Interaction of Cryptococcus neoformans Extracellular Vesicles with the Cell Wall

Cryptococcus neoformans produces extracellular vesicles containing a variety of cargo, including virulence factors. To become extracellular, these vesicles not only must be released from the plasma membrane but also must pass through the dense matrix of the cell wall. The greatest unknown in the area of fungal vesicles is the mechanism by which these vesicles are released to the extracellular space given the presence of the fungal cell wall. Here we used electron microscopy techniques to image the interactions of vesicles with the cell wall. Our goal was to define the ultrastructural morphology of the process to gain insights into the mechanisms involved. We describe single and multiple vesicle-leaving events, which we hypothesized were due to plasma membrane and multivesicular body vesicle origins, respectively. We further utilized melanized cells to “trap” vesicles and visualize those passing through the cell wall. Vesicle size differed depending on whether vesicles left the cytoplasm in single versus multiple release events. Furthermore, we analyzed different vesicle populations for vesicle dimensions and protein composition. Proteomic analysis tripled the number of proteins known to be associated with vesicles. Despite separation of vesicles into batches differing in size, we did not identify major differences in protein composition. In summary, our results indicate that vesicles are generated by more than one mechanism, that vesicles exit the cell by traversing the cell wall, and that vesicle populations exist as a continuum with regard to size and protein composition.     

Cell Wall Alterations Associated with the Hyperproduction of Extracellular Enzymes in Neurospora crassa

A pleiotropic mutation in Neurospora (exo-1), which confers derepression of alpha-amylase, glucoamylase, beta-fructofuranosidase, and trehalase, appears to also affect the composition of the cell wall. Segregants resulting from the backcross of exo-1 to the wild-type strain from which it derived are altered in the ratio of galactosamine to glucosamine in hydrolysates of isolated cell walls. Conidial cell walls exhibit a marked decrease in the amount of galactosamine in both exo-1 and exo-1(+) strains. Increased levels (approximately sevenfold) of amylase are found in conidia of exo-1, as compared with those of exo-1(+).     

Comparative Cell Wall Analyses of Morphological Forms Within the Genus Actinomyces

Comparative cell wall analyses were made of mycelial and smooth forms of Actinomyces bovis and A. israelii to determine the changes which occur in the cell wall composition concurrent with a change in morphology, and to evaluate cell wall analyses as a criterion for taxonomic identification within the genus Actinomyces. Cell walls of the spider forms of A. boyis had little or no aspartic acid and a high hexosamine concentration; cell walls of the smooth forms had a high aspartic acid content and low concentrations of hexosamine. Both forms had large amounts of glutamic acid, alanine, and lysine, as previously reported. A strain of Actinomyces, previously identified as A. naeslundii on the basis of morphology and aerobic growth characteristics, was found to have the basic cell wall composition of A. israelii. When transferred from the Actinomyces maintenance broth to a thioglycolate broth, the cells of this strain passed from a mycelial form through a transient filamentous morphology to become diphtheroidal with continued incubation. Concomitantly, the concentrations of glutamic acid relative to alanine decreased, and the hexosamine content increased. Variation in morphology within the species A. israelii and A. bovis could not be related to any mutual chemical change of their cell walls.     

The Effects of Extracellular Calmodulin on Cell Wall Regeneration of Protoplasts and Cell Division

The effects of anti-calmodulin (CaM) serum, CaM antagonist W7-agaroseand exogenous pure CaM on cell wall regeneration of protoplastsand cell division for Angelica dahurica and other plants werestudied. Anti-CaM serum inhibited cell wall regeneration ofprotoplasts and the first cell division in dose-dependent manner,while the same amount of preimmune serum had a much less inhibitoryeffect than anti-CaM serum. The first cell division was alsoinhibited by CaM antagonist W7-agarose. The addition of exogenouspure CaM enhanced cell wall regeneration of protoplasts andthe cell division for several species of plants, while the sameamount of bovine serum albumin had no obvious effect. CaM wasdetected in the normal culture medium by means of enzyme-linkedimmunosorbent assay. Its content increased with the culturetime. The results suggest that extracellular CaM plays an importantrole in promoting cell wall regeneration of protoplasts andcell division. The possible mechanisms by which extracellularCaM achieves its effects are discussed. (Received February 24, 1994; Accepted November 14, 1994)     

