Microdistribution of sulfate-reducing bacteria in sediments of a hypertrophic lake and their response to the addition of organic matter

To clarify the ecological significance of the association of sulfate-reducing bacteria (SRB) with sediment particle size, SRB utilizing lactate (l-SRB), propionate (p-SRB) and acetate (a-SRB) were examined with different sizes of sediment particles in a hypertrophic freshwater lake using the anaerobic plate count method. The numbers ofl-SRB anda-SRB were 10⁴–10⁵ colony forming units (CFU) per ml in the 0–3 cm layer and 10²–10³ CFU ml⁻¹ in the 10–13 cm layer while the numbers ofp-SRB were one or two orders lower than those ofl-SRB anda-SRB. A sediment suspension was fractionated into four fractions (<1, 1–10, 10–94 and >94 μm). The highest proportions ofl-SRB anda-SRB were found in the 10–94 μm fraction: 66–97% forl-SRB and 53–98% fora-SRB. The highest proportion ofp-SRB was found in the >94 μm fraction (70–74%). These results indicate that most SRB were associated with sediment particles. One isolate from an acetate-utilizing enrichment culture was similar toDesulfotomaculum acetoxidans, a spore-forming sulfate-reducing bacterium. When lactate and sulfate were added to sediment samples,l-SRB anda-SRB in the <10 μm-fraction grew more rapidly than those in whole sediment for the first 2 days. This result suggests that nutrients uptake by free-living and small particle-associated (<10 μm) SRB is higher than that by SRB associated with larger particles.     

Growth factor-induced DNA synthesis in cells that overproduce protein kinase C

In previous studies (Housey et al.: Cell 52:343-354, 1988), our laboratory demonstrated that a cell line R6-PKC3 that stably overproduces high levels of the beta 1 isoform of PKC displayed several abnormalities in growth control, and these phenotypic changes were also markedly enhanced when the cells were exposed to TPA. The present studies indicate that these cells also display marked changes in their response to certain growth factors. A striking finding was that several agents when tested alone in serum-free medium, including EGF, PDGF, TPA, teleocidin, and OAG, stimulated DNA synthesis in quiescent R6-PKC3 cells but had a negligible effect in quiescent R6-C1 cells, a vector control cell line with normal levels of PKC. R6-PKC3 cells also show an exaggerated response to very low concentrations of serum, when compared to R6-C1 control cells. These studies provide direct genetic evidence that alterations in cellular levels of PKC can markedly influence the responses of cells to specific growth factors.     

Deletion of specific protein kinase C subspecies in human melanoma cells

It has been shown that tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), stimulates the proliferation of normal human melanocytes, whereas it inhibits the growth of human melanoma cell lines. The expression of protein kinase C (PKC) subspecies, the major intracellular receptors for TPA, was examined in normal melanocytes and the four melanoma cell lines HM3KO, MeWo, HMV-1, and G361. PKC was partially purified and then separated into subspecies by column chromatography on Mono Q and hydroxyapatite successively, and finally subjected to immunoblot analysis using antibodies specific for the PKC subspecies. Of the PKC subspecies examined, δ-, ϵ-, and ζ-PKC were detected in both normal melanocytes and the four melanoma cell lines. In contrast, both α-PKC and β-PKC were expressed in normal melanocytes, whereas either α-PKC or β-PKC was detected in melanoma cells. Specifically, HM3KO, MeWo, and HMV-1 cells were shown to contain α-PKC but not β-PKC, while G361 cells expressed β-PKC but not α-PKC. The growth of these melanoma cells was suppressed by TPA treatment, and the growth of the G361 cells lacking α-PKC was inhibited more efficiently than the other melanoma cell lines which lacked β-PKC. It was further shown that β-PKC was not detected in freshly isolated human primary or metastatic melanoma tissues. These results suggest that the expression of α-PKC or β-PKC may be altered during the malignant transformation of normal melanocytes and that loss of α-PKC or β-PKC may be related to the inhibitory effect of TPA on the growth of melanoma cells. © 1996 Wiley-Liss, Inc.     

