Macrophage Interaction with Paracoccidioides brasiliensis Yeast Cells Modulates Fungal Metabolism and Generates a Response to Oxidative Stress

Macrophages are key players during Paracoccidioides brasiliensis infection. However, the relative contribution of the fungal response to counteracting macrophage activity remains poorly understood. In this work, we evaluated the P. brasiliensis proteomic response to macrophage internalization. A total of 308 differentially expressed proteins were detected in P. brasiliensis during infection. The positively regulated proteins included those involved in alternative carbon metabolism, such as enzymes involved in gluconeogenesis, beta-oxidation of fatty acids and amino acids catabolism. The down-regulated proteins during P. brasiliensis internalization in macrophages included those related to glycolysis and protein synthesis. Proteins involved in the oxidative stress response in P. brasiliensis yeast cells were also up-regulated during macrophage infection, including superoxide dismutases (SOD), thioredoxins (THX) and cytochrome c peroxidase (CCP). Antisense knockdown mutants evaluated the importance of CCP during macrophage infection. The results suggested that CCP is involved in a complex system of protection against oxidative stress and that gene silencing of this component of the antioxidant system diminished the survival of P. brasiliensis in macrophages and in a murine model of infection.     

Paracoccidioides brasiliensis: Conversion of Yeastlike Forms into Mycelia in Submerged Culture

Details of the sequential morphological changes occurring during yeastlike to mycelial-form conversion of the dimorphic pathogen Paracoccidioides brasiliensis are described and illustrated by photomicrographs. Conversion of yeastlike to hyphal morphology was initiated by changing the temperature of incubation from 37 to 23 C. Production by the parent yeastlike cells of elongated buds developing into hyphae started to be conspicuous after 24 hr of incubation at 23 C. After 120 hr of incubation, growth was almost exclusively filamentous. Direct transformation of parent yeastlike cells into hyphae was not observed. Dry weight increased continuously during the conversion process in spite of the gradual disappearance of the parent yeastlike cells. Concurrent studies showed that changes in ribonucleic acid and deoxyribonucleic acid content per unit dry weight are about the same whether the yeastlike cells are undergoing conversion at 23 C or growing normally at 37 C, and that deoxyribonucleic acid synthesis is apparently required for bud formation in both cases.     

Cell Wall Glucans of the Yeast and Mycelial Forms of Paracoccidioides brasiliensis

Glucans were isolated from the cell wall of the yeast (Y) and mycelial (M) forms of Paracoccidioides brasiliensis. The alkali-soluble glucan of the Y form had properties of alpha-1,3-glucan. The alkali-insoluble glucan of the M form was identified as a beta-glucan which contains a beta-(1 --> 3)-glycosidic linkage by infrared absorption spectrum, by effect of beta-1,3-glucanase, and by partial acid hydrolysis. The alkali-soluble glucans of the M form were a mixture of alpha- and beta-glucans and the ratio of alpha- to beta-glucan was variable, depending on the preparations.     

Chemical Morphology of Glucan and Chitin in the Cell Wall of the Yeast Phase of Paracoccidioides brasiliensis

Short and thick fibers were observed on the outer surface of the yeast phase of Paracoccidioides brasiliensis, and long and thin fibers were seen on the inner surface. The long fibers disappear with chitinase treatment and are composed of chitin. The short fibers disappear under alkali treatment and are composed of alpha-glucan. Comparisons with alpha-(1 --> 3)-glucan isolated from Aspergillus niger and Polyporus betulinus and with chitin from fungal origin support our point of view.     

Global Gene Expression Analysis of Yeast Cells during Sake Brewing

During the brewing of Japanese sake, Saccharomyces cerevisiae cells produce a high concentration of ethanol compared with other ethanol fermentation methods. We analyzed the gene expression profiles of yeast cells during sake brewing using DNA microarray analysis. This analysis revealed some characteristics of yeast gene expression during sake brewing and provided a scaffold for a molecular level understanding of the sake brewing process.     