Role of Different Sponge Cell Types in Species Specific Cell Aggregation

SEVERAL mouse alloantigens described recently are expressed solely or chiefly on lymphoid cells. This indicates that the programme of selective gene action responsible for cyto-differentiation includes instructions for the synthesis of unique cell surface conformations characteristic of particular cell types^{1, 2}.     

Direct Induction of the Three Pre-implantation Blastocyst Cell Types from Fibroblasts

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Comparison of Extracellular Polysaccharide Produced by Pullularia pullulans with Polysaccharides in the Cells and Cell Wall

The purified swep hydrolase appeared homogeneous on SDS disc electrophoresis, and the molecular weights of the enzyme and the subunit were 280,000 and 70,000, respectively. The isoelectric point of this enzyme was pH 4.8.     

不同载气对细胞生长的影响

目的：研究不同类型载气对于汽提发酵的影响。方法：分别二氧化碳和氮气作为汽提载气,测定不同通气量（0.2m^3/h、0.4m3/h、0.6m^3/h）下细胞的生长。结果：相比二氧化碳,以氮气作为载气,细胞生长速度提高1.3倍左右;而且随着通气量增大,这种现象更明显。结论：氮气更适宜作为汽提发酵的载气。     

Separation of Different Cell Types by Rotating Electric Fields

A new method is proposed for separating different cell typesby application of a rotating electric field technique. Plasmamembrane electrical properties and cell size are critical parametersfor cell separation. A suitable chamber is described and thetheoretical and experimental aspects of cell movement are presented.Kalancho? daigremontiana mesophyll protoplasts traveled witha velocity of about 3–7 µm/s on the agar surfaceat a field strength of 3.5?10³ V/m. (Received April 17, 1985; Accepted September 3, 1985)     

Involvement of Cell Surface Carbohydrates in the Sexual Cell Fusion of Dictyostelium discoideum

In order to analyze the molecular mechanism of sexual cell fusion between cells of HM1 and NC4 (opposite mating type strains in Dictyostelium discoideum ), monoclonal antibodies were raised against partially-purified gp 70, a fusion-related protein of HM1 cells. The antibodies were screened for activity to inhibit cell fusion and 9 hybridoma clones were obtained. One of the fusion-blocking monoclonal antibodies, mAb1G7, was used for further analysis. It recognized nearly ten bands in an immunoblot of fusion competent HM1 cells, but no bands when HM1 membrane proteins had been deglycosylated. These results suggest the importance of carbohydrates in the cell fusion process. To confirm this possibility, effects of sugars or lectins on cell fusion were examined. Although inhibition by the sugars was incomplete, Con A, WGA, LCA, strongly inhibited cell fusion. Furthermore, tunicamycin inhibited the acquisition of fusion competence in HM1 cells, indicating the importance of N-linked glycosylation of proteins in cell fusion. All above results suggest that N-linked carbohydrates on HM1 cell surface are involved in the sexual cell fusion of D. discoideum .     

Auxin-Induced Changes in the Cell Wall Xyloglucans: Effects of Auxin on the Two Different Subtractions of Xyloglucans in the Epicotyl Cell Wall of Vigna angularis

