异柠檬酸裂解酶肽类抑制剂的优化筛选

利用噬菌体肽库筛选与计算机模拟分子对接技术, 优化异柠檬酸裂解酶肽类抑制剂的筛选. 先通过噬菌体肽库筛选出与异柠檬酸裂解酶（ICL）具有高亲和力的结合肽, 再利用Discovery Studio 2.1模拟多肽与ICL蛋白晶体（1F8I）的分子对接, 最后用Fmoc固相合成法合成多肽, 并对其生物活性进行检测. 实验结果表明, 通过噬菌体肽库筛选得到了29条七肽序列, 其中12条可与ICL蛋白晶体成功对接. 体外生物活性检测结果显示, 得到的12条七肽均对ICL的活性有明显抑制作用（抑制率均超过50%）.     

红芪组织培养中次生代谢产物的研究Ⅰ.红芪愈伤组织异柠檬酸裂解酶活性的变化

在红芪不同生长状态的愈伤组织中,异柠檬酸裂解酶（ＩＣＬ）活性差异很大,其中以分化愈伤组织中活性最高．与此相平行在红芪不同培养时期的愈伤组织中,不饱和脂肪酸含量降低,多糖含量升高．由此可以肯定异柠檬酸裂解酶为红芪次生代谢产生多糖过程中的一个重要酶．同时在无菌苗中异柠檬酸裂解酶活性也很高,而多糖没有检测到,说明异柠檬酸裂解酶也参加了植物细胞的初级代谢．我们认为初级和次级代谢过程中某些重要的甚至关键的酶是相同的,只是随着细胞的发育过程其活性发生了变化．     

Reductive glutamine metabolism by IDH1 mediates lipogenesis under hypoxia

Acetyl coenzyme A (AcCoA) is the central biosynthetic precursor for fatty-acid synthesis and protein acetylation. In the conventional view of mammalian cell metabolism, AcCoA is primarily generated from glucose-derived pyruvate through the citrate shuttle and ATP citrate lyase in the cytosol. However, proliferating cells that exhibit aerobic glycolysis and those exposed to hypoxia convert glucose to lactate at near-stoichiometric levels, directing glucose carbon away from the tricarboxylic acid cycle and fatty-acid synthesis. Although glutamine is consumed at levels exceeding that required for nitrogen biosynthesis, the regulation and use of glutamine metabolism in hypoxic cells is not well understood. Here we show that human cells use reductive metabolism of α-ketoglutarate to synthesize AcCoA for lipid synthesis. This isocitrate dehydrogenase-1 (IDH1)-dependent pathway is active in most cell lines under normal culture conditions, but cells grown under hypoxia rely almost exclusively on the reductive carboxylation of glutamine-derived α-ketoglutarate for de novo lipogenesis. Furthermore, renal cell lines deficient in the von Hippel-Lindau tumour suppressor protein preferentially use reductive glutamine metabolism for lipid biosynthesis even at normal oxygen levels. These results identify a critical role for oxygen in regulating carbon use to produce AcCoA and support lipid synthesis in mammalian cells.     

Persistence of Mycobacterium tuberculosis in macrophages and mice requires the glyoxylate shunt enzyme isocitrate lyase

Mycobacterium tuberculosis claims more human lives each year than any other bacterial pathogen. Infection is maintained in spite of acquired immunity and resists eradication by antimicrobials. Despite an urgent need for new therapies targeting persistent bacteria, our knowledge of bacterial metabolism throughout the course of infection remains rudimentary. Here we report that persistence of M. tuberculosis in mice is facilitated by isocitrate lyase (ICL), an enzyme essential for the metabolism of fatty acids. Disruption of the icl gene attenuated bacterial persistence and virulence in immune-competent mice without affecting bacterial growth during the acute phase of infection. A link between the requirement for ICL and the immune status of the host was established by the restored virulence of delta icl bacteria in interferon-gamma knockout mice. This link was apparent at the level of the infected macrophage: Activation of infected macrophages increased expression of ICL, and the delta icl mutant was markedly attenuated for survival in activated but not resting macrophages. These data suggest that the metabolism of M. tuberculosis in vivo is profoundly influenced by the host response to infection, an observation with important implications for the treatment of chronic tuberculosis.     

