A novel class of temperature-sensitive mutants generated by RNAi-mediated knockdown

Temperature-sensitive (ts) mutants are powerful tools with which to investigate gene function, but it has been difficult to generate ts mutants in mammalian cells. Recently, RNA interference (RNAi) has been widely used for loss of function analyses. In addition, in various organisms, hypothermic-temperature-sensitive RNAi has been reported. By using this characteristic of RNAi, we attempted to generate ts mutants in mammalian cells and were able to successfully generate ts mutants of cell cycle regulator cdc2 and ubiquitin-activating enzyme E1. We compared ts mutants previously isolated by mutagenesis with those generated by RNAi knockdown, and observed similar phenotypes. This method enabled us to generate ts mutants (KDts, knockdown temperature-sensitive mutants) of the genes of interest and will be utilized to facilitate understanding of the biological processes regulated by an essential gene in mammalian cells.     

烟曲霉感染时内在化Dectin-1介导巨噬细胞自噬活化

目的 探究在烟曲霉感染时Dectin-1是否内在化表达并介导了巨噬细胞的自噬活化,初步明确其作用机制。方法 采用Western blot法和免疫荧光技术,观察经β-1,3-D-葡聚糖酶消化前后的烟曲霉孢子刺激下,RAW264.7细胞内Dectin-1与LC3Ⅱ的表达与定位,通过DCFH-DA探针检测消化β -葡聚糖对ROS生成的影响。结果 烟曲霉孢子刺激后RAW264.7细胞的Dectin-1与LC3Ⅱ表达水平显著升高,同时二者呈斑点状聚集并共定位于烟曲霉孢子表面;消化β-葡聚糖后Dectin-1与LC3Ⅱ表达量降低,荧光斑点消失,并且ROS的生成受到抑制。结论 烟曲霉感染时Dectin-1提高自身内在化表达并诱导了巨噬细胞自噬功能的活化。     

长片段融合PCR在构建烟曲霉rho1基因回补株中的应用

目的融合PCR是一种常用的构建重组片段或重组质粒的手段,但长片段融合PCR的难度较大。文中将探讨长片段融合PCR过程中引物设计及扩增条件对产物的影响。方法以构建烟曲霉rho 1基因回补株为例,采用融合PCR的方法扩增重组片段(长达6.5 kb),在引物设计时引入不同大小的同源区,并设置不同的扩增体系。结果当设计引物的同源区为35 bp,选用具有高扩增效率、高保真性的DNA聚合酶,以及各片段在融合PCR反应体系中的浓度为15 ng/μL时,实现了长达6.5 kb的片段扩增并完成了烟曲霉rho 1回补株的构建。结论在合适的PCR引物设计、片段浓度配比及聚合酶条件下,长片段融合PCR在丝状真菌的基因敲除及回补株的构建中是一种非常有效的工具。     

烟曲霉再鉴定、标准化CSP分型及体外药物敏感性

目的 了解烟曲霉复合体临床株菌种分布,经典烟曲霉临床株CSP基因型及对常见抗真菌药物敏感性状况.方法 菌株来源:北京大学真菌和真菌病研究中心保存分离自125名患者的162株烟曲霉复合体菌株.通过形态学,最高生长温度及分子生物学测序分步鉴定;对CSP基因进行扩增、测序,采用国际化命名体系进行CSP分型;采用微量液基稀释法测定经典烟曲霉对伊曲康唑(ITC)、两性霉素B(AMB)、伏立康唑(VRC)及卡泊芬净(CAS)的敏感性.结果 所有烟曲霉复合体菌株均为经典烟曲霉;共分为16个CSP基因型,最常见为t04A、t03和t01;分离自4名患者的13株菌对ITC的MICs≥4 μg/mL,其中2株菌AMB和VRC的MICa分别为4μg/mL和16 μg/mL.CAS的MECs最高为4μg/mL,仅1株.结论 未检出烟曲霉相关新种;经典烟曲霉临床株共16个CSP基因型,分布与国际研究结果基本一致,其中5个为新型.我国经典烟曲霉临床株ITC耐药率为3.2％,个别菌株AMB,VRC和CAS耐药.     

