Genetics of Candida albicans.

Candida albicans is among the most common fungal pathogens. Infections caused by C. albicans and other Candida species can be life threatening in individuals with impaired immune function. Genetic analysis of C. albicans pathogenesis is complicated by the diploid nature of the species and the absence of a known sexual cycle. Through a combination of parasexual techniques and molecular approaches, an effective genetic system has been developed. The close relationship of C. albicans to the more extensively studied Saccharomyces cerevisiae has been of great utility in the isolation of Candida genes and development of the C. albicans DNA transformation system. Molecular methods have been used for clarification of taxonomic relationships and more precise epidemiologic investigations. Analysis of the physical and genetic maps of C. albicans and the closely related Candida stellatoidea has provided much information on the highly fluid nature of the Candida genome. The genetic system is seeing increased application to biological questions such as drug resistance, virulence determinants, and the phenomenon of phenotypic variation. Although most molecular analysis to data has been with C. albicans, the same methodologies are proving highly effective with other Candida species.     

Genetics of Candida albicans.

Candida albicans is among the most common fungal pathogens. Infections caused by C. albicans and other Candida species can be life threatening in individuals with impaired immune function. Genetic analysis of C. albicans pathogenesis is complicated by the diploid nature of the species and the absence of a known sexual cycle. Through a combination of parasexual techniques and molecular approaches, an effective genetic system has been developed. The close relationship of C. albicans to the more extensively studied Saccharomyces cerevisiae has been of great utility in the isolation of Candida genes and development of the C. albicans DNA transformation system. Molecular methods have been used for clarification of taxonomic relationships and more precise epidemiologic investigations. Analysis of the physical and genetic maps of C. albicans and the closely related Candida stellatoidea has provided much information on the highly fluid nature of the Candida genome. The genetic system is seeing increased application to biological questions such as drug resistance, virulence determinants, and the phenomenon of phenotypic variation. Although most molecular analysis to data has been with C. albicans, the same methodologies are proving highly effective with other Candida species.     

C1, MBL-MASPs and C1-inhibitor: novel approaches for targeting complement-mediated inflammation

Complement activation is initiated by the pattern-recognition molecules complement component C1q, mannose-binding lectin (MBL) and ficolins (H-, L-, M-ficolin), which typically recognize antibody-antigen complexes or foreign polysaccharides. The associated proteases (C1r, C1s, MASP-1 and MASP-2) then activate the complement system. The serpin C1-inhibitor (C1-inh) blocks activity of all these complexes and has been successfully used in models of disease. Many structures of these components became available recently, including that of C1-inh, facilitating the structure-guided design of drugs targeting complement activation. Here, we propose an approach in which therapeutic proteins are made up of natural protein domains and C1-inh to allow targeting to the site of inflammation and more specific inhibition of complement activation. In particular, engineering a fast-acting C1-inh or fusing it to an 'aiming module' has been shown to be feasible and economical using a humanized yeast expression system. Complement-mediated inflammation has been linked to ischemia-reperfusion injury, organ graft rejection and even neurodegeneration, so targeting this process has direct clinical implications.     

Enzyme immunoassay system for detection of staphylococcal enterotoxin,type C

An enzyme immunoassay (EIA) system for detection of staphylococcal enterotoxin, type C, has been developed. The sensitivity of the system is 1 ng/ml. The optimum EIA parameters have been worked out. The absence of false positive results with heterologous toxins confirms the specificity of the assay system. The possibility of the detection of staphylococcal enterotoxin, type C, in staphylococci isolated from different sources has been shown.     