Bioinformatic Comparison of Bacterial Secretomes

The rapid increasing number of completed bacterial genomes provides a good op-portunity to compare their proteomes. This study was undertaken to specifically compare and contrast their secretomes-the fraction of the proteome with pre-dicted N-terminal signal sequences, both type Ⅰ and type Ⅱ. A total of 176 theoreti-cal bacterial proteomes were examined using the ExProt program. Compared with the Gram-positives, the Gram-negative bacteria were found, on average, to con-tain a larger number of potential Sec-dependent sequences. In the Gram-negative bacteria but not in the others, there was a positive correlation between proteome size and secretome size, while there was no correlation between secretome size and pathogenicity. Within the Gram-negative bacteria, intracellular pathogens were found to have the smallest secretomes. However, the secretomes of certain bacte-ria did not fit into the observed pattern. Specifically, the secretome of Borrelia burgdoferi has an unusually large number of putative lipoproteins, and the signal peptides of mycoplasmas show closer sequence similarity to those of the Gram-negative bacteria. Our analysis also suggests that even for a theoretical minimal genome of 300 open reading frames, a fraction of this gene pool (up to a maximum of 20%) may code for proteins with Sec-dependent signal sequences.     

面包酵母细胞出芽率与产品质量关系研究

速用压榨酵母(Compressed yeast,CY)和活性干酵母(Active dry yeast,ADY)是用于面点制作的主要两类酵母产品。发酵力则是其共同质量指标。目前,国产压榨酵母的发酵力波动很大,活性干酵母不仅发酵力低,而且保质期短。面包酵母的发酵力定义为由一定数量的酵母样品,面粉和水制成的面团在恒温下产生CO_2的能力。本文采用Burrows法测量面包酵母发酵力,面团由0.15克干酵母,20克面粉和15mol脱离子水混合而成,并以该面团在30℃、180min内产生的CO₂总量作为发酵力的表征,文献^[2]表明,面包酵母的发酵力与终细胞群体中的带芽细胞分率(Fraction of budding cells,FBC)有密切的关系,传统的概念是,为提高酵母产品质量,FBC越低愈好。经研究发现,压榨酵母的耐贮存力及活性干酵发酵力与FBC成反比,但对速用压榨酵母而言,其发酵力FBC的正相关关系甚为明显,这与传统有着根本的差异。     

Production of Yeast Cells from Methanol

Using immunochemical technique thermal denaturation of soybean 11S globulin, dissolved in different ionic strength solutions (µ=0~4.0) and heated at 100°C for 5 min, has been quantitatively studied. The curves of the percentage of antigenicity remaining were obtained as a function of salt concentration. The 11S globulin became strongly resistant to thermal denaturation with increasing both KCl and potassium phosphate. The stabilizing effect (in terms of percent antigenicity) was separated into three regions. At ionic strength below 0.7, potassium phosphate had no stabilizing effect while KCl had aslightly effect. The rise in stabilizing effect up to about 50%, near 1.0~1.5 µ, represented a second transition to a different denatured state which retains undissociated molecule. At rises up to 75~95%, near 2.5~3.5µ, a different conformational state resulted in which thermally denatured 11S globulin maintained almost intact native conformation after heating. The selection of an adequate ionic strength of protein solution has enabled preparation of thermally denatured 11S globulins which have desired-residual amounts of structured regions.     

Studies on Rapidly Frozen Suspensions of Yeast Cells by Differential Thermal Analysis and Conductometry

Few, if any, yeast cells survived rapid cooling to -196°C and subsequent slow warming. After rapid freezing, the suspensions absorbed latent heat of fusion between -15° and 0°C during warming, and the relation between the amount of heat absorbed and the concentration of cells was the same as that in equivalent KCl solutions, indicating that frozen suspensions behave thermally like frozen solutions. The amount of heat absorbed was such that more than 80 per cent of the intracellular solution had to be frozen. The conductometric behavior of frozen suspensions showed that cell solutes were still inside the cells and surrounded by an intact cell membrane at the time heat was being absorbed. Two models are consistent with these findings. The first assumes that intracellular freezing has taken place; the second that all freezable water has left the cells and frozen externally. The latter model is ruled out because rapidly cooled cells do not shrink by an amount equal to the volume of water that would have to be withdrawn to prevent internal freezing.     

电穿孔法转化完整酵母的研究

本文用酿酒酵母（Ｓａｃｃｈａｒｏｍｙｃｅｓ ｃｅｒｅｖｉｓｉａｅ）作材料,探讨了电穿孔转化完整酵母的几个条件。其中电场强度及脉冲时间是两个最重要的参数。在２ｋｖ／ｃｍ,９ｍｓ时获得１０＾４转化子／ｕｇＤＮＡ的转化率。转化率还与所采用的菌株与质粒等条件有关。此技术简便迅速。     