New restriction fragment length polymorphism (RFLP) markers for Aspergillus fumigatus

In this study, we isolated and tested restriction fragment length polymorphism (RFLP) markers for Aspergillus fumigatus based on PCR products amplified by the random amplified polymorphic DNA (RAPD) primer R108. Four DNA fragments, Afd, Af5, Af4, and Af4A, were amplified. Fragments Afd and Af5 were 85% and 88% identical at the DNA level to part of the Afut1 retrotransposon from A. fumigatus. Fragment Af4A is a duplication of fragment Af4 and both showed similarity at the amino acid level with endonucleases from other fungal retrotransposons. We used both RAPD with primer R108 and RFLP assays with Afut1, Afd, and Af4A, to determine the genetic relatedness of clinical isolates of A. fumigatus isolated sequentially from four patients colonized with A. fumigatus. The combination of these different methods suggested that the isolates infecting the four patients were not identical.     

Aspergillus fumigatus toxicity and gliotoxin levels in feedstuff for domestic animals and pets in Argentina

Aims:  To evaluate gliotoxin production by Aspergillus fumigatus strains isolated from feedstuff intended for domestic animals and pets, and to determine the amount of gliotoxin in these substrates.
Methods and Results:  A total of 150 feedstuff samples were collected. They were composed of 30 samples each of five different feed types (pigs, poultry, cattle, horse and pets). Aspergillus fumigatus gliotoxin production ability and gliotoxin presence in feedstuff was determined by HPLC. Aspergillus fumigatus strains were isolated from all of the tested samples. Strains from cattle, horses and pet food were able to produce gliotoxin. Corn silage samples intended for cattle did not show gliotoxin contamination. All the other tested samples had gliotoxin levels ranging from 29 to 209 μg g⁻¹. Horse and poultry feed samples had the greatest contamination frequency.
Conclusions:  Feed samples contaminated with gliotoxin are potentially toxic to animals.
Significance and Impact of the Study:  The presence of gliotoxin could affect animal productivity and health. Moreover, there are risks of contamination to farm workers handling improperly stored animal feed. Aspergillus fumigatus strains isolated from different sources should be investigated to determine prevention and control strategies.     

烟曲霉菌热稳定性植酸酶在黑曲霉菌中的分泌性表达

在先前克隆获得烟曲霉菌植酸酶phyA基因并构建了重组质粒的基础上,将该质粒转化黑曲霉菌pyrG基因缺陷株M54;同时制备植酸酶多克隆抗体用于植酸酶的免疫学检测。SDS-PAGE和western-blot结果表明,phyA在黑曲霉菌中获得分泌性表达。表达产物活性测定结果显示,重组植酸酶的表达量为597.6 IU/mL。在90℃加热10 min和100℃加热20 min后,重组植酸酶残余酶活分别为74%和70%,具有较好的热稳定性。实现了烟曲霉菌植酸酶在黑曲霉菌中的分泌性表达,表达产物具较高的生物活性和耐热性。     

Split-marker 重组技术构建泛素C 末端水解酶基因缺失菌株

目的:对烟曲霉(Aspergillus fumigatus)泛素末端水解酶(creB)基因进行敲除。方法:通过氨基酸序列分析软件初步分析烟曲霉CreB蛋白结构.利用split-marker重组技术构建重组片段,并通过PEG-原生质体方法对烟曲霉野生菌株进行转化,采用PCR方法对转化子进行筛选,最后选取初步筛选的转化子进行测序鉴定。结果:结构分析显示烟曲霉CreB蛋白具有泛素特异蛋白酶(ubiquitin-processing protease)UBP亚家族六个结构域。本实验构建了转化片段并转化,在抗性平板中获得了25个Hyg抗性转化子,进一步采用PCR方法筛选到20个转化子,最终通过测序分析获得一株creB基因缺失菌株。结论:Split-marker重组技术是对烟曲霉creB基因进行敲除的快速有效的方法。获得的creB缺失菌株可用于基因功能研究。     

