5—硝基咪唑类药物体外抗无芽孢厌氧菌效果的观察

从多种临床标本中分离并鉴定了200株无芽孢厌氧菌.赛克硝唑(SNZ)、替硝唑(TNZ)和甲硝唑(MNZ)对120株革兰氏阴性厌氧菌的MIC90分别为1～4,1～2和4～8 mg/L,对80株革兰氏阳性厌氧菌的MIC90分别为4～8,4～16和16～32 mg/L.SNZ体外抑制革兰氏阴性厌氧菌作用略弱于TNZ,但抑制革兰氏阳性无厌氧菌较TNZ稍强,MNZ对两类厌氧菌的抑制效果均不如SNZ和TNZ.     

类肌肽4(5)-丙氨酰胺-5(4)-羧酸咪唑的酶促合成及表征

肌肽(β-Ala-L-His)是一种高效抗氧化剂,广泛应用于生物、化工、医药等领域。应用微水相酶促合成类肌肽,效率高价格低,且具有相似性质,开发前景广阔。本研究以L-丙氨酸和4,5-二羧酸咪唑制备类肌肽4(5)-丙氨酰胺-5(4)-羧酸咪唑,正交实验下的最佳合成条件为:四氢呋喃:pH8磷酸缓冲溶液=10:1.6(V/V),L-丙氨酸:4,5-二羧酸咪唑=1:3(m/m),α-胰凝乳蛋白酶:底物=1:200(m/m),35oC下磁力搅拌1.5h。硅胶G60薄层色谱(TLC)分离反应产物,Rf=0.81处出现新斑点;刮下该点纯化后进行紫外扫描,高效液相色谱(HPLC)和核磁共振,紫外光谱253nm处吸收明显增强,310nm处出现新吸收峰;253nm、310nm、330nm高效液相色谱保留时间均为4.0min;13C核磁共振显示8组碳原子。结合胰凝乳蛋白酶的催化机理,得出产物结构为4(5)-丙氨酰胺-5(4)-羧酸咪唑。     

副干酪乳酪杆菌ZY-1内源性质粒分析及其表达系统的构建与应用

高效稳定的乳杆菌表达载体的构建是实现其菌种改良和个性化菌株开发的关键。本研究从副干酪乳酪杆菌(Lacticaseibacillus paracasei) ZY-1中分离出4个内源性质粒并进行功能分析。通过将pLPZ3与pLPZ4的复制子rep,与pNZ5319质粒的氯霉素乙酰转移酶报告基因cat、pUC19的复制子ori构建大肠杆菌-乳酸菌穿梭载体pLPZ3N与pLPZ4N,进一步加上启动子Pldh3和mCherry红色荧光蛋白,获得表达载体pLPZ3E与pLPZ4E。pLPZ3与pLPZ4质粒大小分别为6 289 bp和5 087 bp,GC含量分别为40.94%和39.51%。2个穿梭载体可成功转化至乳酪杆菌属中,pLPZ4N的转化效率(5.23×10²-8.93×10²CFU/μg)略高于pLPZ3N。乳酸菌表达载体pLPZ3E与pLPZ4E转化至副干酪乳酪杆菌S-NB后,成功获得了mCherry红色荧光蛋白的表达。以P_ldh3为启动子构建的重组表达载体pLPZ4E-lacG转化得到的重组菌,其β-半乳糖苷酶酶活性...     

乙酰短杆菌中核苷磷酸化酶的性质研究

目的：以乙酰短杆菌完整细胞为酶源,研究不同条件下核苷磷酸化酶的性质。方法：将乙酰短杆菌湿茵体置于不同保藏温度及在不同种类缓冲溶液中考察其稳定性;在有或无核保护下核苷磷酸化酶对热的稳定性;并设计核苷的磷酸解反应或合成反应,测定核苷磷酸化酶的活力及酶促反应的袁观米氏常数。结果：乙酰短杆菌中的核苷磷酸化酶经低温保藏可以保持较长时间的稳定性;茵体在60℃处理1小时即失去核苷磷酸化酶的活力,但是添加胸腺嘧啶有明显的保护作用;茵体中核苷磷酸化酶的合成能力明显大于磷酸解能力;对尿苷和5-甲基尿苷的表观米氏常数和最大反应速率分别为16．7、11．4mm01／L,0．0063、0．0041mmol／L．min。结论：含核苷磷酸化酶的乙酰短杆菌完整细胞作为酶源,在低温下可以长时间保藏,反应中的碱基对核苷磷酸化酶的抗热性有益,该菌种可以作为工业上核苷磷酸化酶的来源。     