Characterization of the Secretomes of Two Vibrios Pathogenic to Mollusks

Vibrio tapetis causes the brown ring disease in the Japanese clam Ruditapes philippinarum while Vibrio aestuarianus is associated with massive oyster mortalities. As extracellular proteins are often associated with the virulence of pathogenic bacteria, we undertook a proteomic approach to characterize the secretomes of both vibrios. The extracellular proteins (ECPs) of both species were fractionated by SEC-FPLC and in vitro assays were performed to measure the effects of each fraction on hemocyte cellular parameters (phagocytosis and adhesion). Fractions showing a significant effect were subjected to SDS-PAGE, and proteins were identified by nano LC-MS/MS. 45 proteins were identified for V. aestuarianus and 87 for V. tapetis. Most of them belonged to outer membrane or were periplasmic, including porins or adhesins that were already described as virulence factors in other bacterial species. Others were transporter components, flagella proteins, or proteins of unknown function (14 and 15 respectively). Interestingly, for V. aestuarianus, we noted the secretion of 3 extracellular enzymes including the Vam metalloprotease and two other enzymes (one putative lipase and one protease). For V. tapetis, we identified five extracellular enymes, i.e. two different endochitinases, one protease, one lipase and an adhesin. A comparison of both secretomes also showed that only the putative extracellular lipase was common to both secretomes, underscoring the difference in pathogenicity mechanisms between these two species. Overall, these results characterize for the first time the secretomes of these two marine pathogenic vibrios and constitute a useful working basis to further analyze the contribution of specific proteins in the virulence mechanisms of these species.     

Chemical Composition of Giant Cells Induced by Tunicamycin and Normal Mycelia of Penicillium citrinum

Growth of Penicillium citrinum was reduced in the presence of tunicamycin. Under this condition, reduction of yield of cell wall was greater than that of cellular protein.

Chitin content in the cell wall was several times higher in giant cells formed from conidia in the presence of tunicamycin than in normal mycelia, while reducing sugar content, presumably reflecting glucan content, did not significantly differ. Galactosamine, which was present in normal mycelia and absent in conidia, could not be detected in giant cells. The amino acid composition of the cell wall and whole cells of giant cells differed distinctly from that of normal mycelia.

Tunicamycin did not significantly inhibit the synthesis of DNA, RNA and protein as judged by incorporation of radioactive precursors, while cell wall synthesis, as judged by incorporation of radioactive N-acetylglucosamine, glucose and alanine into acid insoluble fraction, was inhibited by more than 40% in the presence of 10 μg/ml of tunicamycin. In fungi tunicamycin probably acts primarily as an inhibitor of cell wall glycoprotein synthesis and not of chitin synthesis.

Cyclic nucleotides level also differed distinctly between giant cells and mycelia.     

Auxin-Induced Expansion Growth of Cells and Protoplasts of Yeast

Using an auxin-responsive mutant of Sacchairomyces ellipsodeus, expansion growth of cells caused by auxin was studied especially in comparison with that of protoplasts.

• 1 Indole-3-acetic acid induced detectable cell expansion growth in 3 hours in a buffered simple solution where no cell division occurred.
• 2 The auxin-induced expansion growth was inhibited by an antiauxin, trans-cinnamic acid.
• 3 Actinomycin D, chloramphenicol and cycloheximide inhibited the auxin-induced cell expansion growth.
• 4 Protoplasts did not expand in response to auxin under the condition where intact cells did.
• 5 The stability of protoplasts was not changed by the low auxin concentration (20 mg/1) which induced cell expansion.
• 6 High concentrations (100–1000 mg/1) of auxin caused protoplasts to burst even under an osmotically stable condition.

     

酵母细胞对高渗环境的适应与胞内甘油累积

甘油是包括酿酒酵母在内的许多种酵母细胞中的主要相容性溶质。为适应在高渗环境下的生存,酵母细胞将在胞内累积甘油。胞内甘油累积的增加可由甘油合成的增强,甘油利用的减弱,细胞膜通透性下降导致的胞内甘油流失的减少以及从环境中吸取更多的甘油而产生。本文综述了酵母细胞对环境渗透压变化的信号传导,高渗诱导的基因表达,环境渗透压升高时酵母细胞内甘油的累积以及甘油合成的限速步骤。     

酿酒酵母感受态细胞的低温保存及酵母菌落PCR-快速筛选鉴定

异源蛋白在酵母中的表达涉及到大量重组子的筛选鉴定.以直接煮沸2～5 min后的酵母菌落水悬浮液为模板,建立了酵母克隆的PCR快速筛选方法.介绍了PCR反应中减少引物二聚体、提高反应特异性及－80℃低温保存酿酒酵母感受态细胞等技巧.