. Purified PVRL3 ectodomain bound to TcdB by pull-down. Pretreatment of cells with a monoclonal antibody against PVRL3 or prebinding TcdB to PVRL3 ectodomain also inhibited cytotoxicity in cell culture. The receptor is highly expressed on the surface epithelium of the human colon and was observed to colocalize with TcdB in both an explant model and in tissue from a patient with pseudomembranous colitis. These data suggest PVRL3 is a physiologically relevant binding partner that can serve as a target for the prevention of TcdB-induced cytotoxicity in C. difficile infection.Clostridium difficile infection (CDI) is the leading cause of antibiotic-associated diarrhea and pseudomembranous colitis in the United States (1, 2). Over the past decade, morbidity and lethality from CDI have increased (3, 4), and the need for new treatment options has become a priority.The pathology associated with CDI is associated with the activities of two large, glucosylating toxins, TcdA and TcdB (5). Upon binding to the colonic epithelium, these toxins induce the fluid secretion, immune cell influx, and tissue damage associated with clinical manifestations of CDI (5). TcdA and TcdB have four functional domains: an N-terminal glucosyltransferase domain (GTD), an autoprotease domain, a pore-forming and delivery domain, and a combined repetitive oligopeptides (CROPS) domain, which extends from around residue 1830 to the C terminus and has been implicated in receptor binding. The toxins enter cells by receptor-mediated endocytosis (6). Acidification of the endosome is thought to trigger a structural change in the delivery domain, allowing for pore formation and translocation of the GTD into the cytosol (7, 8). Activation of the autoprocessing domain by eukaryotic inositol hexakisphosphate results in the release of the GTD into the cell, allowing access to substrates (8). The GTD transfers a glucose from UDP glucose onto the switch I region of Rho family GTPases such as Rho, Rac1, and Cdc42 (9, 10). These modifications cause a cytopathic effect resulting from rearrangement of the actin cytoskeleton and can lead to apoptosis (11). At higher concentrations, TcdB is also capable of inducing the production of reactive oxygen species, resulting in cell death by a necrotic mechanism (12, 13). We speculate that both mechanisms are important in the context of disease; the cytopathic effects promote inflammation and disruption of the tight junctions, whereas the TcdB-induced necrosis contributes to the colonic tissue damage observed in severe cases of CDI.Although TcdA and TcdB are homologs, they appear to perform separate, nonredundant functions (14, 15). TcdA and TcdB are thought to have different receptors, based on sensitivity differences among cell types in vitro (16–19). Multiple receptors for TcdA have been proposed including Gal alpha 1–3Gal beta 1–4GlcNAc, blood antigens I, X, and Y, rabbit sucrase isomaltase, and gp96 (18, 20–22). The TcdA CROPS domain is thought to play a role in binding cell surface carbohydrates (18, 23, 24). Antibodies against the CROPS domains of TcdA and TcdB can block intoxication (25, 26), and excess TcdA CROPS domain can compete with TcdA holotoxin for cell binding (27). At the same time, truncations of TcdA and TcdB that lack the CROPS domains are still capable of intoxicating cells (7, 28, 29) and a homologous toxin from Clostridium perfringens, TpeL, lacks a CROPS domain entirely (29, 30). A receptor for TpeL has been identified (29), suggesting that a receptor-binding site for other large clostridial toxins could exist outside of the CROPS. A recent report indicates that chondroitin sulfate proteoglycan 4 (CSPG4) mediates TcdB-induced cytopathic and apoptotic events in HeLa and HT29 cells (31). CSPG4 does not mediate the necrotic effects that occur at higher TcdB concentrations and CSPG4 binds TcdB outside the CROPS; these observations are consistent with a dual receptor hypothesis. This study represents an independent effort to define the cellular factor(s) responsible for TcdB binding and toxicity.     

DIM联合肿瘤坏死因子相关凋亡诱导配体对前列腺癌细胞的抑制作用

目的 探讨3,3-二吲哚基甲烷(DIM)联合肿瘤坏死因子相关凋亡诱导配体(TRAIL)对前列腺癌细胞的抑制作用.方法 CCK-8法测定DIM、TRAIL及DIM联合TRAIL作用于PC-3细胞后的细胞生存率,同时用流式细胞术测定细胞凋亡率,Western印迹检测caspase-3表达水平的变化.结果 各给药组与对照组比较均能不同程度抑制PC-3细胞增殖并诱导其凋亡,且caspase-3表达上调;DIM与TRAIL联用明显增强诱导凋亡作用.结论 DIM与TRAIL联用均可明显增强PC-3细胞抑制效果和诱导凋亡作用,其凋亡作用机制可能与其上调caspase-3表达有关.