Differential catalytic efficiency of allelic variants of human glutathione S-transferase Pi in catalyzing the glutathione conjugation of thiotepa.

Alkylating agents are extensively used in the treatment of cancer. The clinical usefulness of this class of anticancer drugs, however, is often limited by the emergence of drug-resistant tumor cells. Increased glutathione (GSH) conjugation through catalysis by GSH S-transferases (GSTs) is believed to be an important mechanism in tumor cell resistance to alkylating agents. In the present study, we report that the allelic variants of human Pi class GST (hGSTP1-1), which differ in their primary structures at amino acids in positions 104 and/or 113, exhibit significant differences in their activity in the GSH conjugation of alkylating anticancer drug thiotepa. Mass spectrometry revealed that the major product of the reaction between thiotepa and GSH was the monoglutathionyl-thiotepa conjugate. While nonenzymatic formation of monoglutathionyl-thiotepa was negligible, the formation of this conjugate was increased significantly in the presence of hGSTP1-1 protein. The hGSTP1-1-catalyzed GSH conjugation of thiotepa was time and protein dependent and followed Michaelis-Menten kinetics. The catalytic efficiency of hGSTP1-1(I104, A113) variant was approximately 1.9- and 2.6-fold higher compared with hGSTP1-1(V104,A113) and hGSTP1-1(V104,V113) isoforms, respectively. The results of the present study indicate that the hGSTP1-1 polymorphism may be an important factor in GST-mediated tumor cell resistance to thiotepa, and that subjects homozygous for the hGSTP1-1(I104,A113) allele, which is most frequent in human populations, are likely to be at a greater risk for developing GST-mediated resistance to thiotepa than heterozygotes or homozygotes with valine 104 background.     

喀斯特小流域栓皮栎林土壤螨类群落结构特征

为比较石漠化环境与喀斯特森林土壤螨类的群落结构差异,对贵州喀斯特地区朝营小流域栓皮栎林的土壤螨类群落结构本底进行了研究,经2014年各季节的4次调查,共发现土壤螨类3目54科83属.对螨类属数、个体数量、个体密度、Shannon多样性指数(H)、Margalef丰富度指数(SR)、Pielou均匀性指数(J)、捕食性螨类成熟度指数(MI)、甲螨MGP类群和甲螨营养结构等进行了分析.结果表明:土壤螨类在类群属数和个体数量上均以甲螨亚目的属占优势,夏季和秋季具有丰富的属数、较高的个体密度与多样性,春季和秋季具有丰富的个体数量,群落分布具有明显的表聚性.捕食性螨类夏季生态类群以K选择型为主,其他季节以r选择型为主;甲螨生态类群主要为P型和O型,缝甲螨属、异珠足甲螨属和合若甲螨属等属构成了栓皮栎林土壤螨类的营养功能集团.研究表明,该区山毛榉林与其他地区山毛榉林、其他不同类型森林的土壤螨类主要类群存在差异,其中含丰富属组成的派伦螨科、厉螨科、奥甲螨科和单翼甲螨科以及多奥甲螨属、派伦满属、菌甲螨属和单翼甲螨属等数量上占优势的类群属可作为山毛榉林土壤环境的生物指示.     

抗菌肽VIP在毕赤酵母中的高效表达及鉴定

基于人抗菌肽VIP(Vasoactive intestinal peptide)基因序列,按照毕赤酵母密码子偏好性设计引物;用SOE-PCR法扩增目的基因;然后将目的基因克隆至毕赤酵母分泌型表达载体pPICZαA上,构建VIP分泌表达菌株GS115-p PICZαA-vip。用甲醇诱导96 h收集上清,用质谱进行鉴定,结果显示分泌表达产物与人抗菌肽VIP理论值(3 326.82 Da)完全一致,表明人抗菌肽VIP成功得到分泌表达。琼脂糖凝胶扩散法实验结果显示,重组VIP对大肠杆菌Escherichia coli ATCC25922和金黄色葡萄球菌Staphylococcus aureus ATCC25923都有很强的抗菌活性,MIC(Minimal inhibitory concentration)分别为8 mmol/L和16 mmol/L。进一步细胞毒性和溶血性实验结果显示,重组VIP对正常细胞NCM460和IPEC-J2没有毒性,其对SD大鼠红细胞不具有溶血活性。通过透射电镜观察了VIP的抗菌机制,结果显示VIP主要通过破坏细胞膜的方式抑杀细菌。本研究为人抗菌肽VIP的开发应用和大量生产奠定了基础。     

