麦迪霉素产生菌880103#菌株的选育

麦迪霉素产生菌生米加链霉菌A03菌株经过多次诱变筛选及抗自身代谢物突变,得到了产量正变株;通过推理筛选的方法,应用初级代谢产物生物合成途径的调节突变株,分离得到了一主要组分麦迪霉素A_1高于对照菌株的优良菌株880103。并通过加入前体的方法,使其麦迪霉素A_1组分进一步提高。在试产中,该菌株与对照菌株相比,发酵单位相对提高16％,麦迪霉素 A_1组分相对提高 28％,成品效价相对提高4.7％;且发酵周期短、温度适中、遗传性能稳定、适应性强,是一株优于对照菌株的优质高产菌株。     

通过获得链霉素抗性基因(str)突变株筛选梅岭霉素高产菌株

本文通过链霉素对梅岭霉素（meilingmycin）产生菌南昌链霉菌NS－41－80菌株孢子致死浓度的测定，采用诱变剂EMS四种不同诱变剂量对菌株的孢子进行诱变处理，诱变处理的孢子涂布在含链霉素致死浓度的高氏平板上，获得了大量的链霉素抗性基因（str）突变株。然后从链霉素抗性基因突变株进一步筛选到梅岭霉素高产菌株80－5．11－221，在摇瓶条件下，只产梅岭霉素不产南昌霉素，梅岭霉素活性效价达1500μg/ml，比出发菌株NS－41－80的摇瓶发酵效价855μg/ml提高了77．9％，该菌株连续传代六代进行摇瓶发酵，其F2代和F3代梅岭霉素发酵效价稳定，F4代至F6代随着传代数增加，其梅岭霉素发酵效价急速下降。通过EMS诱变剂量分别与抗药性突变率和链霉素抗性基因突变株产梅岭霉素产量的产势统计分析表明，菌株抗药性突变与产抗生素突变密切相关，产抗生素突变的EMS诱变剂量高于链霉素抗性基因突变诱变剂量。在0．03mol/L的EMS剂量作用下，菌株致死率为99．43％，而抗药性突变率为0．0440％，建立了梅岭霉素产生菌链霉素抗性基因突变筛选方法，为南昌链霉菌高产菌种选育研究作了有益的尝试，并有助于其它链霉菌属的抗生素产生菌育种研究。     

几种不同抗生素对他克莫司产生菌的影响

目的 寻找对他克莫司产生菌有影响的抗生素便于进行优良菌种选育.方法 他克莫司产生菌弗氏链霉菌类群FCZ0311经自然分离得到浅红色、桃红色和灰白色3种主要类犁的菌落.研究了几种不同化学类型抗生素对3种不同类型菌株的生长、菌落形态、培养特征及其它们对他克莫司生物合成的影响.结果 应用链霉素处理浅红型和桃红型菌株,获得了效价较出发菌株提高30%的3株突变株.结论 链霉素处理能够提高浅红型和桃红型菌株的效价,为他克莫司菌种选育提供了一个新的途径.     

A new subspecies of Streptomyces clavuligerus,S.clavuligerus subsp.Jinyunensis subsp.nov.

从我国重庆缙云山土壤中分离到一株链霉菌，编号为３．０４８。其代谢产物中具有抑制β－内酰胺酶活性的物质——克拉维酸。该菌株的形态培养特征和生理生化特征与棒状链霉菌标准菌株ＡＴＣＣ２７０６４相似，但又有差别，故定名为棒状链霉菌缙云亚种。     

链霉菌702抗药性致死突变标志微波诱变筛选研究

目的 筛选出产抗真菌活性物质的高产链霉菌702突变株.方法 分别以链霉菌702菌株为试验材料和以庆大霉素为敏感抗生素建立链霉菌702孢子致死突变标志的微波诱变筛选模型,通过微波对链霉菌702菌株孢子进行不同时间的诱变处理,将诱变处理后的孢予悬液涂布于含致死浓度的庆大霉素的PDA平板培养基上,获得抗庆大霉素突变株,分别挑取单个抗药性突变菌株进行摇瓶初筛和复筛.生物效价测定采用一剂量法.结果 微波处理30s对菌株的致死率可达70.53%,抗药性突变率高达23.13%,获得的抗药性突变株经过摇瓶初筛和复筛,获得高产突变株20-29-47菌株,产抗真菌活件物质的摇瓶发酵单位达到1478μg/ml,比出发菌株发酵单位986μg/ml提高T49.9%.结论 采用抗药性致死突变标志的微波诱变筛选模型可以获得产抗真菌活性物质的链霉菌702高产菌株.     