白假丝酵母的快速分离鉴定

白假丝酵母又称白色念珠菌，是一种单细胞真菌，由于近年来人类在医疗过程中大量使用抗生素、医疗导管等高新技术，导致念珠菌病呈明显上升趋势，研究白假丝酵母致病性的工作也日益增加，快速分离鉴定白假丝酵母就显得尤为重要．从形态学、生理生化反应、免疫、基因探针技术、PCR技术、基因芯片技术等方面综述了分离鉴定白假丝酵母的方法．并对不同分离鉴定手段进行了评价。     

异柠檬酸裂解酶肽类抑制剂结构的修饰

利用Fmoc固相多肽合成法, 按已知异柠檬酸裂解酶抑制剂的直链肽氨基酸序列合成首尾相连的环肽抑制剂. 经色谱纯化和质谱鉴定, 其相对分子质量的实测值与理论值相符. 抑制率实验结果表明, 合成的环肽对异柠檬酸裂解酶有明显抑制作用, 抑制率大于50%. 采用高效液相色谱法分别检测直链肽和环肽在血浆中的半衰期, 实验
结果表明, 环肽在血浆中的半衰期为11 min, 比直链肽的半衰期延长175%.     

Gamma-tubulin is a centrosomal protein required for cell cycle-dependent microtubule nucleation.

gamma-Tubulin is a newly identified member of the tubulin family whose sequence is highly conserved from yeast to man. This minor microtubule protein is localized to the microtubule organizing centres and a mutation in the gene encoding it produces a microtubuleless mitotic arrest in the filamentous fungus Aspergillus nidulans. Here we investigate the in vivo function of gamma-tubulin in mammalian cells using a synthetic peptide to generate a polyclonal antibody that binds to a highly conserved segment of gamma-tubulin. After microinjection into cultured mammalian cells, immunofluorescence localization revealed that this antibody binds to native centrosomes at all phases of the cell cycle. In the presence of the gamma-tubulin antibody, microtubules fail to regrow into cytoplasmic arrays after depolymerization induced by nocodazole or cold. Furthermore, cells injected immediately before or during mitosis fail to assemble a functional spindle. Thus in vivo gamma-tubulin is required for microtubule nucleation throughout the mammalian cell cycle.     

白色念珠菌基因分型方法研究进展

目的：探讨近年来白色念珠菌基因分型方法研究的新进展。方法：查阅相关文献资料，归纳总结。结果：白色念珠菌基因分型是近年来白色念珠菌分子生物学研究中的一个热点和主要方向，其方法很多。常用的分子生物学技术各有千秋，目前倾向于计算机辅助分析，多种分型技术合用。结论：白色念珠菌基因分型方法研究有新进展，对临床念珠菌病的诊断、治疗及分子流行病学研究等具有重要意义，同时，理想的分型技术还有待进一步研究。     

结核分枝杆菌H37Rv异柠檬酸裂解酶基因的克隆表达及活性

以结核分枝杆菌H₃₇Rv基因为模板, PCR反应扩增该菌株的异柠檬酸裂解酶基因（ICL）, 将其克隆入原核表达载体pET28b中, 并将pET28b I
CL转化入大肠杆菌BL21(DE3)中进行诱导表达. 结果表明, ICL蛋白的最佳诱导表达条件为: 温度20 ℃, IPTG终浓度为025 mmol/L, 诱导表达4 h, 在此条件下 ICL实现了高效表达, 以镍离子螯合型琼脂糖凝胶亲和层析柱纯化ICL蛋白, 纯化程度较高. 酶学性质鉴定表明, 实验获得了具有生物学活性的重组蛋白, 重组ICL的比活力为24 μmol/(mg·min).     

