Down-regulation of the expression of PKC1 and SRB1/PSA1/VIG9, two genes involved in cell wall integrity in Saccharomyces cerevisiae, causes flocculation.
(1/1254)
The cell wall integrity determinants PKC1 and SRB1/PSA1/VIG9 of Saccharomyces cerevisiae were expressed under the control of the tightly regulated promoter pMET3. Substitution of the cell-cycle-regulated SRB1/PSA1 native promoter with pMET3 led to faster cell growth, larger cell volumes, and a twofold reduction of the steady-state SRB1/PSA1 mRNA level. In addition, the new pattern of expression of SRB1/PSA1 resulted in a dominant flocculation phenotype at all phases of batch growth. By contrast, expression of PKC1 from pMET3 increased the flocculation capacity of cells only at stationary phase. Methionine-mediated repression of either PSA1/SRB1 or PKC1 resulted in enhanced cell clumping. Cells in which both these genes had been replaced with their respective pMET3-regulated cassettes were highly flocculent under both expression and repression conditions. These results suggest that greater exposure of flocculin on the cell surface, caused by either cell wall distortion (through depletion of Pkc1p) or aberrant regulation of mannosylation (through constitutive production of Srb1p), results in an increased flocculation ability. (+info)
Pyrrolidine dithiocarbamate prevents I-kappaB degradation and reduces microvascular injury induced by lipopolysaccharide in multiple organs.
(2/1254)
Lipopolysaccharide (LPS) is a key mediator of multiple organ injury observed in septic shock. The mechanisms responsible for LPS-induced multiple organ injury remain obscure. In the present study, we tested the hypothesis that the LPS-induced injury occurs through activation of the transcription factor, nuclear factor-kappaB (NF-kappaB). We examined the effects of inhibiting NF-kappaB activation in vivo in the rat on LPS-induced: 1) gene and protein expression of the cytokine-inducible neutrophil chemoattractant (CINC) and intercellular adhesion molecule-1 (ICAM-1); b) neutrophil influx into lungs, heart, and liver; and c) increase in microvascular permeability induced by LPS in these organs. LPS (8 mg/kg, i.v.) challenge of rats activated NF-kappaB and induced CINC and ICAM-1 mRNA and protein expression. Pretreatment of rats with pyrrolidine dithiocarbamate (50, 100, and 200 mg/kg, i.p.), an inhibitor of NF-kappaB activation, prevented LPS-induced I-kappaBalpha degradation and the resultant NF-kappaB activation and inhibited, in a dose-related manner, the LPS-induced CINC and ICAM-1 mRNA and protein expression. Pyrrolidine dithiocarbamate also markedly reduced the LPS-induced tissue myeloperoxidase activity (an indicator of tissue neutrophil retention) and the LPS-induced increase in microvascular permeability in these organs. These results demonstrate that NF-kappaB activation is an important in vivo mechanism mediating LPS-induced CINC and ICAM-1 expression, as well as neutrophil recruitment, and the subsequent organ injury. Thus, inhibition of NF-kappaB activation may be an important strategy for the treatment of sepsis-induced multiple organ injury. (+info)
Arginine biosynthesis in Neisseria gonorrhoeae: enzymes catalyzing the formation of ornithine and citrulline.
(3/1254)
Many of the Neisseria gonorrhoeae strains isolated from patients require arginine for growth in a defined medium. As a basis for genetic studies of these Arg- strains, we examined two biosynthetic enzymes of Arg+ (nonrequiring) gonococci. Cell-free extracts contained (i) glutamate acetyltransferase, which catalyzes the formation of L-ornithine from alpha-N-acetyl-L-ornithine, and (ii) ornithine transcaramylase, which catalyzes the reaction between L-ornithine and carbamyl phosphate, yielding L-citrulline. Arg- strains were unable to utilze alpha-N-acetyl-L-ornithine for growth lacked significant activity of glutamate acetyltransferase, and activity was gained by Arg+ clones derived by DNA-mediated transformation. Some of the Arg- patient isolates were unable to use either alpha-N-acetyl-L-ornithine or L-ornithine in place of arginine, and two separate steps of genetic transformation were required to yield Arg+ cells. Extracts of these doubly auxotrophic cells lacked glutamate acetyltransferase activity, but, unexpectedly, they displayed normal ornithine transcarbamylase activity. This finding illustrates the importance of identifying the products specified by arg loci during genetic studies of arginine auxotrophy. (+info)
Cyclic AMP can decrease expression of genes subject to catabolite repression in Saccharomyces cerevisiae.