中生菌素耐自生产物突变株的选育

以中生菌素产生菌淡紫灰链霉菌海南变种UV-69自然分离获得的A3为出发菌株,经过5h培养制备和紫外诱变60s处理后,利用其自生代谢产物,筛选耐自生产物突变株,获得突变株NV-36,其摇瓶发酵效价比出发菌株提高20%。传代试验表明,该突变株的高产性能遗传特性较稳定。在20吨发酵罐上连续5批验证,平均发酵效价比原生产水平提高35%。     

棒状链霉菌缙云亚种的鉴定

从我国重庆缙云山土壤中分离到一株链霉菌，编号为３．０４８。其代谢产物中具有抑制β－内酰胺酶活性的物质－克拉维酸。该菌株的形态培养特征和生理生化特征与棒着链霉菌标准菌株ＡＴＣＣ２７０６４相似，但又有差别，故定名为棒状链霉菌缙云亚种。     

基因组重排选育麦迪霉素高产菌株

目的以生米卡链霉菌(Streptomyces mycarofaciens)突变株为研究对象,选育麦迪霉素高产菌株。探索并验证基因组重排技术在菌种选育中的重要作用。方法通过2轮多亲株灭活原生质体融合技术实现基因组重排。将来自不同育种方法的5株麦迪霉素高产菌株B64-5、01-GM-1、H-101、H-106和H-108作为第一轮亲本,在质量浓度为3 g.L-1溶菌酶3、2℃水浴60 min条件下制备原生质体,分别采用紫外线照射148 s和52℃加热60 min灭活原生质体,然后采用质量分数35%PEG 4000、37℃保温2 min诱导原生质体融合。融合子经过初筛和复筛,获得产量进一步提高的重组子作为亲本进行第二轮的多亲株灭活原生质体融合实验。用生物学方法测定麦迪霉素效价,用HPLC方法测定麦迪霉素A1含量。结果与结论经过2轮基因组重排实验成功选育出3株高产且遗传稳定的麦迪霉素生产菌株。其中1株菌株GSZ2-32发酵前补加前体X-1后摇瓶产量比原始出发菌株Streptomyces mycarofaciens var.464提高1.1倍,麦迪霉素A1(MDMA1)质量分数含量保持在80%以上。     

氨甲酰妥布霉素高产菌株选育及其发酵特性研究

目的筛选氨甲酰妥布霉素高产菌株并稳定其发酵效价。方法以黑暗链霉菌Ts-228为出发菌株，经自然分离，UV诱变结合耐自身代谢产物处理，采用琼脂块法并结合摇瓶发酵来筛选高产菌，获得了Tt-49新菌株。以摇瓶发酵考察生长特性，罐上发酵绘制代谢曲线。结果效价提高了1．8倍，妥布霉素含量高达68．5％。该菌株生长周期仅4d，传代稳定。种龄18h左右，接种量10％。按照代谢曲线图进行调控，发酵罐的产抗水平达5865u／ml。结论自然分离与诱变相结合，是黑暗链霉菌选育的有效途径。出发菌株经UV诱变和耐受驯育，发酵效价显著提高。溶氧是妥布霉素发酵生产中的重要调控因子。     

剪切环境对南昌链霉菌形态和代谢的影响

通过在摇瓶和发酵罐上考察不同剪切环境对南昌链霉菌的代谢过程和菌丝形态的影响 ,并对某些菌丝形态参数进行了定量分析 ,发现南昌链霉菌对剪切的敏感性。在此基础上通过改变发酵罐上搅拌桨叶类型促进了前期菌体生长 ,并使最终发酵效价提高了 5 2     

