Elimination of the yeastCandida parapsilosis from lymphoid cells and monolayer cells in culture

Summary In our laboratory, airborne yeast contaminants of cell cultures have consistently been of the genusCandida (speciesCandida parapsilosis), which are difficult to control with fungicidal agents. To salvage cell lines that show the presence of this fungus, two effective methods may be employed. In early stages of infection, the addition of activated mouse peritoneal macrophages (5×10⁵ cells/ml) to the culture medium containing 5 μg Fungizone/ml eliminates all spores by phagocytosis. More heavily contaminated cultures can be depleted of fungi by density centrifugation on a layer of 38% Percoll. Remaining single spores, often not detectable by light microscopy, can be removed by the addition of macrophages (2×10⁵/ml) and Fungizone (5 μg/ml) to the culture medium. Contaminated monolayer cells can be freed of blastospores by several washes with balanced salt solution and subsequent culturing for 4 d in medium containing 10 μg Fungizone/ml without any toxic effects to the cells. These procedures can rescue valuable cell lines and hybridomas that would otherwise be lost. This work was supported by Veterans Administration Research Funds.     

A micromethod for the quantitative determination of the viability of Candida albicans hyphae

A micromethod for the quantitative determination of the viability of Candida albicans hypae was devised which takes advantage of the dimorphic nature of C. albicans which grows exclusively in the yeast form when incubated aerobically on Sabouraud dextrose agar at 30°C. When tested by thisd method, all viable, C. albicans hyphae were recognized as microcolonies consisting of one hypha surrounded by several yeast form progeny. In contrast to this, no yeast form progeny emerged from nonviable hypae. By counting appropriate total numbers (200–400) of microcolony-forming hypae and infertile hyphae, it was possible to determine the ratio of viable to nonviable cells in a given hyphal suspension. This micromethod may be used for quantitative assessment of the candidacidal effects of various antimycotic agents or phagocytes C. albicans hyphae whose viability could not have been determined by the conventional plating technique because of the species' high propensity to clump.     

Cloning and expression of Candida albicans ADE2 and proteinase genes on a replicative plasmid in C. albicans and in Saccharomyces cerevisiae.

Summary A plasmid vector (denoted pRC2312) was constructed, which replicates autonomously in Escherichia coli, Saccharomyces cerevisiae and Candida albicans. It contains LEU2, URA3 and an autonomously replicating sequence (ARS) from C. albicans for selection and replication in yeasts, and bla (ampicillin resistance) and ori for selection and replication in E. coli. S. cerevisiae AH22 (Leu^–) was transformed by pRC2312 to Leu^– at a frequency of 1.41 × 10⁵ colonies per g DNA. Transformation of C. albicans SGY-243 (Ura-) to Ura⁺ with pRC2312 resulted in smaller transformant colonies at a frequency of 5.42 × 10³ per g DNA where the plasmid replicated autonomously in transformed cells, and larger transformant colonies at a frequency of 32 per g DNA, in which plasmid integrated into the genome. Plasmid copy number in yeasts was determined by a DNA hybridization method and was estimated to be 15±3 per haploid genome in S. cerevisiae and 2–3 per genome in C. albicans replicative transformants. Multiple tandem integration occurred in integrative transformants and copy number of the integrated sequence was estimated to be 7–12 per diploid genome. The C. albicans ADE2 gene was ligated into plasmid pRC2312 and the construct transformed Ade^– strains of both C. albicans and S. cerevisiae to Ade⁺. The vector pRC2312 was also used to clone a fragment of C. albicans genomic DNA containing an aspartic proteinase gene. C. albicans transformants harboring this plasmid showed a two-fold increase in aspartic proteinase activity. However S. cerevisiae transformants showed no such increase in proteinase activity, suggesting the gene was not expressed in S. cerevisiae.     

Inhibition and killing of fungi by the polyamine oxidase-polyamine system

Both components of the polyamine oxidase (PAO)-polyamine system are known to be present in phagocytes and have thus been postulated to contribute to the antimicrobial activity of these cells. Therefore, the effects of the PAO-polyamine system on three medically important opportunistic fungi were examined. Yeasts of Cryptococcus neoformans, but not Candida albicans blastoconidia or Aspergillus fumigatus conidia, were efficiently killed by the system. Two putative end products of the system, hydrogen peroxide and acrolein, both killed C. neoformans at concentrations attainable with the whole system. However, catalase failed to inhibit activity of the whole system, making hydrogen peroxide an unlikely mediator of killing. Although C. albicans blastoconidia and A. fumigatus conidia were not killed by the PAO-polyamine system, germ tube formation by the former, and hyphal growth by the latter, were markedly inhibited. These data establish that the PAO-polyamine system possesses antifungal activity.     