(4/1254)
External cyclic AMP (cAMP) hindered the derepression of gluconeogenic enzymes in a pde2 mutant of Saccharomyces cerevisiae, but it did not prevent invertase derepression. cAMP reduced nearly 20-fold the transcription driven by upstream activation sequence (UAS1FBP1) from FBP1, encoding fructose-1,6-bisphosphatase; it decreased 2-fold the activation of transcription by UAS2FBP1. Nuclear extracts from cells derepressed in the presence of cAMP were impaired in the formation of specific UASFBP1-protein complexes in band shift experiments. cAMP does not appear to act through the repressing protein Mig1. Control of FBP1 transcription through cAMP is redundant with other regulatory mechanisms. (+info)
Effects of short chain alkanols on the inducible nitric oxide synthase in a glial cell line.
(5/1254)
1. Ethanol inhibits inducible nitric oxide synthase (iNOS) expression in C6 glioma cells by an unknown mechanism. Because relatively high concentrations are needed for inhibition in drug-naive cells (IC50 approximately = to 150 mM), suppression due to cytotoxicity is one possible mechanism that has not been ruled out. Therefore, the present study examined the effects of ethanol and other alkanols on C6 glioma cell viability and iNOS activity to better understand the mechanism for inhibition. 2. iNOS expression was induced in cell culture with lipopolysaccharide and phorbol ester treatment. Nitrite accumulation in culture medium, the in vitro conversion of [3H]-L-arginine to [3H]-L-citrulline, and immunoblotting were used to quantify iNOS induction and activity. Trypan blue exclusion, extracellular release of lactate dehydrogenase, and quantity of total cell protein were used as measures of viability. 3. Short chain alkanols, methanol through 1-heptanol, concentration-dependently inhibited nitrite accumulation. Longer chain alkanols, 1-octanol and 1-decanol, did not except at cytotoxic concentrations. Experiments indicated short chain alkanol inhibition was not due to direct actions on iNOS catalytic activity, but that it transpires during iNOS induction. Immunoblots showed reduced iNOS protein levels. 4. Correlation analysis ruled out iNOS inhibition as being due to decreased cell number, total cell protein, or cell viability. In contrast, there was significant correlation with physical measures of lipophilicity. 5. In conclusion, inhibition of iNOS expression by ethanol and other short chain alkanols is not due to cytotoxicity. Instead, the strong correlation with lipophilicity suggests the inhibition derives from an interaction with unknown hydrophobic cellular sites. (+info)
Role for the leucine-responsive regulatory protein (Lrp) as a structural protein in regulating the Escherichia coli gcvTHP operon.
(6/1254)
The Escherichia coli glycine-cleavage enzyme system (gcvTHP and lpd gene products) provides C1 units for cellular methylation reactions. Both the GcvA and leucine-responsive regulatory (Lrp) proteins are required for regulation of the gcv operon. One model proposed for gcv regulation is that Lrp plays a structural role, bending the DNA to allow GcvA to function as either an activator or a repressor in response to environmental signals. This hypothesis was tested by replacing all but the upstream 22 bp of the Lrp-binding region in a gcvT::lacZ fusion with the I1A site from phage lambda. Integration host factor (IHF) binds the I1A site and bends the DNA about 140 degrees. Shifting the I1A site by increments of 1 base around the DNA helix resulted in IHF-dependent activation and repression of gcvT::lacZ expression that were face-of-the-helix dependent. Activation was also dependent on the GcvA protein, and repression was dependent on both the GcvA and GcvR proteins, demonstrating that the roles for these proteins were not altered. The results are consistent with Lrp playing primarily a structural role in gcv regulation, although they do not completely rule out the possibility that Lrp also interacts with another gcv-regulatory protein or with RNA polymerase. (+info)
Novel alleles of yeast hexokinase PII with distinct effects on catalytic activity and catabolite repression of SUC2.