The role of complement in atherosclerosis

PURPOSE OF REVIEW: Although it has long been recognized that atherosclerotic lesions show evidence of complement activation, the functional roles of the complement system in atherogenesis are not yet fully resolved. This article highlights recent publications on the complement system in the atherosclerosis field. RECENT FINDINGS: There have been a number of recent papers reporting on the association of complement proteins and complement regulators with high density lipoproteins, complement activation by enzymatically-modified LDL, signalling pathways downstream of C3a and C5a receptors and membrane C5b-9 assembly, and the prevention of C5b-9 assembly on endothelial cells via upregulation of CD59 expression in response to arterial laminar flow. C1q has been found to play a protective role in early lesion formation in LDL receptor deficient mice, and Crry-Ig and soluble C1 inhibitor have both been shown to have therapeutic effects in models of vascular injury in ApoE deficient mice. The possibility that the Y402H Factor H polymophism influences atherosclerosis has been supported in a recent paper showing increased risk in white hypertensive individuals. SUMMARY: The articles that have emerged over the last year highlight the relevance of the complement system to the atherosclerosis field.     

The anticoagulant protein C pathway

The anticoagulant protein C system regulates the activity of coagulation factors VIIIa and Va, cofactors in the activation of factor X and prothrombin, respectively. Protein C is activated on endothelium by the thrombin-thrombomodulin-EPCR (endothelial protein C receptor) complex. Activated protein C (APC)-mediated cleavages of factors VIIIa and Va occur on negatively charged phospholipid membranes and involve protein cofactors, protein S and factor V. APC also has anti-inflammatory and anti-apoptotic activities that involve binding of APC to EPCR and cleavage of PAR-1 (protease-activated receptor-1). Genetic defects affecting the protein C system are the most common risk factors of venous thrombosis. The protein C system contains multi-domain proteins, the molecular recognition of which will be reviewed.     

Construction and consequences of directed mutations affecting the hemin receptor in pathogenic Corynebacterium species

Genes encoding an ATP-binding cassette transporter system involved in hemin iron utilization from Corynebacterium ulcerans were cloned and characterized. The genes are homologous to a hemin transport system previously identified in Corynebacterium diphtheriae. Disruption of the hmuT gene, which encodes the putative hemin receptor, resulted in greatly reduced ability of C. ulcerans to use hemin or hemoglobin as an iron source. Inactivation of hmuT in C. diphtheriae by site-specific recombination had no effect on hemin utilization, which suggests that C. diphtheriae has an additional system for transporting hemin.     

Evidence for a regulatory element controlling amino aced transport system L in Chinese hamster ovary cells

We have used the technique of somatic cell hybridization to study the regulation of the neutral amino acid transport system L in Chinese hamster ovary (CHO) cells. The cell line CHO–;tsO25C1 has a temperature-sinsitive mutationin leucyl-tRNA synthetase. At the nonpermissive temperature of 39^oC, CHO–tsO25C1 cells are unable to charge leucyl-tRNA and behave as though starved for leucine by increasing their system L transport activity two- to fourfold. From the temperature-sensitive cell line, we have isolated a regulatory mutant cell, CHO–C11B6, that has constitutively elevated system L transport activity. The CHO–C11B6 cell line retains the temperature-sensitive leucyl-tRNA synthetase mutation, but growth of this cell line is temperature resistant because its increased system L transport activity leads of increased intracellular leucine levels, which compensate for the defective. Hybrid cells formed by fusion of the temperature-sensitive CHO–;tsO25C1 cells the temperature-resistant CHO–C11B6 cells show temperature-sensitive growth and temperature-dependent regulation of leucine transport activity. These data suggest that the system L activity of CHO cells is regulated by a dominant-acting element that is defective or absent in the regulatory mutant CHO–C11B6 cell line.     

Formation and properties of mixed disulfides between thioredoxin reductase from Escherichia coli and thioredoxin: evidence that cysteine-138 functions to initiate dithiol-disulfide interchange and to accept the reducing equivalent from reduced flavin.