氟康唑3日间隔疗法治疗念珠菌性外阴、阴道炎疗效观察

目的探讨氟康唑3日间隔疗法治疗念珠菌性外阴、阴道炎的临床疗效。方法110例患者随机分为两组．治疗组60例采用氟康唑3日间隔疗法（第1日服药150mg后．第4、7日各再服1次）,对照组50例1次单剂量口服氟康唑150mg。结果治疗组和对照组有效率分别为86．67％、72．00％．差异有显著性（χ^2=9．46,P〈0．05）．结论氟康唑3日间隔疗法治疗念珠菌性外阴、阴道炎疗效好。毒副作用小,安全性高。     

白假丝酵母与细菌相互作用机制及其与感染的关系

人体内定植着数目庞大、结构复杂的细菌及真菌等微生物群,它们之间存在复杂的相互作用。既往研究主要集中于细菌或真菌的某个单一物种,但最近研究表明,细菌与真菌之间的相互作用对更好地理解体内的微生态系统至关重要。白假丝酵母(又称白念珠菌)是人备体中最常见的机会性致病真菌,通常被认为是人类正常菌群的一部分。白念珠菌与细菌的相互作用近年来备受关注,其协同和拮抗作用有助于维持不同物种间复杂的平衡关系。了解白念珠菌与细菌的相互作用,不仅可加深对微生物致病机制的理解,还可为预防和治疗白念珠菌或细菌感染及新型抗菌药物研发提供新途径。本文就白念珠菌与细菌共存时的相互作用机制及其对人类健康、疾病的影响进行综述,从而为控制念珠菌或细菌感染提供新的策略。     

新疆地区白念珠菌基因型分析及其体外药物敏感性研究

目的研究新疆地区汉族和维吾尔族患者来源的50株白念珠菌的基因型及其对两性霉素B、5-氟胞嘧啶、米卡芬净、伊曲康唑、氟康唑和咪康唑的体外敏感性。方法采用PCR法扩增白念珠菌rDNA 25S的Ⅰ类内含子包含区,根据扩增产物的大小判断基因型(A型为450 bp,B型为840 bp,C型为450 bp和840 bp)。采用CLSI M27-A液基微量稀释法测定50株白念珠菌对上述6种抗真菌药的体外敏感性。结果 50株菌分为3种基因型:A型30株,B型和C型各10株。所有菌株对两性霉素B、5-氟胞嘧啶、米卡芬净和咪康唑的MIC值较低,MIC范围依次为0.25～0.5μg/mL,0.125～0.5μg/mL,≤0.03μg/mL,0.25～8μg/mL;对伊曲康唑和氟康唑的MIC值较高,MIC范围分别为0.25～8μg/mL,0.5～64μg/mL。B型和C型对5-氟胞嘧啶的MIC值均为0.125μg/mL,对伊曲康唑和氟康唑的耐药率分别为84%、70%。不同族别来源的菌株基因型比较无显著差异(P>0.05),不同基因型菌株的抗真菌药物敏感性比较也无显著差异(P>0.05)。结论新疆地区白念珠菌分A,B,C三种基因型。汉...     

酵母表面展示脂肪酶合成己二酸二异辛酯

展示酶的酵母细胞既具有固定化酶的优点,又有制备简单、成本较低的特点.采用表面展示南极假丝酵母脂肪酶B (Candida antarctica lipase B,CALB)的毕赤酵母细胞催化合成己二酸二异辛酯(Diisooctyl adipate,DIOA),对该反应体系进行优化,并实现了初步工艺放大制备.经条件优化后,在10mL反应体系中,DIOA的产率可达85.0％.该工艺放大到200mL反应体系时,DIOA产率可达97.8％.经减压蒸馏,DIOA纯度可达到98.2％.该酵母表面展示脂肪酶在合成绿色润滑油己二酸二异辛酯中具有良好应用前景.     

氟康唑对热带念珠菌活性氧和线粒体膜电位的影响

为了探讨氟康唑作用机制,观察它对热带念珠菌作用后存活率、活性氧(Reactive oxygen species,ROS)、线粒体膜电位(Mitochondrial membrane potential,△Ψm)和细胞周期的变化。参照NCCLS M27-A方案的微量稀释法测定氟康唑对热带念珠菌的最低抑菌浓度(MIC);热带念珠菌与不同浓度氟康唑共同培养后用流式细胞术(Flow cytometry,FCM)分析热带念珠菌存活率、ROS、线粒体膜电位△Ψm和细胞周期的变化。结果表明,氟康唑作用后,热带念珠菌氟康唑耐药株的存活率、ROS、线粒体膜电位△Ψm和细胞周期各期比例均没有明显变化;而热带念珠菌氟康唑敏感株的存活率和线粒体膜电位△Ψm明显下降,ROS明显升高,而且大部分热带念珠菌阻滞于G2/M期,并出现明显凋亡峰,呈一定的时间剂量依赖关系。自由基清除剂谷胱甘肽抑制热带念珠菌ROS的产生,阻止细胞周期G2/M期阻滞和降低凋亡。由此可见,氟康唑可能通过刺激热带念珠菌产生过多ROS,并使线粒体膜电位△Ψm下降,从而诱导热带念珠菌凋亡。     

白念珠菌钙稳态系统及钙信号途径的研究进展

白念珠菌是临床重要的条件致病菌,其胞内的钙稳态及钙信号途径与宿主侵染、压力应答等诸多生理过程紧密相关。研究该菌的钙稳态系统及钙信号调控网络,对明确白念珠菌的侵染机制与耐药机理,以及开发具有新靶点的抗真菌药物具有重要意义。本文对这些内容的研究进展进行了综述。