紫色杆菌内毒素对小鼠粒-巨系祖细胞(CFU-GM)的动员

本文研究了低毒性的紫色杆菌内毒素对小鼠 CFU-GM 的动员作用,并对其作用机制作了初步探讨。小鼠周围血中 CFU-GM 数随着静脉注射内毒素量的增加而升高。高峰出现在注射后的第三天,为对照组的2.1倍。同时骨髓 CFU-GM 数逐渐下降。脾脏不影响内毒素的动员作用。但在去除肾上腺的小鼠身上,内毒素失去动员作用。结果表明紫色杆菌内毒素是一种比较安全、有效的干细胞动员剂。其动员机制很可能是通过肾上腺或皮质类固醇激素的介导而产生作用。     

一种来源于脆弱类杆菌的α-半乳糖苷酶的理化性质及其酶解B型红细胞的工艺研究

利用α-半乳糖苷酶去除红细胞表面的B抗原是制备通用O型红细胞的有效方法.本文在克隆表达纯化脆弱类杆菌来源的α-半乳糖苷酶的基础上对其理化性质进行了研究,该酶的分子量为64908Da,等电点在7.12～7.30之间,最适温度为41℃,最适pH为5.6～6.0,其理化性质适合用于B型红细胞的血型改造;为了确定高效、快速、温和的酶解条件,本文对酶解B型红细胞的工艺进行了优化.通过研究缓冲液对酶与红细胞结合的影响,确定了最佳酶解缓冲液为250mmol/L甘氨酸和3mmol/LNaCl,pH6.8;酶解的最适红细胞压积为40%,酶解温度为26℃,酶解时间为1h.利用优化的酶解工艺获得的B-ECORBCs形态及结构功能指标均正常,流式细胞结果证明其B抗原和H抗原标记率与O型红细胞相当,说明制备B-ECORBCs的工艺已成熟.这种工艺具有酶用量少、酶解条件温和、制备过程简单和时间短等优势,具有很好的临床应用前景.     

细菌线性质粒的复制研究

细菌的线性质粒是近年来基础微生物学研究的一个新热点。线性质粒的复制与环状质粒相比,其最突出的特点就是它的5'末端填补机制——填补因为5'末端引物被切除所留下的缺口。根据线性质粒末端结构的不同将其分为两大类:一类具有共价闭合的发夹末端,另一类具有共价结合的末端蛋白。就细菌线性质粒复制的研究现状作一综述,重点介绍了具有两种不同末端结构的线性质粒5'末端填补机制。     

圈卷产色链霉菌硝基烷类氧化酶的分离纯化及酶学性质

圈卷产色链霉菌硝基烷类氧化酶基因naoA在大肠杆菌中获得了成功表达,从含有重组质粒pNA101(pET23b∷naoA)的工程菌株BL21(DE3)中分离纯化了硝基烷类氧化酶,SDSPAGE检测为均一。对纯酶进行了酶学性质及动力学研究。底物为1硝基丙烷、2硝基丙烷和硝基乙烷时,在04mol/L的磷酸缓冲液中,酶的最适反应pH值为7～8,最适反应温度为48℃～56℃。室温保存6d后,酶的活性保持了43.3%,但对60℃以上的高温敏感。硫醇化合物如巯基乙醇、还原型谷胱甘肽不同程度地抑制酶活性,特别是NADH,其浓度为1mmol/L时,酶活性几乎全部丧失。以1硝基丙烷为底物时,NaoA的Km为357mmol/L,Vmax为0199μmol/(μg.min)。     

吡啶酮类衍生物对5-脂氧合酶的抑制作用

合成了一系列3-羟基-4(1H)-吡啶酮类衍生物,并研究了它们对5-脂氧合酶的抑制作用. 发现6-取代-3-羟基-4(1H)-吡啶酮化合物(2a～2e)对5-脂氧合酶具有显著抑制作用, 特别是6-苯硫基-1-苯基-2-甲基-3-羟基-4(1H)-吡啶酮(2a)的抑制效果最好(IC₅₀=2.52 μmol/L). 6-位没有取代基的羟基吡啶酮类化合物对5-脂氧合酶却没有抑制作用. 讨论了6-取代-3-羟基-4(1H)-吡啶酮类化合物对5-脂氧合酶的抑制作用机制.     