Mutation of one of the cysteine residues in the redox active disulfide of thioredoxin reductase from Escherichia coli results in C135S with Cys138 remaining or C138S with Cys135 remaining. The expression system for the genes encoding thioredoxin reductase, wild-type enzyme, C135S, and C138S has been re-engineered to allow for greater yields of protein. Wild-type enzyme and C135S were found to be as previously reported, whereas discrepancies were detected in the characteristics of C138S. It was shown that the original C138S was a heterogeneous mixture containing C138S and wild-type enzyme and that enzyme obtained from the new expression system is the correct species. C138S obtained from the new expression system having 0.1% activity and 7% flavin fluorescence of wild-type enzyme was used in this study. Reductive titrations show that, as expected, only 1 mol of sodium dithionite/mol of FAD is required to reduce C138S. The remaining thiol in C135S and C138S has been reacted with 5,5'-dithiobis-(2-nitrobenzoic acid) to form mixed disulfides. The half time of the reaction was <5 s for Cys138 in C135S and approximately 300 s for Cys135 in C138S showing that Cys138 is much more reactive. The resulting mixed disulfides have been reacted with Cys32 in C35S mutant thioredoxin to form stable, covalent adducts C138S-C35S and C135S-C35S. The half times show that Cys138 is approximately fourfold more susceptible to attack by the nucleophile. These results suggest that Cys138 may be the thiol initiating dithiol-disulfide interchange between thioredoxin reductase and thioredoxin.     

C1q蛋白家族的结构、分布、分类和功能

C1q蛋白家族由众多含C1q结构域的蛋白组成, 从细菌到高等哺乳动物中都有分布。这类蛋白由一条信号肽、胶原样区(Collage-like region, CLR)和C1q球状结构域(Globular C1q domain, gC1q)组成。C1q蛋白家族根据其结构特点, 可分为三大类分子：C1q、C1q-like和ghC1q。C1q是补体经典途径的起始分子, 能够识别免疫复合物, 启动补体系统经典途径; 此外, 作为一种模式识别受体分子(Pattern recognition receptor, PRR), 它可以结合种类繁多的配体。C1q-like蛋白的结构类似于C1q分子, 含有CLR和gC1q结构域, 在水蛭中参与神经系统的修复, 在脊椎动物中实现从凝集素到免疫球蛋白结合分子的功能转变, 参与补体系统的激活。ghC1q蛋白只具有gC1q结构域和一段短的N末端序列, 包括分泌型蛋白(sghC1q)和非分泌型蛋白(cghC1q)。sghC1q在无脊椎动物固有免疫系统中发挥重要作用; 脊椎动物中的sghC1q可作为一类新型跨神经元调节因子, 在大脑的许多区域调节突触发育和突触可塑性。cghC1q基因最早可追溯至芽孢杆菌属的细菌中, 具有典型的gC1q果冻卷结构, 说明gC1q结构域有着非常悠久的进化历程且结构高度保守。文章对C1q蛋白家族的结构、分布、分类以及功能进行综述, 以期为从事该领域研究的科研人员提供有益参考。     

Methodologic problems in the biotyping of Candida albicans

The biotyping of 284 C. albicans strains has been carried out in accordance with the system of three tests, proposed by F.C. Odds and A. B. Abbott. The reliability of the epidemiological conclusions made as the result of this work has been analyzed. The independence of the signs of C. albicans used in biotyping and the asymmetrical character of the test for its sensitivity to 5-fluorocytosine have been shown. The change of this test for a more symmetrical one is proposed. The study has shown that in the process of prolonged storage with periodic subculturing the proportion of C. albicans strains resistant to pH 1.40 and possessing proteolytic activity is decreased. The distribution of different biotypes among C. albicans strains isolated from candidiasis patients and from carriers has proved to be the same.     

RcsA-dependent and -independent growth defects caused by the activated Rcs phosphorelay system in the Escherichia coli pgsA null mutant

In the Escherichia coli pgsA null mutant, which lacks the major acidic phospholipids, the Rcs phosphorelay signal transduction system is activated, causing thermosensitive growth. The mutant grows poorly at 37 degrees C and lyses at 42 degrees C. We showed that the poor growth at 37 degrees C was corrected by disruption of the rcsA gene, which codes for a coregulator protein that interacts with the RcsB response regulator of the phosphorelay system. However, mutant cells still lysed when incubated at 42 degrees C even in the absence of RcsA. We conclude that the activated Rcs phosphorelay in the pgsA null mutant has both RcsA-dependent and -independent growth inhibitory effects. Since the Rcs system has been shown to positively regulate the essential cell division genes ftsA and ftsZ independently of RcsA, we measured cellular levels of the FtsZ protein, but found that the growth defect of the mutant at 42 degrees C did not involve a change in the level of this protein.     