丙烯酰胺诱导人白血病HL-60和NB_4细胞hprt基因的分子突变谱

为了研究丙烯酰胺的遗传毒理作用 ,采用单细胞克隆培养 ,双向筛选计数 ,多重PCR扩增与电泳分析 ,研究了诱导HL 6 0和NB4 两种细胞hprt基因突变率及分子突变谱 .发现只有丙烯酰胺高剂量组 (70 0mg·L- 1)才对两种细胞有明确的致hprt基因突变作用 ;丙烯酰胺诱发突变主要由点突变和缺失两部分组成 (40 .0 %～ 6 6 .7% ,33.3%～ 6 0 .0 % ) ,而自发突变几乎全是点突变 (90 .0 %以上 ) ,两种细胞均无全基因缺失型 ;缺失突变可以发生于hprt基因上的每个外显子 (除外显子 7/ 8以外 ) ,较集中于基因的 3′末端 ,且诱发突变中绝大多数是点突变与单个外显子缺失 (93.3% ,86 .1% ) ,两种细胞情况类似 .结果提示 ,丙烯酰胺具有较弱的诱导hprt基因突变的作用 ,且诱发突变与自发突变的分子图谱不一样 ,这可能与其作用机理有关     

Differential roles of Arg97, Asp293, and Arg108 in enzyme stability and substrate specificity of CYP2C9

CYP2C9 metabolizes a wide range of drugs, many of which are negatively charged at physiological pH. Therefore, it has been thought that complementarily charged amino acid(s) are critically involved in substrate binding. Previous studies have implicated arginine residues at positions 97, 105, and 108 and aspartate at position 293 in the normal catalytic function of the enzyme. To elucidate the role of these amino acids in the substrate specificity of CYP2C9, a series of mutants were constructed and analyzed for functional activity, thermal stability, and ligand binding. Charge-modifying mutations at positions 97, 105, and 293 decreased catalytic activity toward diclofenac, (S)-warfarin, and pyrene in a substrate-independent manner with Arg105 the least, and Arg97 the most, sensitive amino acids in this regard. Decreases in functional activity paralleled thermal instability of the mutants, suggesting that loss of function reflects more generalized structural changes rather than the absence of a specifically charged amino acid at these three positions. The R108H mutant was inactive toward all three substrates because of unexpected nitrogen ligation to the heme. Conversely, the R108F mutant exhibited substrate-dependent catalytic behavior, with almost complete loss of activity toward (S)-warfarin and diclofenac, but preservation of pyrene metabolism. In addition, the R108F mutation abrogated the Type I difference spectra induced by flurbiprofen and benzbromarone, obligate anions at physiological pH. These data identify critical roles for Arg97 and Asp293 in the structural stability of the enzyme and demonstrate a selective role for Arg108 in the binding and metabolism of negatively charged substrates of CYP2C9.     

Characterizing the Free‐Energy Landscape of MDM2 Protein–Ligand Interactions by Steered Molecular Dynamics Simulations

Inhibition of p53–MDM2 interaction by small molecules is considered to be a promising approach to re‐activate wild‐type p53 for tumor suppression. Several inhibitors of the MDM2–p53 interaction were designed and studied by the experimental methods and the molecular dynamics simulation. However, the unbinding mechanism was still unclear. The steered molecular dynamics simulations combined with Brownian dynamics fluctuation–dissipation theorem were employed to obtain the free‐energy landscape of unbinding between MDM2 and their four ligands. It was shown that compounds 4 and 8 dissociate faster than compounds 5 and 7 . The absolute binding free energies for these four ligands are in close agreement with experimental results. The open movement of helix II and helix IV in the MDM2 protein‐binding pocket upon unbinding is also consistent with experimental MDM2‐unbound conformation. We further found that different binding mechanisms among different ligands are associated with H‐bond with Lys51 and Glu25. These mechanistic results may be useful for improving ligand design.     

Homology modeling studies on human galactose-1-phosphate uridylyltransferase and on its galactosemia-related mutant Q188R provide an explanation of molecular effects of the mutation on homo- and heterodimers

We have created theoretical models of the three-dimensional dimeric structure of human galactose-1-phosphate uridylyltransferase as well as of homo- and heterodimers carrying the Q188R mutation by using comparative modeling procedures. These mutants are associated to the most frequent form of the genetic disease galactosemia. We have analyzed the impact of this mutation both on enzyme-substrate interactions as well as on interchain interactions in the heterodimers and in the homodimer. We suggest a molecular explanation for the altered function, caused by different enzyme-substrate interactions, and for the partial dominant negative effect of the mutant allele that is present in heterozygotes for this gene, related to a substantial loss of interchain hydrogen bonds. These results can be considered a starting point for a more extensive characterization at the molecular level of the other mutations linked to this genetic disease.     