基于iTRAQ技术对植物乳杆菌FS5-5的耐盐特性分析

【目的】对大酱中耐盐性较好的植物乳杆菌进行蛋白质组学研究,为植物乳杆菌盐胁迫应激机制的研究提供实验数据。【方法】本项研究以筛选自东北传统农家大酱的耐盐性较好的植物乳杆菌FS5-5为研究对象,绘制了其在0%、6.0%、7.0%和8.0%(W/V)Na Cl浓度下的生长曲线,并利用i TRAQ技术研究了其在0%、6.0%、7.0%和8.0%(W/V)Na Cl浓度下的蛋白质表达情况。【结果】植物乳杆菌FS5-5在0%、6.0%、7.0%和8.0%(W/V)Na Cl浓度下到达对数生长期中期的时间点分别为5、10、12和12 h;以差异倍数在1.2倍以上且P0.05为筛选条件对6.0%、7.0%和8.0%(W/V)Na Cl浓度下与0%进行差异蛋白质的筛选,共筛选出1271个差异蛋白质。这些差异蛋白质主要参与糖代谢、氨基酸代谢、脂肪酸代谢、核苷酸代谢、应激反应、转运、PTS系统和核糖体代谢等。【结论】植物乳杆菌在高盐浓度下生长与能量合成蛋白质、应激蛋白质以及相容性溶质转运蛋白质的表达上调有密切关系。     

d-Xylose catabolism in Bacteroides xylanolyticus X5-1

The xylose metabolism of Bacteroides xylanolyticus X5-1 was studied by determining specific enzyme activities in cell free extracts, by following ¹³C-label distribution patterns in growing cultures and by mass balance calculations. Enzyme activities of the pentose phosphate pathway and the Embden-Meyerhof-Parnas pathway were sufficiently high to account for in vivo xylose fermentation to pyruvate via a combination of these two pathways. Pyruvate was mainly oxidized to acetyl-CoA, CO₂ and a reduced cofactor (ferredoxin). Part of the pyruvate was converted to acetyl-CoA and formate by means of a pyruvate-formate lyase. Acetyl-CoA was either converted to acetate by a combined action of phosphotransacetylase and acetate kinase or reduced to ethanol by an acetaldehyde dehydrogenase and an ethanol dehydrogenase. The latter two enzymes displayed both a NADH- and a NADPH-linked activity. Cofactor regeneration proceeded via a reduction of intermediates of the metabolism (i.e. acetyl-CoA and acetaldehyde) and via proton reduction. According to the deduced pathway about 2.5 mol ATP are generated per mol of xylose degraded.Abbreviations PPP Pentose phosphate pathway - PKP phosphoketolase pathway     

Spread and evolution of natural plasmids harboring transposon Tn5

Abstract: The presence of transposon Tn 5 was studied in 730 Enterobacteriaceae strains from clinical and sewage origin. From these strains, twenty-five conjugative plasmids harboring transposon Tn 5 were isolated. These plasmids were compared with pJR67 and pRYC119, the only previously studied plasmids harboring Tn 5 . A phylogenetic tree of the evolution of all different plasmids was proposed. Irrespective of their bacterial host and geographical place of isolation, some of the plasmids were shown to be identical. All of them can be included in only eight different prototypical plasmid species. Twenty-two plasmids (88%) carried an IncI1 incompatibility determinant as judged form DNA hybridization experiments. The presence of some other common resistance genes suggested that these plasmids are descendants of a common ancestor. These IncI1 plasmids could be grouped in six prototypical species. The results presented here suggest that Tn 5 spread in nature may be dependent on the conjugative ability of the IncI plasmids harboring the transposon, rather than on the efficiency of Tn 5 transposition between different replicons.     

PIP5KIC在自噬体形成过程中的重要作用

自噬（autophagy）是一种在真核生物中十分保守的溶酶体依赖性降解途径,它通过形成双层膜结构包裹胞内堆积的蛋白质和受损细胞器并将其运送到溶酶体中进行降解。在实验中发现,一型磷脂酰肌醇4-磷酸5-激酶C亚型（type I phosphatidylinositol 4-phosphate 5-kinase isoform C,PIP5KIC）会参与到自噬过程中。在哺乳动物细胞中,敲低一型磷脂酰肌醇4-磷酸5-激酶C亚型会造成欧米茄体（omegasome）的形状异常,进而造成自噬水平的降低。同样,在酵母中敲掉其同源物磷脂酰肌醇5-激酶Mss4后也会导致类似的现象。因此,推测一型磷脂酰肌醇4-磷酸5-激酶C亚型在自噬体的生成中起着很重要的作用。     

Plasmid pC present in Salmonella enterica serovar Enteritidis PT14b strains encodes a restriction modification system