In order to study the IAA-induced modifications of the cellwall of azuki bean (Vigna angularis Ohwi et Ohashi cv. Takara)epicotyl segments, the xyloglucans were subfractionated intotwo components, i.e., 4K-U and 24K xyloglucans, which were obtainedby extraction with 4% KOH solution containing 8 M urea and 24%KOH solution, respectively. The weight-average molecular weightsof 4K-U and 24K xyloglucans were estimated to be 40 x 10⁴ and106 x 10⁴, respectively. Complete acid hydrolysis of 4K-U and24K xyloglucans gave glucose, xylose, galactose and fucose inmole % 48.3 : 33.5 : 13.8 : 4.4 and 45.3 : 30.9 : 19.6 : 4.3,respectively. Treatment of epicotyl segments with IAA (0.1 mM) caused a decreasein the amount of 24K xyloglucans and an increase in 4K-U xyloglucans,whereas the total amount of the two xyloglucans remained constant.Furthermore, IAA treatment caused a decrease in the molecularweight of 24K xyloglucans from 106 x 10⁴ to 78 x 10⁴ withoutcausing changes in their sugar compositions. With 4K-U xyloglucans,IAA caused an increase in the mole % of xylose and a decreasein the mole % of galactose and fucose. ¹ This paper is dedicated to the late Professor Joji Ashida. (Received November 26, 1982; Accepted February 7, 1983)     

基于三类特征融合的O-糖基化位点预测

糖基化是蛋白质翻译后的主要修饰,O-糖基化的固定模式未知,高精度识别O-糖基化位点是机器学习面临的挑战性问题.以迄今最大的人O-糖基化位点Steentoft数据集为基础,本文首次提出了基于位置的卡方差表特征χ~2-pos,融合伪氨基酸序列进化信息Pse PSSM以及无方向的k间隔氨基酸对组分Undirected-CKSAAP表征序列,构建5个正负样本均衡的支持向量机分类器,经加权投票,独立测试准确率、Matthew相关系数及ROC曲线下面积,分别达到了89.62%、0.79、0.96,明显优于文献报道结果.χ~2-pos、Pse PSSM与Undirected-CKSAAP三种特征的融合在蛋白质糖基化、磷酸化等位点预测中有广泛应用前景.     

Extracellular Carbohydrates and Polysaccharides of the Alga Chlorella pyrenoidosa Chick S-39

In the growing culture of the thermophilic alga Chlorella pyrenoidosa Chick S-39, the amount of extracellular carbohydrates in the medium reached 5–17% of their content in the cells and 20–40% of the total content of extracellular organic matter. Experiments with the enrichment and synchronous algal cultures showed that the accumulation of extracellular carbohydrates and polysaccharides in the media occurred due to their release from the cells, rather than to cell lysis, and depended on cell photosynthetic activity and reproduction. Chromatographic determination of free sugars revealed the presence of saccharose, glucose, and fructose in the culture medium. Extracellular carbohydrates in C. pyrenoidosa cultures were represented mainly by water-soluble polysaccharides containing galactose, mannose, arabinose, xylose, ribose, fucose, and rhamnose.     

Binding Properties of the Neural Cell Adhesion Molecule to Different Components of the Extracellular Matrix

A soluble form of the neural cell adhesion molecule (N-CAM) was obtained from 100,000-g supernatants of crude brain membrane fractions by incubation for 2 h at 37 degrees C. The isolated N-CAM, consisting of one polypeptide chain with a molecular mass of 110 kilodaltons (N-CAM 110), was studied for its binding specificity to different components of the extracellular matrix (ECM). N-CAM 110 bound to different types of collagen (collagen types I-VI and IX). The binding efficiency was dependent on salt concentration and could be called specific according to the following criteria: (a) Binding showed substrate specificity (binding to collagens, but not to other ECM components, such as laminin or fibronectin). (b) Binding of N-CAM 110 to heat-denatured collagens was absent or substantially reduced. (c) Binding was saturable (Scatchard plot analyses were linear with KD values in the range of 9.3-2.0 X 10(-9) M, depending on the collagen type and buffer conditions). Binding of N-CAM 110 to collagens could be prevented in a concentration-dependent manner by the glycosaminoglycans heparin and chondroitin sulfate. N-CAM 110 also interacted with immobilized heparin, and this interaction could be prevented by heparin and chondroitin sulfate. Thus, in addition to its role in cell-cell adhesion, N-CAM is a binding partner for different ECM components, an observation suggesting that it also serves as a substrate adhesion molecule in vivo.