从人血液中克隆过氧化氢酶基因

目的 从人血液中克隆过氧化氢酶基因.方法 以新鲜的人血液为材料,提取全血RNA,运用RT-PCR方法扩增过氧化氢酶基因,构建pMD18T/CAT克隆载体,并进行了不同来源过氧化氢酶的氨基酸序列同源分析.结果 从人血液中成功扩增出过氧化氢酶基因,获得了重组载体PMD-18T/CAT,氨基酸序列分析的同源性达80% 以上.结论 从人血液中可以很方便地克隆出过氧化氢酶基因.     

一种新槲寄生蛋白A链的基因克隆及序列分析

目的克隆槲寄生蛋白具有N-糖苷酶活性的毒性A链。方法利用硫酸铵沉淀、亲和色谱和离子交换色谱法,从吉林产槲寄生[Viscum coloratum(Komar．)Nakai]中提取、分离、纯化其中具有半乳糖结合活性的蛋白质成分。采用SDS-PAGE分析纯化的蛋白样品,蛋白条带转印至PVDF膜后,Edman降解法测定肽链的N端序列。由测序结果设计引物,利用RT-PCR方法克隆一种蛋白A链的基因。结果从槲寄生中纯化出两种高毒性的蛋白质新成分CM-1、CM-2,其中CM-1 A链被克隆并测序,其与白果槲寄生蛋白A链的同源性较高。结论一种新槲寄生蛋白A链的基因被成功克隆。     

Gene regulation and host adaptation mechanisms in Candida albicans.

The yeast Candida albicans is a harmless member of the normal microflora on the mucosal surfaces of most healthy persons, but it can cause severe opportunistic infections in immunosuppressed patients. To become a successful human commensal and pathogen, C. albicans has evolved host adaptation mechanisms on different levels. The regulated expression of virulence and other genes in response to environmental signals allows an optimal adaptation to new host niches during the course of an infection. In addition, C. albicans is able to switch between different cell types in a reversible and apparently random fashion. Phenotypic switching involves the coordinated regulation of phase-specific genes, and the resulting generation of selected, pre-programmed cell types may represent an additional strategy to adapt to certain host environments. Finally, C. albicans produces genetically altered variants at a high rate. This microevolution ensures survival when the pathogen encounters new adverse conditions, as exemplified by the development of stable drug-resistant variants under the selection pressure caused by antimycotic therapy. Thus, rather than the possession of single dominant virulence factors, it is its remarkable versatility that makes C. albicans the most important fungal pathogen of humans.     

Candida albicans and selenium

Although low selenium levels have been recorded in infants, no specific human disorder has been linked to low selenium status. The incidence of thrush, the common enteric fungal infection caused by Candida albicans, has increased markedly with antibiotic therapy and research has provided evidence that its colonization leads to competition for Coenzyme Q10 (CoQ10) in the host. Furthermore it is now known that ubiquinones are essential in heart muscle for oxidative phosphorylation in the mitochondrial respiratory chain and considered that glutathione peroxidase (GSHPx) in the mitochondria protects ubiquinone from oxidation.     

Cloning and Sequencing of Hepatitis B Virus Pre-S and S Gene Regions

The polymerase chain reaction (PCR) was used to amplify the pre-S1, pre-S2 and S gene regions of hepatitis B virus (HBV). Sera from three different patients were used as the source of HBV DNA. The resulting 1.2-kb amplification product was cloned into the plasmid pI B130. Restriction enzyme analysis revealed that there are two BamHI sites located about 300 bp apart within the S gene. DNA sequencing revealed a greatest homology to the HBV adw subtype.     