巴氏杆菌糖酵解酶的黏附作用研究

巴氏杆菌（主要是多杀性巴氏杆菌）可以引起多种动物疫病（巴氏杆菌病）,同时也引起人类感染发病。[目的] 研究巴氏杆菌糖酵解酶对宿主细胞（兔肾细胞）和两种常见分子[纤连蛋白（fibronectin,Fn）和血浆纤维蛋白溶解酶原（plasminogen,Plg）]的黏附作用。[方法] 采用原核表达系统对多杀性巴氏杆菌的糖酵解酶进行表达并纯化及制备多克隆抗体,通过菌体表面蛋白定位检测、黏附与黏附抑制等实验探究巴氏杆菌糖酵解酶的黏附作用。[结果] 菌体表面蛋白检测结果显示除烯醇化酶和丙酮酸激酶外的7个糖酵解酶在多杀性巴氏杆菌表面存在。这7个糖酵解酶均能黏附兔肾细胞,但仅有磷酸葡萄糖异构酶的多克隆抗体能对多杀性巴氏杆菌黏附宿主细胞产生抑制作用。Far Western blotting结果显示9个糖酵解酶均能结合宿主Fn和Plg。招募抑制实验结果显示磷酸葡萄糖异构酶、醛缩酶、磷酸甘油酸变位酶的抗体对多杀性巴氏杆菌结合Fn和Plg都有抑制作用,磷酸果糖激酶、丙糖磷酸异构酶、甘油醛-3-磷酸脱氢酶、磷酸甘油激酶抗体仅对多杀性巴氏杆菌结合Fn或Plg有抑制作用。[结论] 多杀性巴氏杆菌糖酵解酶成员葡萄糖异构酶、磷酸果糖激酶、醛缩酶、丙糖磷酸异构酶、甘油醛-3-磷酸脱氢酶、磷酸甘油激酶、磷酸甘油酸变位酶在多杀性巴氏杆菌黏附宿主细胞或分子过程中发挥作用。该研究的完成将加深巴氏杆菌病分子发病机制的认识,并为巴氏杆菌病的诊断标识筛选、新型疫苗创制和药物靶标筛选等提供基础数据。     

Molecular characterization and expression analysis of a KIFC1-like kinesin gene in the testis of Eumeces chinensis

The member of the kinesin-14 subfamily, KIFC1, is a carboxyl-terminal motor protein that plays an important role in the elongation of nucleus and acrosome biogenesis during the spermiogenesis of mammals. Here, we had cloned and sequenced the cDNA of a mammalian KIFC1 homologue (termed ec-KIFC1) from the total RNA of the testis of the reptile Eumeces chinensis. The full-length sequence was 2,339 bp that contained a 216 bp 5′-untranslated region (5′UTR), a 194 bp 3′-untranslated region (3′UTR) and a 1,929 bp open reading frame that encoded a special protein of 643 amino acids (aa). The calculated molecular weight of the putative ec-KIFC1 was 71 kDa and its estimated isoelectric point was 9.47. The putative ec-KIFC1 protein owns a tail domain from 1 to 116 aa, a stalk domain from 117 to 291 aa and a conserved carboxyl motor domain from 292 to 642 aa. Protein alignment demonstrated that ec-KIFC1 had 45.6, 42.8, 44.6, 36.9, 43.7, 46.4, 45.1, 55.6 and 49.8 % identity with its homologues in Mus musculus, Salmo salar, Danio rerio, Eriocheir sinensis, Rattus norvegicus, Homo sapiens, Bos taurus, Gallus gallus and Xenopus laevis, respectively. Tissue expression analysis showed the presence of ovary, heart, liver, intestine, oviduct, testis and muscle. The phylogenetic tree revealed that ec-KIFC1 was more closely related to vertebrate KIFC1 than to invertebrate KIFC1. In situ hybridization showed that the ec-KIFC1 mRNA was localized in the periphery of the nuclear membrane and the center of the nucleus in early spermatids. In mid spermatids, the ec-KIFC1 had abundant expression in the center of nucleus, and was expressed in the tail and the anterior part of spermatids. In the late spermatid, the nucleus gradually became elongated, and the ec-KIFC1 mRNA signal was still centralized in the nucleus. In mature spermatids, the signal of the ec-KIFC1 gradually became weak, and was mainly located at the tail of spermatids. Therefore, the ec-KIFC1 probably plays a critical role in the spermatogenesis of E. chinensis.     

Novel Approach for Improving the Productivity of Antibiotic-Producing Strains by Inducing Combined Resistant Mutations

We developed a novel approach for improving the production of antibiotic from Streptomyces coelicolor A3(2) by inducing combined drug-resistant mutations. Mutants with enhanced (1.6- to 3-fold-higher) actinorhodin production were detected at a high frequency (5 to 10%) among isolates resistant to streptomycin (Str^r), gentamicin (Gen^r), or rifampin (Rif^r), which developed spontaneously on agar plates which contained one of the three drugs. Construction of double mutants (str gen and str rif) by introducing gentamicin or rifampin resistance into an str mutant resulted in further increased (1.7- to 2.5-fold-higher) actinorhodin productivity. Likewise, triple mutants (str gen rif) thus constructed were found to have an even greater ability for producing the antibiotic, eventually generating a mutant able to produce 48 times more actinorhodin than the wild-type strain. Analysis of str mutants revealed that a point mutation occurred within the rpsL gene, which encodes the ribosomal protein S12. rif mutants were found to have a point mutation in the rpoB gene, which encodes the β-subunit of RNA polymerase. Mutation points in gen mutants still remain unknown. These single, double, and triple mutants displayed in hierarchical order a remarkable increase in the production of ActII-ORF4, a pathway-specific regulatory protein, as determined by Western blotting analysis. This reflects the same hierarchical order observed for the increase in actinorhodin production. The superior ability of the triple mutants was demonstrated by physiological analyses under various cultural conditions. We conclude that by inducing combined drug-resistant mutations we can continuously increase the production of antibiotic in a stepwise manner. This new breeding approach could be especially effective for initially improving the production of antibiotics from wild-type strains.     

