Biochemical characterization of indole prenyltransferases: filling the last gap of prenylation positions by a 5-dimethylallyltryptophan synthase from Aspergillus clavatus

The putative prenyltransferase gene ACLA_031240 belonging to the dimethylallyltryptophan synthase superfamily was identified in the genome sequence of Aspergillus clavatus and overexpressed in Escherichia coli. The soluble His-tagged protein EAW08391 was purified to near homogeneity and used for biochemical investigation with diverse aromatic substrates in the presence of different prenyl diphosphates. It has shown that in the presence of dimethylallyl diphosphate (DMAPP), the recombinant enzyme accepted very well simple indole derivatives with L-tryptophan as the best substrate. Product formation was also observed for tryptophan-containing cyclic dipeptides but with much lower conversion yields. In contrast, no product formation was detected in the reaction mixtures of L-tryptophan with geranyl or farnesyl diphosphate. Structure elucidation of the enzyme products by NMR and MS analyses proved unequivocally the highly regiospecific regular prenylation at C-5 of the indole nucleus of the simple indole derivatives. EAW08391 was therefore termed 5-dimethylallyltryptophan synthase, and it filled the last gap in the toolbox of indole prenyltransferases regarding their prenylation positions. K(m) values of 5-dimethylallyltryptophan synthase were determined for L-tryptophan and DMAPP at 34 and 76 μM, respectively. Average turnover number (k(cat)) at 1.1 s(-1) was calculated from kinetic data of L-tryptophan and DMAPP. Catalytic efficiencies of 5-dimethylallyltryptophan synthase for L-tryptophan at 25,588 s(-1)·M(-1) and for other 11 simple indole derivatives up to 1538 s(-1)·M(-1) provided evidence for its potential usage as a catalyst for chemoenzymatic synthesis.     

Persistent Antibody Responses but Declining Cytotoxic T-Lymphocyte Responses to Multiple Human Immunodeficiency Virus Type 1 Antigens in a Long-Term Nonprogressing Individual with a Defective p17 Proviral Sequence and No Detectable Viral RNA Expression

Long-term nonprogressor AD-18 has been infected with human immunodeficiency virus type 1 (HIV-1) for at least 16 years. During the past 5 years, he has had undetectable levels of plasma viremia, and HIV-1 cannot be isolated from him. Sequencing of proviral DNA indicates that the only HIV-1 sequences that can be identified in AD-18 have gross defects in the p17-encoding regions of the gag gene (Y. Huang, L. Zhang, and D. D. Ho, Virology 240:36–49, 1998). However, AD-18 has strong, sustained antibody responses to several HIV-1 antigens, including p17. Cytotoxic T-lymphocyte responses to Env and Gag antigens have gradually diminished over the past 4 years, at a time when the titers of antibodies to the same proteins have remained stable. We discuss what these observations might mean for the generation and maintenance of immunological memory.     

Protein expression profiles in the epidermis of cyclooxygenase-2 transgenic mice by 2-dimensional gel electrophoresis and mass spectrometry

Exposure of murine skin to tumor-promoting agents such as 12-O-tetradecanoyl-phorbol-13-acetate (TPA) causes up-regulation of cyclooxygenase-2 (COX-2) and increased prostaglandin (PG) synthesis. Pharmacological inhibition of COX-2 significantly reduces skin tumor development. However, we previously demonstrated that K14.COX-2 transgenic (TG) mice that overexpressed COX-2 in the epidermis were unexpectedly resistant to tumor development under the classical 7,12-dimethylbenz[a]anthracene-TPA protocol. In the present study, we employed a proteomic approach of 2-dimensional gel electrophoresis (2-DE) and mass spectrometry to profile differentially expressed proteins in the epidermis of K14.COX-2 TG and wild-type control mice. Various 2-DE approaches were used to identify the maximum number of differentially expressed proteins: 20 for untreated samples, 3 for acetone-treated samples, and 22 for TPA-treated samples. These proteins include 14-3-3 sigma, numerous actin fragments, actin filament related proteins cofilin-1 and destrin, galectin-3, galectin-7, prohibitin, S100A6, S100A9, and many others. The differential expression of galectin-3, galectin-7, S100A9 was validated by Western blot analysis and/or immunohistochemical analysis. The current data suggest that some of the differentially expressed proteins might increase apoptosis and cell cycle arrest, which, in turn, may provide insight into the role of COX-2 in skin tumorigenesis.     

