新生隐球菌基因组DNA不同抽提方法的比较

目的 DNA是进行分子生物学研究的重要基础。在本研究中,我们建立了2种简单快速抽提基因组DNA的方法并可用作PCR扩增的模板。通过比较4种不同的DNA抽提方法以确定哪种更适合进行下一步的基因分析。方法这4种方法是:玻璃珠法,酶法,3%SDS法和氯化苄法。玻璃珠法是用玻璃珠在混漩器上剧烈振荡破碎细胞壁;3%SDS法是将细胞在含10mmol/LDTT的3%SDS溶液中加热,然后用5mmol/LKAc和异丙醇抽提,DNA的产量通过A260测定。结果 3%SDS溶解法、经典酶法、玻璃珠法和氯化苄法的DNA产量分别为0.4154±0.0367、0.8484±0.0756、1.2636±0.2040、0.4070±0.0339(g/L×108CFU/mL)。结论玻璃珠法是最敏感、重复性好、简单、费用合理的抽提方法 。     

新生隐球菌荚膜研究现状

新生隐球菌是一种专性需氧条件致病菌,它的细胞壁外包绕着一个多糖荚膜,是其主要毒性因子之一。荚膜主要包含两种多糖-葡萄糖醛酸木糖和半乳糖甘露聚糖,此外还有少部分的甘露糖蛋白。这些多糖分子除构成多糖荚膜外,同时也参与新生隐球菌与宿主之间的免疫反应。该文对新生隐球菌荚膜的结构、生物合成、免疫反应及针对荚膜的抗真菌治疗等方面作一综述,旨在为新生隐球菌相关疾病的研究提供新的思路。     

新生隐球菌的生态学,流行病学,分子生物学及临床研究

在我国，对新生隐球菌已进行了较系统的研究，生态学方面，由鸽粪分离的环境株具有表型的多态性，包括新生变种的Ａ、Ｄ血清型及尿素酶阴性株，而这些多态性菌株均已在临床发现。分子生物学方面，Ｇ＋Ｃｍｏｌ％和核型已被进行分析。由ＰＦＧＥ分析所得到的有意义的信息是两个变种和５种血清型新生隐球菌株具有明显不同的核型谱。临床方面，研制的一种新的可同时检测酚氧化酶和尿素酶的培养基可用于该菌的临床鉴定。使用一种高渗培养     

艾滋病合并新生隐球菌感染的研究进展

新生隐球菌感染是全世界艾滋病患者死亡的主要原因,尤其是在撒哈拉以南非洲地区发病率最高[1]。新生隐球菌除了容易感染HIV个体外,还易感染其他免疫功能低下的个体,如造血系统恶性肿瘤、器官移植后服用免疫抑制剂及免疫缺陷病患者。格特隐球菌主要侵犯免疫功能正常的个体,但也感染免疫功能低下患者如合并艾滋病毒的患者[2]。     

新生隐球菌感染与细胞因子的相互关系

新生隐球菌是重要的条件致病真菌,可导致免疫抑制患者产生致命的新生隐球菌性脑膜炎;继而在脑部损伤部位有多种细胞因子的表达和炎症细胞的浸润,形成了一个细胞因子网络,介导一系列致病机制和防御机制的产生。为明确细胞因子与新生隐球菌在致病和防御过程中的相互作用,本文就新生隐球菌与细胞因子的关系进行综述。     

新生隐球菌线粒体的快速抽提和电镜观察

通过NovoZym234酶溶壁和低渗机械振荡破壁相结合,应用差速离心法分离并纯化了对数生长期的新生隐球菌线粒体,然后从经DNaseI处理的线粒体制备液中分离纯化线粒体DNA;并对差速离心中所获得的菌体、原生质体、线粒体三部分沉淀进行了透射电镜观察,结果均证明了我们所抽提的DNA是纯净的,适用于酶切分析和PCR分析研究,由此成功地建立了快速有效分离和纯化线粒体DNA的方法。 Abstract:Cryptococcus neoformans may be grown to the exponential phase,are broken by a combination of NovoZym234 and mechanical means,and mitochondrial DNA was extracted from DNaseI-treated mitrochondrial preparetion by differential centrifugation.Three pellets,including yeast cell,protoplasts,mitochondrial,were examined by transmission electronic microscopy.The resulting mtDNA is sufficicently pure for restriction endonucleases analysis and PCR in further studying.A rapid and effective method for the preparation of the mtDNA of C.neoformans was established.     