Attenuation of Virulence and Changes in Morphology in Candida albicans by Disruption of the N-Acetylglucosamine Catabolic Pathway

A Candida albicans mutant with mutations in the N-acetylglucosamine (GlcNAc) catabolic pathway gene cluster, including the GlcNAc-6-phosphate deacetylase (DAC1), glucosamine-6-phosphate deaminase (NAG1), and GlcNAc kinase (HXK1) genes, was not able to grow on amino sugars, exhibited highly attenuated virulence in a murine systemic candidiasis model, and was less adherent to human buccal epithelial cells in vitro. No germ tubes were formed by the mutant after induction with GlcNAc, but the mutant exhibited hyperfilamentation under stress-induced filamentation conditions. In addition, the GlcNAc catabolic pathway played a vital role in determining the colony phenotype. Our results imply that this pathway is very important because of its diverse links with pathways involved in virulence and morphogenesis of the organism.     

大鼠大脑组织中谷氨酸脱羧酶GAD67基因的克隆和序列测定

目的克隆大鼠脑组织中谷氨酸脱羧酶GAD67基因.方法采用RT-PCR方法,大鼠脑组织中的mRNA逆转录成cDNA,再以cDNA为模板,扩增谷氨酸脱羧酶GAD67基因片段,克隆入T载体,并经序列测定.结果 RT-PCR法扩增出一特异产物与预期长度1795bp相符,T载体克隆测序与大鼠谷氨酸脱羧酶GAD67 100%同源.结论采用RT-PCR和T载体技术获得了大鼠脑组织中的谷氨酸脱羧酶GAD67基因克隆,为该基因的体外表达打下基础.     

Effects of ploidy and mating type on virulence of Candida albicans

Candida albicans is the most common fungal pathogen of humans. The recent discovery of sexuality in this organism has led to the demonstration of a mating type locus which is usually heterozygous, although some isolates are homozygous. Tetraploids can be formed between homozygotes of the opposite mating type. However, the role of the mating process and tetraploid formation in virulence has not been investigated. We describe here experiments using a murine model of disseminated candidiasis which demonstrate that in three strains, including CAI-4, the most commonly used strain background, tetraploids are less virulent than diploids and can undergo changes in ploidy during infection. In contrast to reports with other strains, we find that MTL homozygotes are almost as virulent as the heterozygotes. These results show that the level of ploidy in Candida albicans can affect virulence, but the mating type configuration does not necessarily do so.     

大鼠大脑组织中谷氨酸脱羧酶GAD67基因的克隆和序列测定

目的　克隆大鼠脑组织中谷氨酸脱羧酶GAD6 7基因 .方法　采用RT -PCR方法 ,大鼠脑组织中的mRNA逆转录成cDNA ,再以cDNA为模板 ,扩增谷氨酸脱羧酶GAD6 7基因片段 ,克隆入T载体 ,并经序列测定 .结果　RT -PCR法扩增出一特异产物与预期长度 1795bp相符 ,T载体克隆测序与大鼠谷氨酸脱羧酶GAD6 710 0 %同源 .结论　采用RT -PCR和T载体技术获得了大鼠脑组织中的谷氨酸脱羧酶GAD6 7基因克隆 ,为该基因的体外表达打下基础 .     

Cloning and Characterization of CAD1/AAF1, a Gene from Candida albicans That Induces Adherence to Endothelial Cells after Expression in Saccharomyces cerevisiae

Adherence to the endothelial cell lining of the vasculature is probably a critical step in the egress of Candida albicans from the intravascular compartment. To identify potential adhesins that mediate the attachment of this organism to endothelial cells, a genomic library from C. albicans was used to transform a nonadherent strain of Saccharomyces cerevisiae. The population of transformed yeasts was enriched for highly adherent clones by repeated passages over endothelial cells. One clone which exhibited a fivefold increase in endothelial cell adherence, compared with S. cerevisiae transformed with vector alone, was identified. This organism also flocculated. The candidal DNA fragment within this adherent/flocculent organism was found to contain a single 1.8-kb open reading frame, which was designated CAD1. It was found to be identical to AAF1. The predicted protein encoded by CAD1/AAF1 contained features suggestive of a regulatory factor. Consistent with this finding, immunoelectron microscopy revealed that CAD1/AAF1 localized to the cytoplasm and nucleus but not the cell wall or plasma membrane of the transformed yeasts. Because yeasts transformed with CAD1/AAF1 both flocculated and exhibited increased endothelial cell adherence, the relationship between adherence and flocculation was examined. S. cerevisiae expressing either of two flocculation phenotypes, Flo1 or NewFlo, adhered to endothelial cells as avidly as did yeasts expressing CAD1/AAF1. Inhibition studies revealed that the flocculation phenotype induced by CAD1/AAF1 was similar to Flo1. Thus, CAD1/AAF1 probably encodes a regulatory protein that stimulates endothelial cell adherence in S. cerevisiae by inducing a flocculation phenotype. Whether CAD1/AAF1 contributes to the adherence of C. albicans to endothelial cells remains to be determined.     