Blockade of L-type voltage-gated Ca channel inhibits ischemia-induced neurogenesis by down-regulating iNOS expression in adult mouse

Neurogenesis in the adult mammalian hippocampus may contribute to repairing the brain after injury. The signals that regulate neurogenesis in the dentate gyrus following ischemic stroke insult are not well known. We have previously reported that inducible nitric oxide synthase (iNOS) expression is necessary for ischemia-stimulated neurogenesis in the adult dentate gyrus. Here, we show that mice subjected to 90 min of middle cerebral artery occlusion (MCAO) significantly increased the number of new neurons and up-regulated iNOS expression in the dentate gyrus. Blockade of the L-type voltage-gated Ca(2+) channel (L-VGCC) prevented neurogenesis in the dentate gyrus and subventricular zone (SVZ), and down-regulated iNOS expression in the dentate gyrus after cerebral ischemia. This study suggests that Ca(2+) influx through L-VGCC is involved in ischemia-induced neurogenesis by up-regulating iNOS expression.     

人白介素6受体基因在痘苗病毒载体系统中的表达

人ＩＬ－６受体是一个在各种细胞上广泛表达的跨膜糖蛋白分子,是ＩＬ－６发挥细胞效应所必需的。本文通过将ＩＬ－６ＲｃＤＮＡ重组到痘苗病毒的ＴＫ基因中构建成重组痘苗病毒ＶＩＬ６Ｒ。细胞原位杂交和ＡＰＡＡＰ染色结果表明,感染ＶＩＬ６Ｒ后的Ｖｅｒｏ细胞中,ＩＬ－６Ｒ在ｍＲＮＡ和蛋白水平上均呈现较强的表达。Ｗｅｓｔｅｒｎｂｌｏｔ分析所表达的分子量为８０ｋＤ,表明所表达的产物是糖基化的。ＩＬ－６结合试验表明,表达的膜ＩＬ－６Ｒ能够结合ｒＩＬ－６,说明它是有功能的。利用ＶＩＬ６Ｒ免疫小鼠后,能够刺激较强的抗体产生。从而为进一步研究ＩＬ－６Ｒ的信号传导和构效关系提供了基础。     

甲羟戊酸通路对细胞生长调控作用的研究进展

甲羟戊酸(MVA)通路对细胞生长具有重要的调节作用,MVA及其衍生物通过对蛋白质异戊烯化和N糖基化修饰而影响Ras蛋白、生长因子及受体的功能、细胞内信号转导和细胞的生长。MVA通路参与血管活性物质生成的调节是其调节细胞生长的另一机制。MVA生成的限速酶羟甲基戊二酸单酰辅酶A(HMGCoA)则受MVA通路衍生物的反馈抑制。HMGCoA还原酶抑制剂通过抑制MVA及其衍生物的生成而抑制细胞的生长和增殖。     

纽氏钝绥螨、尼氏钝绥螨及其猎物—桔全爪螨生态学特性的比较研究

本文从天敌与猎物的种群内禀增长力,天敌对猎物不同密度的功能反应及数值反应三个方面来评价和比较纽氏钝绥螨、尼氏钝绥螨时其猎物——枯全爪螨的控制作用。在五种温度下,两种捕食螨的种群内禀增长力都大于桔全爪螨。它们对猎物的功能反应属HollingⅡ型。尼氏钝绥螨的捕食量大于纽氏钝绥螨,在25℃时两种捕食螨捕食量最大,应用Rogers的模型能较好地对试验结果进行模拟。尼氏钝绥螨对桔全爪不同虫态的取食不存在选择效应,纽氏钝绥螨则嗜食若螨和幼螨。两种捕食螨对桔全爪螨的数值反应表明,仅供给桔全爪螨雌成螨作为食物,对两种捕食螨都不利,尤其对尼氏钝绥螨更为明显。综上所述,两种捕食螨能比较有效地控制桔全爪螨种群,当猎物密度较高时,尼氏钝绥螨控制效果优于纽氏钝绥螨,但纽氏钝绥螨控制效果优于纽氏钝绥螨,但纽氏钝绥螨田间种群数量比尼氏钝绥螨稳定。柑桔园中存在其它补充食物时对这两种捕食敌有利。     

复杂疾病靶基因识别与网络重建的计算系统生物研究

复杂疾病相关靶基因的识别、构建疾病驱使相关基因网络及进行疾病机制研究,是功能基因组学研究中非常重要的科学问题。文章以计算系统生物学的观点和三维的角度,综述了基于生物谱（SNP遗传谱、芯片表达谱和2D-PAGE蛋白质谱等）的复杂疾病靶基因识别、多水平（SNPs虚拟网络、基因调控网络、蛋白质互作网络等）遗传网络逆向重构方法,及不同水平的网络之间在生物学和拓扑学上的纵向映射关系,并给出复杂疾病靶基因识别与网络关系的计算系统生物方法研究的未来展望。