小鼠烟曲霉角膜炎的实验研究

目的 比较伊曲康唑和氟康唑对烟曲霉的体外抗菌活性,观察伊曲康唑对小鼠烟曲霉角膜炎的治疗作用.方法 通过角膜基质注射法建立烟曲霉角膜炎小鼠模型.造模后观察角膜病变,取角膜病变处分泌物做真菌镜检、真菌培养以证实造模成功.用药基法检测伊曲康唑和氟康唑对烟曲霉的最低抑菌浓度( MIC)和最低杀菌浓度(MFC).对烟曲霉角膜炎小鼠给予伊曲康唑治疗,治疗结束行临床评分、炎性评分、菌落形成单位测定以评价疗效.结果 伊曲康唑对烟曲霉的MIC和MFC分别为6.25 μg/mL、12.5 μg/mL;氟康唑对烟曲霉的MIC和MFC分别为500 μg/mL、1 000 μg/mL.伊曲康唑治疗组临床评分、炎性评分和测定的菌落数较对照组均明显减少(P＜0.05).结论 伊曲康唑对烟曲霉的体外抗菌活性优于氟康唑,并且对烟曲霉性角膜炎有明显疗效.     

地塞米松对过氧化氢体外氧化杀伤烟曲霉的影响

目的研究地塞米松对过氧化氢（H2O2）体外杀伤烟曲霉（Aspergillus fumigatus,A.fumigatus）的影响。方法用不同浓度的地塞米松（0 mg/mL,0.02 mg/mL,0.2 mg/mL）处理烟曲霉孢子,按照处理时间不同分为A（0.5 h）,B（2 h）,C（7 h）,D（16 h）4组。用测定H2O2杀伤真菌的标准方法——斑点法分别测定各组烟曲霉孢子对H2O2的氧化杀伤敏感性。结果在H2O2浓度为1.5 mmol/L下,未用地塞米松处理的孢子（阴性对照）氧化杀伤敏感性为5×101（生长良好）。A（0.5 h）组：地塞米松0.02 mg/mL处理后孢子的氧化杀伤敏感性为5×10^3;地塞米松0.2 mg/mL处理后孢子的氧化杀伤敏感性为5×10^4。B（2 h）组：地塞米松0.02 mg/mL和0.2 mg/mL处理后孢子的氧化杀伤敏感性均为5×10^3。C（7 h）和D（16 h）组：不同浓度地塞米松处理后氧化杀伤敏感性与阴性对照没有差别（均为5×10^1）。结论地塞米松使H2O2在体外杀伤烟曲霉的能力增强,这种作用仅发生在地塞米松接触烟曲霉孢子的早期（〈2 h）。     

产植酸酶菌株的筛选及其植酸酶基因的克隆

从土壤中筛选到产植酸酶活性较高的烟曲霉菌株WY-2,其植酸酶最适pH为5.5,最适温度为55℃。通过对烟曲霉WY-2植酸酶基因进行PCR扩增,获得了一个1.5kb大小的特异性产物,将其克隆到载体pMD18-T中。测序结果分析表明,该基因片段含有植酸酶基因完整的阅读框架(ORF),基因全长1459bp,其中包含一个61bp的内含子,编码465个氨基酸,有7个潜在的糖基化位点,5′端有一编码26个氨基酸的信号肽序列。该基因与已报道的烟曲霉ATCC34625植酸酶基因有91%同源性,编码的氨基酸序列同源性为91%。     

烟曲霉FADB基因参与菌株耐药的潜在机理

FAD结合的氧化还原酶编码基因FADB (GenBank ID:Afu4g14630)的编码产物在烟曲霉中为一种与FAD结合的氧化还原酶,可能参与真菌的呼吸。为了探究其具体功能,本研究通过克隆烟曲霉FADB基因,构建烟曲霉FADB基因的敲除株,了解该基因对烟曲霉药物敏感性、渗透压、氧化压力物质敏感性的作用机理。采用高通量基因替换方法构建出FADB基因的敲除盒,筛选出符合的烟曲霉FADB敲除株ΔFADB。观察该突变株与野生株AF293对药物敏感性、氧化压力、渗透压物质敏感性的变化。利用E-test法检测ΔFADB对3种唑类药物(泊沙康唑、伊曲康唑、伏立康唑)的敏感性,结果证实ΔFADB对泊沙康唑的敏感性明显提高。在渗透压和氧化压力的培养试验中,与野生株AF293相比较,ΔFADB对氧化剂D-山梨醇和甲萘醌更敏感。加入泊沙康唑后,ΔFADB比野生株AF293产生的活性氧更多。说明烟曲霉FADB基因参与泊沙康唑抗烟曲霉机制并起到一定作用,该基因诱导的氧化应激反应可能是抗真菌药物杀伤真菌的作用机制之一,本研究为烟曲霉唑类耐药机制的研究提供了新的理论基础。     