温度对茶尺蠖核型多角体病毒增殖动态的影响

多角体计数、对流免疫电泳、单向免疫扩散及火箭免疫电泳测定的结果表明:26℃适于茶尺蠖核型多角体病毒(EoNPV)的增殖。多角体含量或其相对值随着时间的推移而增长,并渐趋于平稳,两者间呈Logistic曲线关系。单位体重或单头幼虫所含的多角体数量(y_1或y_2)、扩散环直径(y_3)和火箭峰值(y_4)与时间(t)的关系式分别为:y_1=(8.1481)/(1 EXP(9.4210-0.0608t))×10~9PIB/克;y_2=(6.1596)/(1 EXP(5.4809-0.0376t))×10~8PIB/头,y_3=(1.4)/(1 EXP(2.710-0.015t))cm;y_4=(3.52)/(1 EXP(4.580-0.040t))cm。但30℃下,EoNPV增殖严重受抑制,饲毒后24～168小时内难以测出多角体,其后多角体含量也极显著低于26℃,乃至难以被三种免疫测定法测出。     

GADD45α mediates arsenite‐induced cell apoptotic effect in human hepatoma cells via JNKs/AP‐1‐dependent pathway

Arsenite (As(III)), an effective chemotherapeutic agent for the acute promyelocytic leukemia (APL) and multiple myeloma (MM), might be also a promise for the therapy of other cancers, including the solid tumors. However, the molecular bases of arsenite‐induced cytotoxicity in the tumor cells have not been fully defined. In this study, we have disclosed that arsenite effectively induces the apoptotic response in the HepG2 human hepatoma cells by triggering GADD45α induction and the subsequent activation of JNKs/AP‐1 cell death pathway. However, signaling events relating to GADD45α/JNKs/AP‐1 pathway activation have not been observed in HL7702 human diploid hepatic cells under the same arsenite exposure condition. Our results thus have illustrated the selective pro‐apoptotic role of arsenite in the hepatoma cells by activating GADD45α‐dependent cell death pathway whereas with little effect on the normal hepatic cells. The approaches to up‐regulate GADD45α levels might be helpful in improving the chemotherapeutic action of arsenite on certain solid tumors including hepatoma. J. Cell. Biochem. 109: 1264–1273, 2010. © 2010 Wiley‐Liss, Inc.     

Association of the epithelial sodium channel with Apx and alpha-spectrin in A6 renal epithelial cells.

Recent molecular cloning of the epithelial sodium channel (ENaC) provides the opportunity to identify ENaC-associated proteins that function in regulating its cell surface expression and activity. We have examined whether ENaC is associated with Apx (apical protein Xenopus) and the spectrin-based membrane cytoskeleton in Xenopus A6 renal epithelial cells. We have also addressed whether Apx is required for the expression of amiloride-sensitive Na(+) currents by cloned ENaC. Sucrose density gradient centrifugation of A6 cell detergent extracts showed co-sedimentation of xENaC, alpha-spectrin, and Apx. Immunoblot analysis of proteins co-immunoprecipitating under high stringency conditions from peak Xenopus ENaC/Apx-containing gradient fractions indicate that ENaC, Apx, and alpha-spectrin are associated in a macromolecular complex. To examine whether Apx is required for the functional expression of ENaC, alphabetagamma mENaC cRNAs were coinjected into Xenopus oocytes with Apx sense or antisense oligodeoxynucleotides. The two-electrode voltage clamp technique showed there was a marked reduction in amiloride-sensitive current in oocytes coinjected with antisense oligonucleotides when to compared with oocytes coinjected with sense oligonucleotides. These studies indicate that ENaC is associated in a macromolecular complex with Apx and alpha-spectrin in A6 cells and suggest that Apx is required for the functional expression of ENaC in Xenopus epithelia.     

生物素对地衣芽孢杆菌生长和乳酸形成的影响

生物素对地衣芽孢杆菌生长和乳酸形成的影响胡尚勤（重庆师范学院生物系重庆６３００４７）地衣芽孢杆菌（Ｂａｃｉｌｕｓｌｉｃｇｅｎｉｆｏｒｍｉｓ）是我国９０年代首次从正常待产妇阴道拭子分离出来的一个无毒新菌株。现在国内已经用它来生产“整肠生”活菌剂。它是一...