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)     

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。     

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.     

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)     

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.     

Cellular Localization and Expression of Template-Activating Factor I in Different Cell Types

Template-activating factors I (TAF-I) α and β have been identified as chromatin remodeling factors from human HeLa cells. TAF-Iβ corresponds to the protein encoded by thesetgene, which was found in an acute undifferentiated leukemia as a fusion version with thecangene via chromosomal translocation. To determine the localization of TAF-I, we raised both polyclonal and monoclonal antibodies against TAF-I. The proteins that react to the antibodies are present not only in human cells but also in mouse, frog, insect, and yeast cells. The mouse TAF-I homologue is ubiquitous in a variety of tissue cells, including liver, kidney, spleen, lung, heart, and brain. It is of interest that the amounts of TAF-Iα and β vary among hemopoietic cells and some specific cell types do not contain TAF-Iα. The level of the TAF-I proteins does not change significantly during the cell cycle progression in either HeLa cells synchronized with an excess concentration of thymidine or NIH 3T3 cells released from the serum-depleted state. TAF-I is predominantly located in nuclei, while TAF-I that is devoid of its acidic region, the region which is essential for the TAF-I activity, shows both nuclear and cytoplasmic localization. The localization of TAF-I in conjunction with the regulation of its activity is discussed.     

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.     

The Variability of oxLDL-induced Cytotoxicity on Different Types of Cell Lines

The epidemiologic studies indicated an association of obesity with increased incidence of colorectal, breast and ovarian cancer. Further studies found a positive correlation between increased serum oxLDL and an increased risk of the three cancers. In contrast, our previous studies found a negative correlation between the serum oxLDL levels and the risk of leukemia and esophageal cancer. Identification of the variability of cytotoxicity of oxLDL-induced on different types of cell lines is important for understanding the mechanism of oxLDL involved in the tumorigenesis. In the present study, we investigated the effective impacts of oxLDL on the proliferation and apoptosis for the human umbilical vein endothelial cells (HUVEC) and two cancer cell lines (EC-9706 and K562/AO2 with multi-drug resistance). HUVEC, K562/AO2 and EC-9706 cell lines were cultured in the presence of oxLDL, and cell proliferation was tested by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, apoptosis and cell cycle by flow cytometer, mRNA expression by RT-PCR and protein expression by Western blot. OxLDL could inhibit proliferation and apoptosis of the three cell lines; however, there were significant differences of effective action on the viability and apoptosis. The dose of oxLDL-induced cytotoxicity on HUVEC was higher than that on the two tumor cells. The antibody of lectin-like oxLDL receptor-1 (LOX-1-ab) can block oxLDL-induced cytotoxicity. Cells apoptosis is mediated by reducing Bcl-2 and increasing Bax and caspase-3 mRNA and protein expression. This study showed the dose of oxLDL-induced cytotoxicity on HUVEC was higher than that on K562/AO2 and EC-9706 tumor cells. The antibody of LOX-1 receptor can block the oxLDL-induced cytotoxicity.     

基质金属蛋白酶与血管壁细胞外基质重建

细胞外基质（ECM）不仅维持血管壁的完整性,而且还为血管细胞传递增殖,迁移,分化和凋亡的调控信号,基质金属蛋白酶（MMP）及其内源性抑制剂（TIMP）通过调节ECM合成与降解之间的动态平衡,使ECM维持正常的结构与功能。在高血压,动脉粥样硬化与血管再狭窄的发生与发展过程中,MMP和TIMP的合成与分泌出现异常,由此所引起的ECM合成与降解失调使ECM迅速发生重建。     

Effectiveness of Different Frankia Cell Types as Inocula for the Actinorhizal Plant Casuarina

The soil bacterium Frankia of the Actinomycetales, capable of forming N₂-fixing symbiotic root nodules on a diverse array of actinorhizal plants, has several morphological forms when grown in pure culture. Fresh hydrated preparations of whole cells, hyphae, and spores were all infective on seedlings of Casuarina at different dilutions. Desiccated hyphae showed no infection capacity, while desiccated spores remained infective, although at a reduced level. On the basis of most-probable-number statistics, spore suspensions were 3 orders of magnitude more infective than hyphae.     

Staphylococcus aureus Mutants Lacking the LytR-CpsA-Psr Family of Enzymes Release Cell Wall Teichoic Acids into the Extracellular Medium

The LytR-CpsA-Psr (LCP) proteins are thought to transfer bactoprenol-linked biosynthetic intermediates of wall teichoic acid (WTA) to the peptidoglycan of Gram-positive bacteria. In Bacillus subtilis, mutants lacking all three LCP enzymes do not deposit WTA in the envelope, while Staphylococcus aureus Δlcp mutants display impaired growth and reduced levels of envelope phosphate. We show here that the S. aureus Δlcp mutant synthesized WTA yet released ribitol phosphate polymers into the extracellular medium. Further, Δlcp mutant staphylococci no longer restricted the deposition of LysM-type murein hydrolases to cell division sites, which was associated with defects in cell shape and increased autolysis. Mutations in S. aureus WTA synthesis genes (tagB, tarF, or tarJ2) inhibit growth, which is attributed to the depletion of bactoprenol, an essential component of peptidoglycan synthesis (lipid II). The growth defect of S. aureus tagB and tarFJ mutants was alleviated by inhibition of WTA synthesis with tunicamycin, whereas the growth defect of the Δlcp mutant was not relieved by tunicamycin treatment or by mutation of tagO, whose product catalyzes the first committed step of WTA synthesis. Further, sortase A-mediated anchoring of proteins to peptidoglycan, which also involves bactoprenol and lipid II, was not impaired in the Δlcp mutant. We propose a model whereby the S. aureus Δlcp mutant, defective in tethering WTA to the cell wall, cleaves WTA synthesis intermediates, releasing ribitol phosphate into the medium and recycling bactoprenol for peptidoglycan synthesis.