单核细胞抗烟曲霉感染作用的研究进展

烟曲霉是临床常见的机会性致病真菌,好发于免疫低下人群,常因吸入孢子导致肺部感染,治疗困难。烟曲霉的致病性与其较强的生存能力有关,但主要取决于宿主免疫状态,尤其是天然免疫功能。单核细胞是天然免疫的重要效应细胞,可分化为树突细胞和巨噬细胞。依据其表面分子分型,单核细胞功能上分别以吞噬抗原、呈递抗原和杀伤抗原为特点,在抗烟曲霉感染中具有较高的抵御能力和诊断价值。现阶段研究基于不断发展的荧光标记、灌流培养、光谱成像、基因敲除等技术,通过体外细胞实验和小鼠体内实验,观察到单核细胞与烟曲霉相互作用。在烟曲霉孢子入侵早期,单核细胞即启动基因表达,直接杀灭孢子,也可通过增强中性粒细胞杀孢子活性间接参与抗感染免疫调节,其中白细胞介素家族可能是关键信号分子。     

Aspergillus fumigatus catalases: cloning of an Aspergillus nidulans catalase B homologue and evidence for at least three catalases

The presence of catalases in the water soluble fractions of three Aspergillus fumigatus strains was investigated using non-denaturing and denaturing polyacrylamide gel electrophoresis and Western analysis. Using non-denaturing polyacrylamide gel electrophoresis and staining for catalase activity, three separate catalases were identified. An A. fumigatus catalase gene (catB) was cloned from genomic DNA using the Aspergillus niger catR gene as a probe. Polyclonal antibodies were raised to a glutathione S-transferase-CatB fusion product expressed in Escherichia coli. Western analysis indicated that, under denaturing conditions, the polyclonal antibody recognised a 90-kDa band and under non-denaturing conditions, two separate bands were identified. These results indicate that A. fumigatus in addition to CatB, produces at least two other catalases, one of which is similar in size to CatB. The polyclonal antibody was also used to observe catalase expression in mice, experimentally infected with A. fumigatus. Staining was observed heterogeneously throughout the fungal hyphae. This result indicates that catalase is produced by A. fumigatus during invasive aspergillosis.     

Endogenous Inhibitors for Spore Germination in Lygodium japonicum and Their Inhibitory Effects on Pollen Germinations in Camellia japonica and Camellia sinensis

Several kinds of anthracyclines having γ-rhodofnycinone as the aglycone were isolated from Streptomyces cosmosus TMF 518, and their derivatives were prepared by chemical modification. We tested their differentiation inducing activity in Friend leukemia cells and clarified their structure activity relationship as follows: 1) The aglycone, γ-rhodomycinone, had no differentiation inducing activity but was cytotoxic; 2) the compounds with two sugar chains at both C7 and C10 had more potent differentiation inducing activity than those with only a sugar chain at C-10; 3) cosmomycin C was the most favorable candidate for an anticancer agent of all anthracyclines tested, because the value of ED₅₀ (cytotoxicity)/ED₅₀ (differentiation) was as high as 3000; and 4) the increase in differentiation inducing activity and cytotoxicity was not always in parallel.     

Isolation of a novel lipase producing fungal isolate Aspergillus fumigatus and production optimization of enzyme

Abstract

Fungal lipases occupy a place of prominence among biocatalysts owing to their novel, multifold applications and resistance to high temperature and other operational conditions. In the present study, Aspergillus fumigatus isolated from oil-contaminated soil produced good amount of lipase activity with galactose (1%) as carbon source and peptone (0.1%) as nitrogen source after 72?h of incubation in the production medium at 45?°C and pH 10.0. The isolated enzyme was found to give its optimum reaction temperature at 40?°C and pH 9.0 with the substrate used as p-nitrophenyl benzoate. The activity of lipase was inhibited by the presence of metal ions. A 6.68-fold increase for lipase production was obtained by one variable at a time. Based on the findings of present study, lipase of A. fumigatus is a potential lipase and a candidate for industrial applications such as bioremediation, detergent, leather and pharmaceutical industries.