10种中草药乙醇提取物抗真菌作用研究

以白色念珠菌为指示菌,测定了黄连等10种中草药的抗真菌作用.实验结果显示,不同的中草药其抗真菌作用不同,其中黄连和黄芩2种药物对白色念珠菌的抑制作用效果最好,其次是五蓓子和知母,而甘草对白色念珠菌抑菌作用较差.在一定质量浓度范围内,黄连提取物的抗真菌作用随药液浓度的增大而增强,呈剂量依赖性.其最低抑菌质量浓度为1mg/mL.将抑菌成分不同的中草药进行两两配伍,考察其对白色念珠菌的协同抑制作用,实验结果显示,除五蓓子和丹参的混合药液对白色念珠菌具有协同抑制作用外,其余3组中草药的配伍,并无协同抑制作用.     

SEC65 gene product is a subunit of the yeast signal recognition particle required for its integrity.

Protein targeting to the endoplasmic reticulum (ER) in mammalian cells is catalysed by the signal recognition particle (SRP), which consists of six protein subunits and an RNA subunit. Saccharomyces cerevisiae SRP is a 16S particle, of which only two subunits have been identified: a protein subunit, SRP54p, which is homologous to the mammalian SRP54 subunit, and an RNA subunit, scR1 (ref. 3). The sec65-1 mutant yeast cells are temperature-sensitive for growth and defective in the translocation of several secreted and membrane-bound proteins. The DNA sequence of the SEC65 gene suggests that its product is related to mammalian SRP19 subunit and may have a similar function. Here we show that SEC65p is a subunit of the S. cerevisiae SRP and that it is required for the stable association of another subunit, SRP54p, with SRP. Overexpression of SRP54p suppresses both growth and protein translocation defects in sec65-1 mutant cells.     

Role of Ppt1 in multiple stress responses in Candida albicans

To study the function of CaPptl, we deleted PPT1 gene from the Candida albicans genome by sequentially replacing the entire coding region with the selectable markers ARG4 and HIS1. The results showed that the deletion of Pptl did not affect the hyphal formation of C. albicans under serum induction and caused enhanced sensitivity to DNA damage, Calcofluor white and salt- induced stress. We also found that Pptl was not required for the phenotypic response of cells treated with the genotoxins, methylmethane sulfonate and hydroxyurea. Flow cytometric analyses indicated that pptlA cells and wild-type cells showed similar G2/M arrest profiles when exposed to DNA damage stress. Pptl was not required for the activation of the DNA damage response pathway, as indicated by normal phosphorylation of Rad53 and Rfa2 in pptlA cells under DNA damage stress. We suggest that Pptl plays important roles in response to various stress conditions in C. albicans.     

HIV相关性口腔白色念珠菌rDNA基因型研究

目的:了解HIV感染人群和健康人群口腔白色念珠菌基因型的分布情况。方法:运用CA-INT特异性引物聚合酶链反应方法扩增白色念珠菌包含1类内含子的编码rRNA的25S rDNA区,根据扩增条带大小进行基因分型,并应用χ2检验对54株HIV感染人群和54株健康人群口腔白色念珠菌基因型别进行分析。结果:CA-INT特异性引物PCR法可将108株口腔白色念珠菌分为A、B和C型,以A型最多见,两组人群口腔白色念珠菌基因型分布基本相同,但HIV感染人群口腔白色念珠菌A型所占构成比高于健康人群,差别有统计学意义。结论:HIV感染人群和健康人群口腔白色念珠菌均具有基因多态性,CA-INT特异性引物PCR法是较好的口腔白色念珠菌种内菌株间鉴定的方法。     

竹醋液抗真菌的体外敏感性对比试验研究

基于沙保弱氏培养基,采用试管法和打孔法测定了竹醋液对6种致病真菌的最低抑菌浓度、最低杀菌浓度和抑菌圈直径.以常用市售抗真菌药作为对照,证实了竹醋液在体外有较显著的抗真菌和杀菌作用.     