Candida albicans and atopic dermatitis

The role of sensitization and exposure to Candida albicans in atopic dermatitis (AD) was studied with skin-prick tests, yeast cultures and immunoblotting in 156 young adults with AD attending the Department of Dermatology, University of Turku, during 1983–89. Eighteen patients with allergic rhinitis without eczema and 39 non-atopics were included as controls. Parameters associated with severe AD were simultaneous anti-C. albicans IgE and saprophytic C. albicans growth. A statistically significant correlation between C. albicans sensitization (specific IgE antibodies) and AD symptoms was observed only in patients with saprophytic C. albicans exposure. No correlation between C. albicans-specific IgE and AD severity was shown in patients without gastrointestinal growth. Furthermore, severe eczema was seldom seen in patients without saprophytic C. albicans growth. The most important IgE-binding components of C. albicans in immunoblotting were 27 and 46 kD proteins and mannan, a polysaccharide. IgG and IgA antibodies to C. albicans, mainly towards C. albicans mannan, were found in practically all 70 sera studied. These results suggest a continuous exposure and induction of IgE antibodies by C. albicans in AD patients. Severe phases of AD in colonized patients are associated with IgE synthesis against C. albicans. These findings suggest a role for C. albicans in the exacerbations of AD but the clarification of this subject needs double-blind placebo-controlled treatment trials.     

Isolation of a variant of Candida albicans.

During the course of Candida albicans antigen production, a variant of this organism was encountered which did not produce hyphae at 37 degrees C. Presented here are some of the characteristics of this variant. It produces hyphae at 25 degrees C on cornmeal agar and synthetic medium plus N-acetylglucosamine and Tween 80. At 37 degrees C, it does not produce hyphae on these media, although C. albicans normally does produce hyphae under these circumstances. In liquid synthetic medium, this variant does not produce hyphae at 37 degrees C. The variant strain was analyzed for DNA, RNA, protein content, and particle size. After 50 to 70 h in balanced exponential-phase growth, particle size distribution was narrow, and there were no differences in the DNA, RNA, or protein content per particle in the two strains. When balanced exponential-phase cultures were brought into stationary phase, both strains contained the same amount of DNA per cell.     

人单核细胞趋化蛋白－1（MCP－1）cDNA的克隆和序列测定

人单核细胞趋化蛋白－１（ＭＣＰ－１）是由多种细胞产生的一种趋化因子。它不但能趋化单核细胞还能激活单核细胞，在机体防御、炎症恢复和抗肿瘤等方面起重要作用。本文利用ＲＴ－ＰＣＲ方法克隆了人ＭＣＰ－１ｃＤＮＡ，经核苷酸序列测定，除第１２位密码子为ＴＧＴ与国外报道的ＴＧＣ不同、但编码相同的氨基酸半胱氨酸外，其余序列完全相同。     

Modifications of a Candida albicans biotyping system.

Methods of preparing and plating inocula onto media for the Odds and Abbott Candida albicans biotyping system (F. C. Odds and A. B. Abbott, Sabouraudia 18:301-317, 1980; Odds and Abbott, Sabouraudia 21:79-81, 1983) were altered to utilize inexpensive and commercially available supplies and equipment. The modified system correlates well with the reference system.     