The ancient origin of the complement system

The complement system has been thought to originate exclusively in the deuterostomes. Here, we show that the central complement components already existed in the primitive protostome lineage. A functional homolog of vertebrate complement 3, CrC3, has been isolated from a 'living fossil', the horseshoe crab (Carcinoscorpius rotundicauda). CrC3 resembles human C3 and shows closest homology to C3 sequences of lower deuterostomes. CrC3 and plasma lectins bind a wide range of microbes, forming the frontline innate immune defense system. Additionally, we identified CrC2/Bf, a homolog of vertebrate C2 and Bf that participates in C3 activation, and a C3 receptor-like sequence. Furthermore, complement-mediated phagocytosis of bacteria by the hemocytes of horseshoe crab was also observed. Thus, a primitive yet complex opsonic complement defense system is revealed in the horseshoe crab, a protostome species. Our findings demonstrate an ancient origin of the critical complement components and the opsonic defense mechanism in the Precambrian ancestor of bilateral animals.     

UV-light induced structural and functional changes of protein C4 of the complement system

The influence of UV-light in the dose range of 75.5 - 2265 J/m2 on the structural and functional state of the C4 factor of the complement system has been studied by methods of hemolytic and acid-base titration, electrophoresis in polyacrylamide gel, infra-red and ultra-violet spectrophotometry. It was found that the UV-irradiation in doses of 75.5 and 755 J/m2 induces the photoactivation of the C4 protein due to changes in structures III and IV of the protein, which is accompanied by an increase in the number of aromatic amino acids and ionogenic groups on the protein molecule surface. The maximum dose of UV-irradiation (2265 J/m2) has a destructive effect on C4 protein, reducing its activity in the cascade of hemolytic reactions of the complement system. On the basis of the data obtained, a scheme of possible structural and functional photomodifications of C4 protein has been designed.     

Aggregation behaviors of gelatin with cationic gemini surfactant at air/water interface

The dilational rheological properties of gelatin with cationic gemini surfactant 1,2-ethane bis(dimethyl dodecyl ammonium bromide) (C(12)C(2)C(12)) at air/water interface were investigated using oscillating barriers method at low frequency (0.005-0.1 Hz), which was compared with single-chain surfactant dodecyltrimethyl ammonium bromide (DTAB). The results indicate that the maximum dilational modulus and the film stability of gelatin-C(12)C(2)C(12) are higher than those of gelatin-DTAB. At high concentration of C(12)C(2)C(12) or DTAB, the dilational modulus of gelatin-surfactant system becomes close to that corresponding to pure surfactant, suggesting gelatin at interface is replaced by surfactant. This replacement is also observed by surface tension measurement. However, it is found that gelatin-C(12)C(2)C(12) system has two obvious breaks but gelatin-DTAB has not in surface tension isotherms. These phenomena are ascribed to the double charges and strong hydrophobicity of C(12)C(2)C(12). Based on these experimental results, a mechanism of gelatin-surfactant interaction at air/water interface is proposed.     

