Rapid and efficient identification of cysteine-rich peptides by random screening of a venom gland cDNA library from the hexathelid spider Macrothele gigas.

We identified novel 10 multi-cysteine peptides, namely Magi 7-16, from the spider Macrothele gigas by simple random cDNA screening of the venom gland. Mass analysis of the crude venom detected the mass numbers of the cross-linked forms of all peptides, confirming their presence in the venom. Magi 11, a C-terminus amidated peptide, was chemically synthesized and was indistinguishable from the native peptide proving the feasibility of the method for peptide identification. Moreover, toxicological assays showed diverse lethal or paralytic activities of these peptide toxins on mice and/or insects.     

High-Efficiency Cloning of DNA Sequences Complementary to Mouse Neuroblastoma Polyadenylated RNA

A cDNA library was efficiently synthesized from mouse neuroblastoma poIy(A)⁺RNA. Several modifications of the oligo(dC)(dG) tailing procedure were used. After first strand synthesis, a dATP tail was added to the 3′-end of the cDNA. The second strand was primed for synthesis with oligo(dT). Blunt ends were produced on the cDNA by treatment with S1 nuclease. Size-enriched fractions of high molecular weight DNAs were obtained by passing the cDNA over a Sepharose CL-4B column. The optimal tailing time for each cDNA fraction was individually tested. Tailing reactions used terminal deoxynucleotidyl transferase and annealing reactions used a (G)-tailed PstI cut pBR322. E. coli K12 RR1 cells were transformed and 2.5–5 × 10⁶ transformants per μg cDNA insert were obtained for each size fraction. The transformants had an average insert size of 1200 base pairs and were 98% ampicillin sensitive. Our modifications in the method for cDNA library synthesis had 3 advantages. (1) Homopolymer-primed cDNA treated with S1 nuclease allowed the blund ends to be tailed sychronously. This allowed a higher transformation efficiency without loss of 5′-sequences. (2)Time tailing determined the most efficient tail length and optimized the transformation efficiency in each size fraction. (3) A Sephadex G-50 mini-column was used to desalt and dry nitrogen was used to concentrate the ds cDNA instead of the usual ethanol precipitation. This resulted in almost 100% recovery of synthesized products at each step of this procedure.     

药用真菌杨树菇肽脯氨酰顺反异构酶基因及抗肿瘤活性初步鉴定

目的：鉴定克隆杨树菇抗肿瘤相关蛋白基因活性。方法：构建cDNA文库,随机测序得到脯氨酰顺反异构酶（Peptidyl-prolyl cis/trans isomerase,PPIase）基因,并对其进行生物信息学分析,通过检测其在HeLa细胞中表达引起的细胞凋亡及衰老的变化,初步鉴定其抗肿瘤活性。结果：得到杨树菇PPIase cDNA序列,其氨基酸序列在真菌中十分保守。PPIase基因在真核细胞中表达没有引起肿瘤细胞的凋亡和衰老。结论：杨树菇PPIase序列具有PPIase保守序列模式,是PPIase家族的成员。     

Genes for systemic vascular complications are differentially expressed in the livers of Type 2 diabetic patients

Aims/hypothesis Type 2 diabetes is characterised by excessive hepatic glucose production and frequently leads to systemic vascular complications. We therefore analysed the relationship between the gene expression profile in the liver and the pathophysiology of Type 2 diabetes.Methods Liver biopsy samples were obtained from twelve patients with Type 2 diabetes and from nine non-diabetic patients. To assay gene expression globally in the livers of both groups, we made complementary DNA (cDNA) microarrays consisting of 1080 human cDNAs. Relative expression ratios of individual genes were obtained by comparing cyanine 5-labelled cDNA from the patients with cyanine 3-labelled cDNA from reference RNA from the liver of a non-diabetic patient.Results On assessing the similarities of differentially expressed genes, the gene expression profiles of the twelve diabetic patients formed a separate cluster from those of the non-diabetic patients. Of the 1080 genes assayed, 105 (9.7%) were up-regulated and 134 (12%) were down-regulated in the diabetic livers (p<0.005). The genes up-regulated in the diabetic patients included those encoding angiogenic factors such as vascular endothelial growth factor, endothelin and platelet-derived growth factor. They also included TGF superfamily genes such as TGFA and TGFB1 as well as bone morphogenetic proteins. Among the down-regulated genes in the diabetic patients were molecules defending against stress, e.g. flavin-containing monooxygenase and superoxide dismutase.Conclusions/interpretation These findings suggest that livers of patients with Type 2 diabetes have gene expression profiles indicative of an increased risk of systemic vascular complications.Electronic Supplementary Material Supplementary material is available in the online version of this article at Abbreviations aRNA antisense RNA - BMP bone morphogenetic protein - cDNA complementary DNA - Cy cyanine - FMO flavin-containing monooxygenase - NASH non-alcoholic steatohepatitis - PDGF platelet-derived growth factor - SSC standard saline citrate - SOD superoxide dismutase - VCAM vascular cell adhesion molecule - VEGF vascular endothelial growth factor     

Different hypersensitivities against homologous proteins of MGL_1304 in patients with atopic dermatitis

Background

Atopic dermatitis (AD) is exacerbated by sweating, and the skin of most patients with AD are resided by Malassezia (M.) fungi. Recently, MGL_1304 produced by Malassezia globosa was identified as the major histamine releasing antigen in human sweat.

丙型肝炎5''''非编码区末端快速基因扩增方法的建立及应用

目的 建立快速获得丙型肝炎（HCV）基因组5’非编码区（5’uTR）真末端序列的分子生物学方法。方法 逆转录后利用末端聚合酶（TOT）进行加尾反应，再利用套式聚合酶链反应（PCR）扩增出目的末端基因的cDNA片段，A-T克隆，用限制性内切酶片段长度多态性分析（RVLP）与PCR鉴定重组子，全自动序列分析仪测定插入子序列。结果 cDNA末端快速扩增技术（RACE）获得5株HCV5’UTR克隆，包括3株全长克隆和2株缺失克隆。2株缺失克隆，一条在5’末端缺失53个碱基，另一条缺失144个碱基。结论 RACE技术快速、有效、实用，可有效获得丙型肝炎病毒基因组的5’非编码区末端序列。