(7/1254)
In the yeast Saccharomyces cerevisiae, glucose or fructose represses the expression of a large number of genes. The phosphorylation of glucose or fructose is catalysed by hexokinase PI (Hxk1), hexokinase PII (Hxk2) and a specific glucokinase (Glk1). The authors have shown previously that either Hxk1 or Hxk2 is sufficient for a rapid, sugar-induced disappearance of catabolite-repressible mRNAs (short-term catabolite repression). Hxk2 is specifically required and sufficient for long-term glucose repression and either Hxk1 or Hxk2 is sufficient for long-term repression by fructose. Mutants lacking the TPS1 gene, which encodes trehalose 6-phosphate synthase, can not grow on glucose or fructose. In this study, suppressor mutations of the growth defect of a tps1delta hxk1delta double mutant on fructose were isolated and identified as novel HXK2 alleles. All six alleles studied have single amino acid substitutions. The mutations affected glucose and fructose phosphorylation to a different extent, indicating that Hxk2 binds glucose and fructose via distinct mechanisms. The mutations conferred different effects on long- and short-term repression. Two of the mutants showed very similar defects in catabolite repression, despite large differences in residual sugar-phosphorylation activity. The data show that the long- and short-term phases of catabolite repression can be dissected using different hexokinase mutations. The lack of correlation between in vitro catalytic hexokinase activity, in vivo sugar phosphate accumulation and the establishment of catabolite repression suggests that the production of sugar phosphate is not the sole role of hexokinase in repression. Using the set of six hxk2 mutants it was shown that there is a good correlation between the glucose-induced cAMP signal and in vivo hexokinase activity. There was no correlation between the cAMP signal and the short- or long-term repression of SUC2, arguing against an involvement of cAMP in either stage of catabolite repression. (+info)
Regulation of expression of the fructan hydrolase gene of Streptococcus mutans GS-5 by induction and carbon catabolite repression.
(8/1254)
The polymers of fructose, levan and inulin, as well as sucrose and raffinose, are substrates for the product of the fruA gene of Streptococcus mutans GS-5. The purpose of this study was to characterize the DNA immediately flanking fruA, to explore the regulation of expression of fruA by the carbohydrate source, and to begin to elucidate the molecular basis for differential expression of the gene. Located 3' to fruA was an open reading frame (ORF) with similarity to beta-fructosidases which was cotranscribed with fruA. A transcriptional initiation site, located an appropriate distance from an extended -10-like promoter, was mapped at 165 bp 5' to the fruA structural gene. By the use of computer algorithms, two overlapping, stable stem-loop sequences with the potential to function as rho-independent terminators were found in the 5' untranslated region. Catabolite response elements (CREs), which have been shown to govern carbon catabolite repression (CCR) by functioning as negative cis elements in gram-positive bacteria, were located close to the promoter. The levels of production of fruA mRNA and FruA were elevated in cells growing on levan, inulin, or sucrose as the sole carbohydrate source, and repression was observed when cells were grown on readily metabolizable hexoses. Deletion derivatives containing fusions of fruA promoter regions, lacking sequences 5' or 3' to the promoter, and a promoterless chloramphenicol acetyltransferase gene were used (i) to demonstrate the functionality of the promoter mapped by primer extension, (ii) to demonstrate that CCR of the fru operon requires the CRE that is located 3' to the promoter region, and (iii) to provide preliminary evidence that supports the involvement of an antitermination mechanism in fruA induction. (+info)