核糖体工程技术选育米尔贝霉素高产菌株

本实验室中保存的一株从土壤中分离得到的米尔贝链霉菌(Streptomyces milbemycinicus)27号菌株所产米尔贝霉素A3和A4的初始产量分别为61.0和23.8μg/mL。以该菌株为出发菌株，采用核糖体工程技术，结合紫外诱变技术，对米尔贝霉素产生菌米尔贝链霉菌进行诱变，并以链霉素耐受为筛选压力进行筛选。经过诱变后对单菌落进行摇瓶复筛，其中正突变株中米尔贝霉素产量最高的菌株编号是R2-6-5，其米尔贝霉素A3和A4的产量分别是105.2和38.8μg/mL，较原始菌株分别提高了72.5%和63.3%，且遗传稳定。最后，对产量变化较大的11株突变株基因组中rsmG基因和rspL基因进行突变位点分析，发现在rspL基因内均未发生突变，rsmG基因均发生突变。本研究表明，链霉素抗性降低的突变菌株确实都在相关基因内发生突变，且核糖体工程结合紫外诱变的诱变方式效果良好，能够快速有效的提高米尔贝链霉菌生物合成米尔贝霉素的能力。     

Variation in gut microbiota strongly influences individual rodent phenotypes.

Understanding genetic and phenotypic differences between experimentalanimals (both within a single population and between populationsheld at different sites) is potentially important in explainingvariability in responses to drugs and other stressors. Populationchanges in endogenous and exogenous metabolism in laboratoryanimal colonies have often been ascribed to genetic drift inthe experimental animals themselves. This undoubtedly occursin laboratory rodents; however, phenotypic drift in animal coloniescan have an immediate and potentially greater short term impacton normal physiological variation and mammalian metabolism,as exemplified by Robosky et al. in this issue of ToxicologicalSciences. They describe clear and stable metabolic differentiationof     

Fidelity of DNA polymerase alpha partially purified from a mutator mutant and wild-type mouse FM3A cells

Several mutator mutants were isolated from a cultured mouse cell line (FM3A). The mutants exhibited a high rate of spontaneous mutation at three genetic loci of drug resistance. To investigate a possible link between mutator phenotype and fidelity of DNA replication, DNA polymerase alpha was partially purified from the wild-type and the mutator mutant (Fmut 1) showing the highest mutation rate. Using a combination of synthetic template and primer, the ratio of incorporation of incorrect to correct nucleotides was determined. The results indicated that the DNA polymerase alpha from the mutator mutant showed a slightly higher rate of misincorporation, 1.4 and 1.6 times, than that of the wild-type.     

SNP新颖方法和个体化用药方案的研究进展

编码多种药物代谢酶和转运体基因多态性引起个体问药物代谢差异,将影响药物有效性和药物副作用的发生。单核苷酸多态性涉及到所有遗传变异的类型,正确地SNP基因分型方法能帮助检测遗传多态性、遗传紊乱、病原体耐药。个体化用药打破了传统给药的方式,以个体特征为依据进行个体问用药。因此,SNP分型方法成为识别疾病敏感性和药物反应之间生物联系的关键因素。精确的基因分型方法对疾病的诊断和预后起着重要的作用。目前为止,常用测序、变性高效液相色谱（DHPLC）、单链构象多态性分析、变性梯度凝胶电泳等进行基因分型。但是,他们的适用范围得到限制,山于本身纯在的缺陷。本文总结了基因多态性检测的几种新型方法及比较了几种个体化用药方案的优缺点。     

Genetically defined hyperlipidemia

The unraveling of genetic defects associated with disorders in lipid metabolism has contributed to the understanding of lipoprotein metabolism and the pathophysiological consequences of a particular mutation. The translation, however, of a single genetic defect into the individual's risk of cardiovascular disease and subsequent treatment strategies is an extremely complex issue that involves the identification of multiple additional determinants, including genetic, metabolic and environmental factors. The discovery of these factors, including genetic determinants of drug efficacy, provides insight into the interaction between regulatory systems traditionally thought to be unrelated and may, in the future, lead to a more complete diagnostic and therapeutic appreciation of the individual patient.