Cell killing by HIV-1 protease

The human immunodeficiency virus protease (HIV-1 PR) was expressed both in the yeast Saccharomyces cerevisiae and in mammalian cells. Inducible expression of HIV-1 PR arrested yeast growth, which was followed by cell lysis. The lytic phenotype included loss of plasma membrane integrity and cell wall breakage leading to the release of cell content to the medium. Given that neither poliovirus 2A protease nor 2BC protein, both being highly toxic for S. cerevisiae, were able to produce similar effects, it seems that this lytic phenotype is specific of HIV-1 PR. Drastic alterations in membrane permeability preceded the lysis in yeast expressing HIV-1 PR. Cell killing and lysis provoked by HIV-1 PR were also observed in mammalian cells. Thus, COS7 cells expressing the protease showed increased plasma membrane permeability and underwent lysis by necrosis with no signs of apoptosis. Strikingly, the morphological alterations induced by HIV-1 PR in yeast and mammalian cells were similar in many aspects. To our knowledge, this is the first report of a viral protein with such an activity. These findings contribute to the present knowledge on HIV-1-induced cytopathogenesis.     

Mutants in the S. cerevisiae PKC1 gene display a cell cycle-specific osmotic stability defect

The PKC1 gene of Saccharomyces cerevisiae encodes a homologue of the Ca(2+)-dependent isozymes of mammalian protein kinase C (Levin, D.cE., F. O. Fields, R. Kunisawa, J. M. Bishop, and J. Thorner. 1990. Cell. 62:213-224). Cells depleted of the PKC1 gene product display a uniform phenotype, a behavior indicating a defect in the cell division cycle (cdc). These cells arrest division after DNA replication, but before mitosis. Unlike most cdc mutants, which continue to grow in the absence of cell division, PKC1-depleted cells arrest growth with small buds. We created conditional alleles of PKC1 to explore the nature of this unusual cdc defect. In contrast to PKC1-depleted cells, all of the conditional pkc1 mutants isolated were suppressed by the addition of CaCl2 to the medium, suggesting that the mutant enzymes could be activated by Ca2+. Arrest of growth and cell division in the conditional mutants was accompanied by cessation of protein synthesis, rapid loss of viability, and release of cellular material into the medium, suggesting cell lysis. This conclusion was supported by the observation that a pkc1 deletion mutant was capable of proliferation in osmotically stabilized medium, but underwent rapid cell lysis when shifted to hypo-osmotic medium. We have incorporated these observations into a model to explain the cdc-specific arrest of pkc1 mutants.     

Cells that overproduce protein kinase C are more susceptible to transformation by an activated H-ras oncogene.

We recently developed rat fibroblast cell lines that stably overproduce high levels of the beta 1 form of protein kinase C (PKC). These cells display several disorders in growth control and form small microscopic colonies in agar. In the present study we demonstrate that one of these cell lines, R6-PKC3, is extremely susceptible to transformation by an activated human bladder cancer c-H-ras oncogene (T24). Compared with control cell line R6-C1, T24-transfected R6-PKC3 cells yielded a 10-fold increase in the formation of large colonies in agar. Cell lines established from these colonies displayed a highly transformed morphology, expressed the T24-encoded p21 ras protein, continued to express high levels of PKC, and were highly tumorigenic in nude mice. These results provide genetic evidence that PKC mediates some of the effects of the c-H-ras oncogene on cell transformation. Data are also presented suggesting that optimum synergistic effects between c-H-ras and PKC require critical levels of their respective activities. These findings may be relevant to the process of multistage carcinogenesis in tissues containing cells with an activated c-H-ras oncogene.     

A structured mechanistic model of the kinetics of enzymatic lysis and disruption of yeast cells

A structured, mechanistic model has been built for the kinetics of yeast cell lysis by microbial cell lytic enzymes, based on an understanding of the two-layer yeast cell wall structure and the properties of yeast-lytic enzyme systems. The model predicts the release of protein, peptides and carbohydrates from four cell structures: the outer and inner wall layers, the cytosol and organelles or proteins present in particles; it also predicts organelle or particle lysis or solubilization and the breakdown of released proteins to peptides. Applications of the model to design and optimization of selective product release are discussed.     