Salmonella enterica serovar Enteritidis (S. Enteritidis) possesses plasmids of different sizes and roles. Besides the serovar-specific virulence plasmid present in most field strains, S. Enteritidis can harbour plasmids of low molecular mass whose biological role is poorly understood. We therefore sequenced plasmid pC present in S. Enteritidis strains belonging to phage type PT14b. The size of plasmid was determined to be 5,269 bp and it was predicted to encode four open reading frames (ORFs). The first two ORFs were found (initial 3,230 bp) to be highly homologous to rom and mbeA genes of ColE1 plasmid of Escherichia coli. Proteins encoded by the other two ORFs were 99% homologous to a restriction methylase and restriction endonuclease encoded by plasmid pECO29 of a field strain of E. coli. Using insertional mutagenesis we confirmed experimentally that the plasmid pC-encoded restriction modification system was functional and could explain the high resistance of S. Enteritidis PT14b strains to phage infection.     

Role of neuraminidase-dependent adherence in Bacteroides fragilis attachment to human epithelial cells

Of 50 B. fragilis strains isolated from clinical samples we have demonstrated that 24 (48%) possess an adhesin that mediates a neuraminidase-dependent attachment of B. fragilis to mammalian epithelial cells, but does not mediate any association with human polymorphonuclear leucocytes. This ligand interacts with a mammalian cell receptor that contains a galactoside residue, exposed after neuraminidase pretreatment. Our results suggest a possible role for cell associated neuraminidase in mediating a two step adherence mechanism.     

Plasmid incidence in the subgroups of two bacterial communities

The plasmid incidence of two bacterial communities from soil and freshwater was determined by endogenous plasmid isolation. The overall plasmid incidence for the communities was about 10%, while the frequency of plasmid-containing members in different subgroups ranged from 0% to 100%. Both communities included a minor population where all members contained several plasmids.     

Restored fitness leads to long-term persistence of resistant Bacteroides strains in the human intestine

Acquired antibiotic resistance typically confers a cost to the bacteria, but these costs can be reduced by genetic compensation over time. The fitness of two Bacteroides thetaiotaomicron clones consecutively isolated in vivo was studied using an in vitro pair-wise competition method. The isolates derived from faecal samples of two clindamycin-exposed healthy volunteers and the two B. thetaiotaomicron clone types could be followed up to 18 months in these two subjects. The two clones were originally susceptible to clindamycin and lacked erm genes; however, after 7 days of clindamycin administration they carried the erm (erythromycin methylase)(G) or (F) gene, respectively, and expressed phenotypic clindamycin resistance. The initial cost of acquired resistance was high as seen in the in vitro pair-wise competition experiments. At 2 weeks post-administration, no growth disadvantage was detected for isolates of either of the two clones in the in vitro experiments and this regained fitness remained for isolates collected up to 18 months. Competition analysis of an in vitro isolated erm(G) positive transconjugant also demonstrated an initial reduction of fitness that was restored over time. The results indicate that the biological cost associated with a resistance gene can rapidly be compensated during in vivo growth. Thus, once the resistant clone has gained its resistance determinant it will be difficult to eliminate.     

MRSA感染的血清学分型及质粒研究

探明MRSA感染的血清学特点及质粒分布 ,为其预防和治疗提供科学依据。做血浆凝固抑制试验—中和法和质粒DNA提取用碱裂解法。结果血浆凝固酶Ⅱ型的检出阳性率为 5 4.0 % ( 47/ 87) ,Ⅳ型为 2 7.5 % ( 2 4/ 87) ,其余为Ⅶ和Ⅲ型。按菌株来源分析 :病房工作人员和住院患者的MRSA以Ⅱ型居多 ,Ⅳ型次之 ,有明显的偏重集中趋势 ,而门诊患者MRSA血浆凝固酶型别分布散乱。多数菌株有质粒 ,分布复杂。表明沈阳地区流行的主要血浆凝固酶型别为Ⅱ型和Ⅳ型。     

Screening for the presence of the insertion sequence IS1 in the genus Bacteroides

Abstract A specific DNA probe, containing a conserved region of the insertion sequence IS1, was hybridised to dot blots of total genomic DNA from 2 oral and 5 intestinal Bacteroides spp. Using Escherichia coli K12 as a positive control and Pseudomonas aeruginosa as a negative control, DNA homologous to the probe could not be detected in Bacteroides corporis, Bacteroides intermedius, Bacteroides ovatus, Bacteroides vulgatus, Bacteroides thetaiotaomicron or 2 strains of Bacteroides fragilis . The total DNA included plasmid DNA of 30.2, 42.7 and 42.7 MDa from B. fragilis, B. intermedius and B. corporis , respectively.
IS1 is commonly found in members of the Enterobacteriaceae, and it was concluded that the 2 groups of bacteria are not closely related.