外源基因在莱茵衣藻叶绿体中的表达

把莱茵衣藻(Chlamydomonas reinhardtii)叶绿体作为生物反应器来表达外源基因具有广阔的应用前景。人们利用莱茵衣藻叶绿体表达体系已成功表达多种重组蛋白,其中包括人类药用蛋白。综述了莱茵衣藻叶绿体转化的方法、影响外源基因表达的主要因素以及外源基因在莱茵衣藻叶绿体表达研究进展。     

<Superscript>1</Superscript>H, <Superscript>15</Superscript>N,and <Superscript>13</Superscript>C resonance assignments of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> extracellular adherence protein domain 4

The pathogenic bacterium Staphylococcus aureus has evolved to actively evade many aspects of the human innate immune system by expressing a series of secreted inhibitory proteins. Among these, the extracellular adherence protein (Eap) has been shown to inhibit the classical and lectin pathways of the complement system. By binding to complement component C4b, Eap is able to inhibit formation of the CP/LP C3 pro-convertase. Secreted full-length, mature Eap consists of four ~98 residue domains, all of which adopt a similar beta-grasp fold, and are connected through a short linker region. Through multiple biochemical approaches, it has been determined that the third and fourth domains of Eap are responsible for C4b binding. Here we report the backbone and side-chain resonance assignments of the 11.3 kDa fourth domain of Eap. The assignment data has been deposited in the BMRB database under the accession number 26726.     

1,2-Diacylglycerols do not potentiate the action of phospholipases A2 and C in human platelets

1,2-Diacylglycerol has recently been reported to potentiate the ability of phospholipases A and C to hydrolyze phospholipids in a cell-free system. The present study has been undertaken to investigate whether 1,2-diacylglycerol can also perform this function in intact cells using the platelet as a test system. Exogenous 1-oleoyl-2-acetyl-glycerol ( OAG ) and 1,2- didecanoylglycerol , at concentrations sufficient to produce maximal phosphorylation of a 40,000 dalton protein, caused no significant formation of [3H]inositol phosphates and [32P]phosphatidic acid (products of phospholipase C activation) or [14C]arachidonic acid metabolites and lysophosphatidyl[3H]inositol (products of phospholipase A2 activation). These data therefore imply that 1,2-diacylglycerols do not potentiate the actions of phospholipases A2 and C in intact platelets at concentrations that are physiologically relevant.     

An adenine nucleotide-phosphoenolpyruvate counter-transport system in C3 and C4 plant chloroplasts

A rapid counter-exchange between ATP and phosphoenolpyruvate (PEP) has been demonstrated in pea and maize mesophyll chloroplasts. Chloroplasts preloaded with either [14C] ATP or [14C] PEP readily exchange the radioactive compound with the externally added anions, ATP or PEP, whereas, cold external Pi counter-transports only with internal [14C] PEP. Flooding the system with cold Pi, however, will significantly reduce the counter-transport of external cold PEP with internal [14C] ATP. This ATP-PEP exchange is also markedly decreased by lowering the incubation temperature. The results indicate that the ATP-PEP counter-exchange could represent a key transport system in plant chloroplasts and may be particularly important in the photosynthesis of C4 plants. Furthermore, they provide information required to elucidate the mechanism of the ATP-PEP counter-transport system.     

Chemotaxis and flagellar genes of Chromobacterium violaceum

The availability of the complete genome of the Gram-negative beta-proteobacterium Chromobacterium violaceum has increasingly impacted our understanding of this microorganism. This review focuses on the genomic organization and structural analysis of the deduced proteins of the chemosensory adaptation system of C. violaceum. C. violaceum has multiple homologues of most chemotaxis genes, organized mostly in clusters in the bacterial genome. We found at least 67 genes, distributed in 10 gene clusters, involved in the chemotaxis of C. violaceum. A close examination of the chemoreceptors methyl-accepting chemotaxis proteins (MCPs), and the deduced sequences of the members of the two-component signaling system revealed canonical motifs, described as essential for the function of the deduced proteins. The chemoreceptors found in C. violaceum include the complete repertoire of such genes described in bacteria, designated as tsr, tar, trg, and tap; 41 MCP loci were found in the C. violaceum genome. Also, the C. violaceum genome includes a large repertoire of the proteins of the chemosensory transducer system. Multiple homologues of bacterial chemotaxis genes, including CheA, CheB, CheD, CheR, CheV, CheY, CheZ, and CheW, were found in the C. violaceum genome.