Two putative MAP kinase genes, ZrHOG1 and ZrHOG2, cloned from the salt-tolerant yeast Zygosaccharomyces rouxii are functionally homologous to the Saccharomyces cerevisiae HOG1 gene.
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The salt-tolerant yeast Zygosaccharomyces rouxii can adjust its osmotic balance when responding to osmotic shock by accumulating glycerol as the compatible osmolyte. However, the mechanism of glycerol production in Z. rouxii cells and its genetic regulation remain to be elucidated. Two putative mitogen-activated protein (MAP) kinase genes, ZrHOG1 and ZrHOG2, were cloned from Z. rouxii by their homology with HOG1 from Saccharomyces cerevisiae. The deduced amino acid sequences of ZrHog1p and ZrHog2p indicated close homology to that of Hog1p and contained a TGY motif for phosphorylation by MAP kinase kinase. When ZrHOG1 or ZrHOG2 was expressed in an S. cerevisiae hog1delta null mutant, the salt tolerance and osmotic tolerance characteristics of wild-type S. cerevisiae were restored. In addition, the aberrant cell morphology and low glycerol content of the hog1delta null mutant were corrected, indicating that ZrHog1p and ZrHog2p have functions similar to Hog1p. While the transcription of the glycerol-3-phosphate dehydrogenase gene (GPD1) of the ZrHOG1-harbouring S. cerevisiae mutant was similar to that of wild-type S. cerevisiae, the ZrHOG2-harbouring strain showed prolonged GPD1 transcription. Both Zrhog1delta and Zrhog2delta Z. rouxii null mutants showed a decrease in salt tolerance compared to the wild-type strain. The present study suggested the presence of a high-osmolarity glycerol response (HOG) pathway in Z. rouxii similar to that elucidated in S. cerevisiae. Two putative MAP kinase genes in Z. rouxii appeared to be significant in either osmotic regulation or ion homeostasis. (+info)
Salt-sensitivity classification in normotensive adults.
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The objectives of this study were to assess the reliability, sensitivity and specificity of salt-sensitivity classification in normotensive adults and to determine the predictive power of four clinical indices for salt-sensitivity. A total of 66 healthy, normotensive, free-living adults were administered 11-day salt-sensitivity diagnostic dietary salt challenges on two occasions to permit assessment of classification test-retest reliability. An oral glucose tolerance test, an acute saline loading test, gustatory testing and determination of salivary flow and sodium concentration were carried out to assess (by correlation analysis) their predictive power for salt-sensitivity. Following these procedures, 21 participants followed a reduced-sodium diet for 4 months, during which blood pressure was monitored monthly to allow evaluation of salt-sensitivity classification sensitivity and specificity. Regression was used to develop a predictive model for salt-sensitivity. Salt-sensitivity classification was not highly reliable (kappa-value=0.38), sensitive (0.73) or specific (0.60). No single index was highly predictive of classification status, but a model composed of five indices accounted for 92% of the variance in blood pressure response to acute salt challenge. The dietary salt challenge procedure used here for salt-sensitivity classification of normotensive adults had low test-retest reliability. While a battery of easily measured attributes may facilitate rapid salt-sensitivity classification, such a diagnosis provides only limited insight regarding blood pressure responsiveness to chronic dietary salt restriction in normotensive adults. (+info)
Effects of osmotic stress on Methanococcus thermolithotrophicus: 13C-edited 1H-NMR studies of osmolyte turnover.
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In vivo NMR studies of the thermophilic archaeon Methanococcus thermolithotrophicus, with sodium formate as the substrate for methanogenesis, were used to monitor formate utilization, methane production, and osmolyte pool synthesis and turnover under different conditions. The rate of formate conversion to CO2 and H2 decreased for cells adapted to higher external NaCl, consistent with the slower doubling times for cells adapted to high external NaCl. However, when cells grown at one NaCl concentration were resuspended at a different NaCl, formate utilization rates increased. Production of methane from 13C pools varied little with external NaCl in nonstressed culture, but showed larger changes when cells were osmotically shocked. In the absence of osmotic stress, all three solutes used for osmotic balance in these cells, l-alpha-glutamate, beta-glutamate, and Nepsilon-acetyl-beta-lysine, had 13C turnover rates that increased with external NaCl concentration. Upon hyperosmotic stress, there was a net synthesis of alpha-glutamate (over a 30-min time-scale) with smaller amounts of beta-glutamate and little if any of the zwitterion Nepsilon-acetyl-beta-lysine. This is a marked contrast to adapted growth in high NaCl where Nepsilon-acetyl-beta-lysine is the dominant osmolyte. Hypoosmotic shock selectively enhanced beta-glutamate and Nepsilon-acetyl-beta-lysine turnover. These results are discussed in terms of the osmoadaptation strategies of M. thermolithotrophicus. (+info)
Different signalling pathways contribute to the control of GPD1 gene expression by osmotic stress in Saccharomyces cerevisiae.