Evaluation of a rapid immunochromatographic assay for identification of Candida albicans and Candida dubliniensis

Candida dubliniensis was first established as a novel yeast species in 1995. It is particularly associated with recurrent episodes of oral candidosis in human immunodeficiency virus (HIV)-infected patients, but it has also been detected at other anatomical sites and at a low incidence level in non-HIV-infected patients. It shares so many phenotypic characteristics with C. albicans that it is easily misidentified as such. No rapid, simple, and commercial test that allows differentiation between C. dubliniensis and C. albicans has been developed, until now. Accurate species identification requires the use of genotype-based techniques that are not routinely available in most clinical microbiology diagnostic laboratories. The present study was designed to evaluate the efficiency of a new test (the immunochromatographic membrane [ICM] albi-dubli test; SR2B, Avrille, France) to differentiate between C. albicans and C. dubliniensis. The organisms evaluated were strains whose identities had previously been confirmed by PCR tests and freshly isolated clinical strains and included 58 C. albicans isolates, 60 C. dubliniensis isolates, and 82 isolates belonging to other species of yeast. The ICM albi-dubli test is based on the principle of immunochromatographic analysis and involves the use of two distinct monoclonal antibodies that recognize two unrelated epitopes expressed by both species or specific to only one species. The assay requires no complex instrumentation for analysis and can be recommended for routine use in clinical microbiology laboratories. Results are obtained within 2 h and 30 min and are easy to interpret. This evaluation demonstrated the good performance of this immunochromatographic test for C. albicans and C. dubliniensis isolated on Sabouraud dextrose agar, CHOROMagar Candida, and CandidaSelect, with sensitivities and specificities ranging from 93.1 to 100%. These parameters decreased, however, to 91.4% when the test was performed with yeast isolated with Candida ID.     

Gene expression in HL60 granulocytoids and human polymorphonuclear leukocytes exposed to Candida albicans

Candida albicans is an opportunistic human pathogen causing both superficial and disseminated diseases. It is a dimorphic fungus, switching between yeast and hyphal forms, depending on cues from its microenvironment. Hyphae play an important role in the pathogenesis of candidiasis. The host's response to Candida infection is multifaceted and includes the participation of granulocytes as key effector cells. The aim of this investigation was to study host gene expression during granulocyte-Candida interaction. Effector cells were generated by the granulocytic differentiation of HL60 cells. The resulting cell population was shown to be morphologically and functionally equivalent to granulocytes and is therefore referred to as HL60 granulocytoids for the purposes of this study. Gene expression profiles were determined 1 h after hosts were infected with C. albicans. Three Candida-granulocytoid ratios were chosen to reflect different degrees of HL60 granulocytoid inhibition of C. albicans. The data demonstrate that at the high pathogen-host ratio, C. albicans modulated the HL60 granulocytoid's response by downregulating the expression of known antimicrobial genes. In addition, looking at the expression of a large number of genes, not all of which have necessarily been implicated in candidastatic or candidacidal mechanisms, it has been possible to describe the physiological response of the HL60 granulocytoid to an infectious challenge with C. albicans. Finally, some of the observed changes in HL60 granulocytoid gene expression were investigated in freshly isolated human polymorphonuclear leukocytes infected with C. albicans. Similar changes were seen in these primary human cells, lending support to the validity of this model.     

Antigenic variability of Candida albicans

The concepts of modern biology lead us to think that all structures are liable to continual changes. Ultrastructural and biochemical methods have been able to objectify such a dynamic in Candida albicans, an opportunistic yeast. A broad analysis of antigens is a reliable way to study the antigenic variations which concern this organism. Numerous information on somatic and metabolic antigens of C. albicans is available at the moment. Paradoxically, if one accepts studies dealing with dimorphism, very few works have shown antigenic variability of this species or investigated the mechanisms involved in such a variability. The few approaches done in this way tend to prove that it may be possible to link together the expression of particular antigens and the behavior of the yeast, particularly when it acts as a pathogen.