Activation of cytosolic phospholipase A2 downstream of the Src-phospholipase D1 (PLD1)-protein kinase C γ (PKCγ) signaling axis is required for hypoxia-induced pathological retinal angiogenesis

In view of understanding the mechanisms of retinal neovascularization, we had reported previously that vascular endothelial growth factor (VEGF)-induced pathological retinal angiogenesis requires the activation of Src-PLD1-PKCγ signaling. In the present work, we have identified cytosolic phospholipase A(2) (cPLA(2)) as an effector molecule of Src-PLD1-PKCγ signaling in the mediation of VEGF-induced pathological retinal angiogenesis based on the following observations. VEGF induced cPLA(2) phosphorylation in a time-dependent manner in human retinal microvascular endothelial cells (HRMVECs). VEGF also induced arachidonic acid (AA) release in a dose-, time-, and cPLA(2)-dependent manner. Depletion of cPLA(2) levels inhibited VEGF-induced HRMVEC DNA synthesis, migration, and tube formation. In addition, the exogenous addition of AA rescued VEGF-induced HRMVEC DNA synthesis, migration, and tube formation from inhibition by down-regulation of cPLA(2). Inhibition of Src, PLD1, or PKCγ attenuated VEGF-induced cPLA(2) phosphorylation and AA release. Consistent with these findings, hypoxia induced cPLA(2) phosphorylation and activity in VEGF-Src-PLD1-PKCγ-dependent manner in a mouse model of oxygen-induced retinopathy. In addition, siRNA-mediated down-regulation of cPLA(2) levels in the retina abrogated hypoxia-induced retinal endothelial cell proliferation and neovascularization. These observations suggest that cPLA(2)-dependent AA release is required for VEGF-induced Src-PLD1-PKCγ-mediated pathological retinal angiogenesis.     

Yapsins are a family of aspartyl proteases required for cell wall integrity in Saccharomyces cerevisiae

The yeast cell wall is a crucial extracellular organelle that protects the cell from lysis during environmental stress and morphogenesis. Here, we demonstrate that the yapsin family of five glycosylphosphatidylinositol-linked aspartyl proteases is required for cell wall integrity in Saccharomyces cerevisiae. Yapsin null mutants show hypersensitivity to cell wall perturbation, and both the yps1Delta2Delta mutant and the quintuple yapsin mutant (5ypsDelta) undergo osmoremedial cell lysis at 37 degrees C. The cell walls of both 5ypsDelta and yps1Delta2Delta mutants have decreased amounts of 1,3- and 1,6-beta-glucan. Although there is decreased incorporation of both 1,3- and 1,6-beta-glucan in the 5ypsDelta mutant in vivo, in vitro specific activity of both 1,3- and 1,6-beta-glucan synthesis is similar to wild type, indicating that the yapsins affect processes downstream of glucan synthesis and that the yapsins may be involved in the incorporation or retention of cell wall glucan. Presumably as a response to the significant alterations in cell wall composition, the cell wall integrity mitogen-activated kinase signaling cascade (PKC1-MPK pathway) is basally active in 5ypsDelta. YPS1 expression is induced during cell wall stress and remodeling in a PKC1-MPK1-dependent manner, indicating that Yps1p is a direct, and important, output of the cell wall integrity response. The Candida albicans (SAP9) and Candida glabrata (CgYPS1) homologues of YPS1 complement the phenotypes of the yps1Delta mutant. Taken together, these data indicate that the yapsins play an important role in glucan homeostasis in S. cerevisiae and that yapsin homologues may play a similar role in the pathogenic yeasts C. albicans and C. glabrata.     

The modulation of growth by HMBA in PKC overproducing HT29 colon cancer cells.

To examine whether protein kinase C (PKC) plays a role in mediating growth inhibitory effects of hexamethylene bisacetamide (HMBA) we compared a control H29 colon cancer cell line to a derivative, HT29-PKC7, that overexpresses high levels of PKC beta 1. We found that although HMBA markedly inhibited the growth of the control cells, no inhibition was seen with the HT29-PKC7 cells. On the other hand the tumor promoter 12-0-tetradecanoyl-phorbol-13 acetate inhibited the growth of HT29-PKC7 cells, but no inhibition was seen with the control cells. Maximum inhibition of the growth of both cell lines was obtained by combined treatment with HMBA and TPA. These results may be relevant to the use of HMBA in combination with other agents in the therapy of specific cancers.     