阴道念珠菌病患者阴道分泌物念珠菌培养鉴定及药敏分析

目的：了解大理地区妇科女性患者阴道分泌物念珠菌检出状况、菌群分布及对常用抗真菌药物的敏感性。方法：取有自觉阴道症状到妇科门诊就诊患者的阴道分泌物进行念珠菌培养、鉴定和药敏试验。结果：192份标本中念珠菌阳性标本55例,检出率28．6％,汉族和少数民族妇科门诊女性患者阴道分泌物念珠菌检出率分别为32．2％和25．7％,差异无显著性（P=0．324）;56株念珠菌中白色念珠菌32株、光滑念珠菌12株、克柔念珠菌5株、热带念珠菌4株、其它2株。56株念珠菌对KETO、FCZ、5-FC和Amb的耐药率最高的是KETO,达73．2％;最低的是Amb,无耐药株。结论：外阴阴道念珠菌病是妇科门诊的常见疾病,白色念珠菌仍是主要致病菌,光滑念珠菌感染逐渐增多。本地区妇科患者阴道异常分泌物检出念珠菌对常用抗真菌药物酮康唑存在普遍耐药现象,对两性霉素B基本无耐药株产生。     

白色念珠菌基因组研究进展

目的:介绍白色念珠菌基因组的最新研究进展.方法:查阅国内外大量关于白色念珠菌基因组研究的文献并进行综合详述.结果:白色念珠菌是第一个完成全基因组测序的致病性真核生物,在基因组序列信息研究上取得了重大进展,在基因功能、转录组、蛋白质组等方面获得了许多重要成果.结论:白色念珠菌的基因组研究成果将对研究白色念珠菌的致病机制、耐药机制以及抗真菌药物开发有重大的意义.     

壳聚糖季铵盐衍生物的合成及其抗菌活性

考察了一种壳聚糖季铵盐衍生物(ch itosan-g-DMAE-BC)的合成以及其抗菌性能,采用震荡法,进行了悬菌定量杀菌实验(suspension quantitative test).结果表明:ch itosan-g-DMAE-BC对大肠杆菌、金黄色葡萄球菌以及白色念珠菌在震荡中作用15 m in后,平均灭杀率分别为99.70%,88.4%,99.21%.说明ch itosan-g-DMAE-BC有较强的灭菌作用.     

Small GTP-binding protein associated with Golgi cisternae

Eukaryotic cells seem to use GTP hydrolysis to regulate vesicular traffic in exocytosis and endocytosis. The best evidence for this comes from studies on the yeast Saccharomyces cerevisiae that have identified two small Ras-related GTP-binding proteins, Sec4p and Ypt1p, which control distinct stages of the secretory pathway. In mammalian cells the effects of a non-hydrolysable GTP analogue, GTP-gamma S, on different transport events have suggested that they also have proteins functionally related to yeast Sec4p and Ypt1p. The rab genes have recently been cloned and sequenced for rat and human and their proteins have highly conserved domains in common with Sec4p and Ypt1p (including a putative effector binding site). They are therefore good candidates for GTP-binding proteins involved in intracellular transport in mammalian cells. One of the Rab proteins (Rab1p) is the mammalian counterpart of Ypt1p (ref. 13). Here we report the localization of the protein Rab6p to the Golgi apparatus in several cell types. By immunolabelling and electron microscopy, Rab6p appears to be concentrated predominantly on the medial and trans cisternae and distributed over their entire surface.     

Conformational diversity in a yeast prion dictates its seeding specificity

A perplexing feature of prion-based inheritance is that prions composed of the same polypeptide can evoke different phenotypes (such as distribution of brain lesions), even when propagated in genetically identical hosts. The molecular basis of this strain diversity and the relationship between strains and barriers limiting transmission between species remain unclear. We have used the yeast prion phenomenon [PSI+]4 to investigate these issues and examine the role that conformational differences may have in prion strains. We have made a chimaeric fusion between the prion domains of two species (Saccharomyces cerevisae and Candida albicans) of Sup35, the protein responsible for [PSI+]. Here we report that this chimaera forms alternate prion strains in vivo when initiated by transient overexpression of different Sup35 species. Similarly, in vitro the purified chimaera, when seeded with different species of Sup35 fibres, establishes and propagates distinct amyloid conformations. These fibre conformations dictate amyloid seeding specificity: a chimaera seeded by S. cerevisiae fibres efficiently catalyses conversion of S. cerevisiae Sup35 but not of C. albicans Sup35, and vice versa. These and other considerations argue that heritable prion strains result from self-propagating conformational differences within the prion protein itself. Moreover, these conformational differences seem to act in concert with the primary structure to determine a prion's propensity for transmission across a species barrier.