新生隐球菌的荚膜多糖合成研究进展

新生隐球菌是一重要的致病真菌,其细胞壁外层的多糖荚膜是第1个被公认的新生隐球菌毒性因子。本文总结了在荚膜生理和生化合成方面的研究进展,介绍了研究新生隐球菌荚膜合成的常用方法以及在新生隐球菌的荚膜代谢途径、生化合成酶、分泌、组装和调节这些广泛的研究领域存在的许多未解决的问题。     

新生隐球菌MIS1基因的siRNA表达载体的构建及鉴定

目的 构建靶向新生隐球菌MIS1基因的siRNA重组表达载体质粒,并进行鉴定.方法 根据GenBank的MIS1基因序列,按照载体要求设计单链引物,克隆到空载体psilencer4.I-CMV neo中,经过LiAc化学法将重组质粒转染到新生隐球菌细胞中并用G418筛选,利用real time PCR鉴定阳性细胞的MIS1基因水平.结果 重组表达质粒Psilencer4,1-CMV-si-MIS1经PCR、双酶切及测序鉴定,结果证明重组表达载体构建成功,其能在mRNA水平显著抑制MIS1的表达.结论 已成功构建新生隐球菌MIS1基因的siRNA表达载体,为深入研究MIsl在隐球菌相关疾病的发生及发展中的作用提供了技术手段.     

利用微卫星标记研究新生隐球菌分子流行病学

目的 研究国内隐球菌临床分离株的遗传多态性和分子流行病学.方法 选择与新生隐球菌遗传相关的9个微卫星标记,分析这9个位点从1993 ～2009年国内分离到的新生隐球菌临床株遗传背景、来源及变异程度.结果 116株被研究的隐球菌临床分离株,主要归属于3个微卫星复合物(MC2,MC3和MC12),其中大部分为MC2(103株).8株菌株属于目前为止未被国内外认识的新复合物(MC12).结论 利用微卫星DNA多态性研究新生隐球菌分子流行病学有较大的应用价值.     

Rapid methods to extract DNA and RNA from Cryptococcus neoformans

Extraction of nucleic acids from the pathogenic yeast Cryptococcus neoformans is normally hampered by a thick and resistant capsule, accounting for at least 70% of the whole cellular volume. This paper presents procedures based on mechanical cell breakage to extract DNA and RNA from C. neoformans and other capsulated species. The proposed system for DNA extraction involves capsule relaxation by means of a short urea treatment and bead beating. These two steps allow a consistent extraction even from strains resistant to other procedures. Yield and quality of DNA obtained with the proposed method were higher than those obtained with two earlier described methods. This protocol can be extended to every yeast species and particularly to those difficult to handle for the presence of a capsule. RNA purification is accomplished using an original lysing matrix and the FastPrep System (Bio101) after a preliminary bead beating treatment. Yields range around 1 mg RNA from 15 ml overnight culture (10(9) cells), RNA appears undegraded, making it suitable for molecular manipulations.     

Characterization of a Phenol Oxidase from Cryptococcus neoformans var. neoformans

In Cryptococcus neoformans, enzymic oxidation of various catechols leads to melanin, a proposed virulence factor. A phenol oxidase enzyme of Cryptococcus neoformans var. neoformans produced at 25 C has been purified from an ultracentrifugal supernatant of an extract of broken cells. Hydrophobic interaction chromatography followed by anion-exchange column chromatography allowed purification of the phenol oxidase. The molecular weight of the enzyme estimated by gel filtration was about 80,000 and a dimeric species (Mw = 160,000) was suggested. The isoelectric point of the protein was approximately 4.1. An NH₂-terminal 31 amino acid sequence was determined using phenol oxidase electroblotted onto a PVDF membrane after nondenaturing gel electrophoresis. Upon searching the Peptide Institute (Osaka) data base, no proteins with high degrees of homology were found.     

Efficient implementation of RNA interference in the pathogenic yeast Cryptococcus neoformans

An improved method has been developed for RNA interference in Cryptococcus neoformans, using opposing promoters to facilitate cloning and RNA interference targeting URA5 to allow selection of cells in which silencing is most effective. These advances significantly reduce the variability of silencing and the effort required for interference plasmid construction.     

Mathematical modeling of pathogenicity of Cryptococcus neoformans

Cryptococcus neoformans (Cn) is the most common cause of fungal meningitis worldwide. In infected patients, growth of the fungus can occur within the phagolysosome of phagocytic cells, especially in non‐activated macrophages of immunocompromised subjects. Since this environment is characteristically acidic, Cn must adapt to low pH to survive and efficiently cause disease. In the present work, we designed, tested, and experimentally validated a theoretical model of the sphingolipid biochemical pathway in Cn under acidic conditions. Simulations of metabolic fluxes and enzyme deletions or downregulation led to predictions that show good agreement with experimental results generated post hoc and reconcile intuitively puzzling results. This study demonstrates how biochemical modeling can yield testable predictions and aid our understanding of fungal pathogenesis through the design and computational simulation of hypothetical experiments.     