Characterization of the yeast (1-->6)-beta-glucan biosynthetic components, Kre6p and Skn1p, and genetic interactions between the PKC1 pathway and extracellular matrix assembly

A characterization of the S. cerevisiae KRE6 and SKN1 gene products extends previous genetic studies on their role in (1-->6)-beta-glucan biosynthesis (Roemer, T., and H. Bussey. 1991. Yeast beta-glucan synthesis: KRE6 encodes a predicted type II membrane protein required for glucan synthesis in vivo and for glucan synthase activity in vitro. Proc. Natl. Acad. Sci. USA. 88:11295-11299; Roemer, T., S. Delaney, and H. Bussey. 1993. SKN1 and KRE6 define a pair of functional homologs encoding putative membrane proteins involved in beta-glucan synthesis. Mol. Cell. Biol. 13:4039-4048). KRE6 and SKN1 are predicted to encode homologous proteins that participate in assembly of the cell wall polymer (1-->6)-beta-glucan. KRE6 and SKN1 encode phosphorylated integral-membrane glycoproteins, with Kre6p likely localized within a Golgi subcompartment. Deletion of both these genes is shown to result in a dramatic disorganization of cell wall ultrastructure. Consistent with their direct role in the assembly of this polymer, both Kre6p and Skn1p possess COOH-terminal domains with significant sequence similarity to two recently identified glucan-binding proteins. Deletion of the yeast protein kinase C homolog, PKC1, leads to a lysis defect (Levin, D. E., and E. Bartlett-Heubusch. 1992. Mutants in the S. cerevisiae PKC1 gene display a cell cycle-specific osmotic stability defect. J. Cell Biol. 116:1221-1229). Kre6p when even mildly overproduced, can suppress this pkc1 lysis defect. When mutated, several KRE pathway genes and members of the PKC1-mediated MAP kinase pathway have synthetic lethal interactions as double mutants. These suppression and synthetic lethal interactions, as well as reduced beta- glucan and mannan levels in the pkc1 null wall, support a role for the PKC1 pathway functioning in cell wall assembly. PKC1 potentially participates in cell wall assembly by regulating the synthesis of cell wall components, including (1-->6)-beta-glucan.     

UFDS系统检测蛋白质SUMO化修饰研究

为了验证UFDS系统（Ubc9 fusion-directed sumoylation, UFDS）是否能够检测蛋白质SUMO(small ubiquitin-related modifier)化修饰,构建了Ubc9和PKCθ的融合表达载体,与SUMO1表达载体共转染293T细胞,通过免疫共沉淀和Western印迹检测Ubc9-PKCθ与SUMO1的相互作用。 结果表明,Ubc9-PKCθ与SUMO1相互作用,且SENP1共表达时导致Ubc9-PKCθ去SUMO化修饰;相较于野生型Ubc9-PKCθ,SUMO化修饰位点突变型Ubc9-PKCθ与SUMO1的相互作用显著减弱;而相较于野生型PKCθ,SUMO化修饰位点突变型PKCθ与SUMO1的相互作用则完全检测不到。 研究结果说明,应用UFDS系统能检测蛋白质的SUMO化修饰,但在鉴定潜在的SUMO化修饰位点时有一定的缺陷。     

A pair of functionally redundant yeast genes (PPZ1 and PPZ2) encoding type 1-related protein phosphatases function within the PKC1-mediated pathway.

The PKC1 gene of Saccharomyces cerevisiae encodes a homolog of mammalian protein kinase C that is required for yeast cell growth. Loss of PKC1 function results in cell lysis due to an inability to remodel the cell wall properly during growth. The PKC1 gene has been proposed to regulate a bifurcated pathway, on one branch of which function four putative protein kinases that catalyze a linear cascade of protein phosphorylation culminating in the activation of the mitogen-activated protein kinase homolog, Mpk1p. Here we describe two genes whose overexpression suppress both an mpk1 delta mutation and a pkc1 delta mutation. One of these genes is identical to the previously identified PPZ2 gene. The PPZ2 gene is predicted to encode a type 1-related protein phosphatase and is functionally redundant with a closely related gene, designated PPZ1. Deletion of both PPZ1 and PPZ2 resulted in a temperature-dependent cell lysis defect similar to that observed for bck1 delta, mkk1,2 delta, or mpk1 delta mutants. However, ppz1,2 delta mpk1 delta triple mutants displayed a cell lysis defect at all temperatures. The additivity of the ppz1,2 delta defect with the mpk1 delta defect, combined with the results of genetic epistasis experiments, suggested either that the PPZ1- and PPZ2-encoded protein phosphatases function on a branch of the PKC1-mediated pathway different from that defined by the protein kinases or that they play an auxiliary role in the pathway. The other suppressor gene, designated BCK2 (for bypass of C kinase), is predicted to encode a 92-kDa protein that is rich in serine and threonine residues. Genetic interactions between BCK2 and other pathway components suggested that BCK2 functions on a common pathway branch with PPZ1 and PPZ2.     