Recovery of Cryptococcus neoformans from the nasopharynx of AIDS patients

Nasopharyngeal swabbings, obtained from AIDS patients, were plated onto Niger seed agar containing antibiotics. Cryptococcus neoformans was isolated from 35 out of 84 patients (41.7%) diagnosed as primary cryptococcal cases before antifungal administration, and 8 out of 86 (9.3%) cryptococcosis patients on antifungal therapy. The fungus could not be isolated from any of 447 samples from 194 AIDS patients not diagnosed with cryptococcosis. These findings are novel in that the presence of C. neoformans in AIDS patients at this site has never been looked at previously. This revised version was published online in June 2006 with corrections to the Cover Date.     

Comparative analysis of the intergenic spacer regions and population structure of the species complex of the pathogenic yeast Cryptococcus neoformans

Cryptococcus neoformans is an opportunistic basidiomycete responsible for the high incidence of cryptococcosis in patients with AIDS and in other immune-compromised individuals. This study, which focused on the molecular structure and genetic variability of the two varieties in the C. neoformans and Cryptococcus gattii species complex, employed sequence analysis of the intergenic spacer regions, IGSI and IGSII. The IGS region is the most rapidly evolving region of the rDNA families. The IGSI displayed the most genetic variability represented by nucleotide base substitutions and the presence of long insertions/deletions (indels). In contrast, the IGSII region exhibited less heterogeneity and the indels were not as extensive as those displayed in the IGSI region. Both intergenic spacers contained short, interspersed repeat motifs, which can be related to length polymorphisms observed between sequences. Phylogenetic analysis undertaken in the IGSI, IGSII and IGSI +5S rRNA + IGSII regions revealed the presence of six major phylogenetic lineages, some of which segregated into subgroups. The major lineages are represented by genotypes 1 (C. neoformans var. grubii), genotype 2 (C. neoformans var. neoformans), and genotypes 3, 4, 5 and 6 represented by C. gattii. Genotype 6 is a newly described IGS genotypic group within the C. neoformans species complex. With the inclusion of IGS subgenotypic groups, our sequence analysis distinguished 12 different lineages. Sequencing of clones, which was performed to determine the presence of multiple alleles at the IGS locus in several hybrid strains, yielded a single IGS sequence type per isolate, thus suggesting that the selected group of cloned strains was mono-allelic at this locus. IGS sequence analyses proved to be a powerful technique for the delineation of the varieties of C. neoformans and C. gattii at genotypic and subgenotypic levels.     

Anticryptococcal activity of Voriconazole against Cryptococcus neoformans var. gatti vs var. neoformans: Comparison with Fluconazole and effect of human serum

Voriconazole (VCZ), a new wide-spectrum antifungal triazole currently in development, was tested for activity against Cryptococcus neoformans (CN) var. gattii and var. neoformans in RPMI-1640 (RPMI) or RPMI plus human serum. In RPMI VCZ was 10-fold more inhibitory than FCZ for both varieties of CN. In the presence of human serum neither VCZ nor FCZ had enhanced activity against CN var. gattii. By contrast, both VCZ and FCZ had significantly increased activity in the presence of serum against CN var. neoformans. The lack of serum-enhancing activity for VCZ or FCZ against CN var. gattii may reflect the in vivo situation and predict less efficacy in CN var. gattii infections. This revised version was published online in June 2006 with corrections to the Cover Date.     

巨噬细胞对新生隐球菌B3501标准株的吞噬作用

目的检测巨噬细胞对新生隐球菌活力的影响。方法新生隐球菌标准株B3501与小鼠巨噬细胞系J774细胞共孵育后,检测其出芽率,并通过电镜观察B3501在J774细胞内的超微结构。结果被吞噬的B3501超微结构完好,J774细胞对B3501菌株的吞噬指数在5.67%±1.29%~8.76%±3.09%,而B3501菌在J774细胞内的出芽率较高,可达46.85%±6.63%,出芽率随共孵育时间延长而下降,但4hrs组和8hrs组无明显差别(P>0.05)。超微结构观察显示细胞内的新生隐球菌细胞壁完整。结论虽然巨噬细胞存在着胞内和胞外的抗隐球菌活性,但新生隐球菌仍可在其细胞内外存活并繁殖。