Protoplast formation and regeneration of dehydrodivanillin-degrading strains of Fusobacterium varium and Enterococcus faecium

Two strains of rumen anaerobes isolated from dehydrodivanillin-degrading cultures were identified as Fusobacterium varium and Enterococcus faecium. These organisms degraded dehydrodivanillin synergistically to 5-carboxymethylvanillin and vanillic acid. Specific conditions for protoplast formation and cell wall regeneration for both bacteria were determined, under strictly anaerobic conditions, to be as follows. The cell wall of each bacterium in yeast extract medium was loosened by adding penicillin G during early log-phase growth. The cell wall of F. varium was lysed by lysozyme (1 mg/ml) in glycerol (0.2 M)-phosphate buffer (0.05 M; pH 7.0). The addition of NaCl (0.08 M) with lysozyme was necessary for lysis of E. faecium in this solution. Almost all cells were converted to protoplasts after 2 h of incubation at 37 degrees C. Regeneration of both protoplasts was 20 to 30% on an agar-containing yeast extract medium.     

酵母菌细胞自溶突变株的研究

细胞壁是酵母菌的重要细胞器 ,参与细胞内外多方面的生理生化过程 ,如细胞絮凝、信号转导、致病性等 ,在决定细胞结构完整性方面起着重要的作用。酵母细胞壁是由 β - 1 ,3-葡聚糖、β - 1 ,6-葡聚糖、甘露聚糖蛋白及几丁质等相互交链构成的复杂的双层网状结构[1] 。细胞壁组成或结构的改变会使细胞产生对温度或低渗透压的敏感性 ,在相应的条件下发生自溶 ,使细胞内容物释放到胞外。产生上述效应的突变株称为温度敏感自溶突变株和低渗透敏感自溶突变株[2～ 4 ] 。对此类突变株的研究一方面有利于进一步阐明酵母菌细胞壁代谢及组装的调控机制…     

Protoplast formation and regeneration of dehydrodivanillin-degrading strains of Fusobacterium varium and Enterococcus faecium.

Two strains of rumen anaerobes isolated from dehydrodivanillin-degrading cultures were identified as Fusobacterium varium and Enterococcus faecium. These organisms degraded dehydrodivanillin synergistically to 5-carboxymethylvanillin and vanillic acid. Specific conditions for protoplast formation and cell wall regeneration for both bacteria were determined, under strictly anaerobic conditions, to be as follows. The cell wall of each bacterium in yeast extract medium was loosened by adding penicillin G during early log-phase growth. The cell wall of F. varium was lysed by lysozyme (1 mg/ml) in glycerol (0.2 M)-phosphate buffer (0.05 M; pH 7.0). The addition of NaCl (0.08 M) with lysozyme was necessary for lysis of E. faecium in this solution. Almost all cells were converted to protoplasts after 2 h of incubation at 37 degrees C. Regeneration of both protoplasts was 20 to 30% on an agar-containing yeast extract medium.     

Phage formation in Staphylococcus muscae cultures; further observations on the relationship between virus release and cellular lysis

1. In the synthetic medium of Fildes contaming hydrolyzed casein virus release is not correlated with observable cellular lysis under conditions of a very low multiple infection. 2. In cells with a high multiple infection, lysis occurs much sooner than in cells with a low multiple infection and virus release is correlated with cellular lysis. The experiments indicate that it is the virus particle itself which accelerates lysis under these conditions. 3. A non-dialyzable fraction has been isolated from yeast, the addition of which results in cellular lysis occurring at a sooner than usual interval and being correlated with virus release in cells having a very low multiple infection. 4. By varying the concentration of the yeast fraction, it is possible to lyse the cells at varying times under conditions of a low multiple infection.