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Yeast cells respond to a shift to higher osmolarity by increasing the cellular content of the osmolyte glycerol. This response is accompanied by a stimulation of the expression of genes encoding enzymes in the glycerol production pathway. In this study the osmotic induction of one of those genes, GPD1, which encodes glycerol-3-phosphate dehydrogenase, was monitored in time course experiments. The response is independent of the osmolyte and consists of four apparent phases: a lag phase, an initial induction phase, a feedback phase and a sustained long-term induction. Osmotic shock with progressively higher osmolyte concentrations caused a prolonged lag phase. Deletion of HOG1, which encodes the terminal protein kinase of the high osmolarity glycerol (HOG) response pathway, led to an even longer lag phase and drastically lower basal and induced GPD1 mRNA levels. However, the induction was only moderately diminished. Overstimulation of Hog1p by deletion of the genes for the protein phosphatases PTP2 and PTP3 led to higher basal and induced mRNA levels and a shorter lag phase. The protein phosphatase calcineurin, which mediates salt-induced expression of some genes, does not appear to contribute to the control of GPD1 expression. Although GPD1 expression has so far not been reported to be controlled by a general stress response mechanism, heat-shock induction of the GPD1 mRNA level was observed. However, unregulated protein kinase A activity, which strongly affects the general stress response, only marginally altered the mRNA level of GPD1. The osmotic stimulation of GPD1 expression does not seem to be mediated by derepression, since deletion of the SSN6 gene, which encodes a general repressor, did not significantly alter the induction profile. A hypoosmotic shock led to a transient 10-fold drop of the GPD1 mRNA level. Neither the HOG nor the protein kinase C pathway, which is stimulated by a decrease in external osmolarity, is involved in this effect. It was concluded that osmotic regulation of GPD1 expression is the result of an interplay between different signalling pathways, some of which remain to be identified. (+info)
In vivo transcription of the Escherichia coli oxyR regulon as a function of growth phase and in response to oxidative stress.
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Simultaneous expression of seven genes in Escherichia coli was measured by a reverse transcription-multiplex PCR fluorescence procedure. Genes studied were (i) oxyR (transcriptional regulator); (ii) katG, dps, gorA, and ahpCF (controlled by OxyR); (iii) sodA (controlled by SoxRS); and (iv) trxA (not related to OxyR or SoxRS). Except for trxA, transcription of all genes was activated during the course of growth of wild-type bacteria, though notable variations were observed with respect to both the time and extent of activation. Whereas oxyR, katG, dps, and gorA were activated during exponential growth, ahpCF and sodA were stimulated in stationary phase. Maximal induction ranged from 4.6- to 86.5-fold, for gorA and dps, respectively. Treatment with H2O2 stimulated expression of the genes (katG, dps, ahpCF, and gorA) previously identified as members of the OxyR regulon, except for oxyR itself. Induction by H2O2 was a remarkably rapid and reversible process that took place in an OxyR-dependent and sigmaS-independent manner. NaCl induced expression of the genes controlled by OxyR, including the oxyR locus. This transcriptional up-regulation was preserved in a strain with the DeltaoxyR::kan mutation, but it was abolished (ahpCF) or significantly reduced (oxyR and dps) in a strain with the rpoS::Tn10 mutation, potentially reflecting positive transcriptional regulation of the oxyR regulon by sigmaS. Expression of trxA was not increased either by H2O2 stress or by a shift to high-osmolarity conditions. (+info)
Rapid conditions for the cleavage of oligodeoxyribonucleotides from cis-diol-bearing universal polymer supports and their deprotection.
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Two sets of deprotection conditions have been evolved for the deprotection of oligodeoxyribonucleotides and their cleavage from commercially available cis -diol group-bearing universal polymer supports. In the first case, oligodeoxyribonucleotides anchored on the universal support were subjected to one of the standard deprotection conditions followed by treatment with aqueous 0.5 M sodium chloride + 0.2 M sodium hydroxide solution for 30 min at room temperature. In the second case, oligonucleotides bound to the universal support were treated with methanolic sodium hydroxide solution under microwave radiation to obtain fully deprotected oligomers within 4 min. Under both conditions, the cleavage of oligonucleotides from the support and their deprotection occurred quantitatively without any side product formation. The cleaved oligonucleotides were found to be identical in all respects (retention time on HPLC and biological activity in PCR) to the corresponding standard oligo-nucleotides. (+info)
A reexamination of the angiotoxicity of superselective injection of DMSO in the swine rete embolization model.
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BACKGROUND AND PURPOSE: There are a variety of embolization applications for non-adhesive, liquid agents. We reevaluated the potential microvascular angiotoxicity of superselective infusions of dimethyl sulfoxide (DMSO) using very long infusion rates in a previously described animal model. METHODS: Twenty-six swine underwent percutaneous femoral puncture for superselective catheterization of the artery of the rete while being continuously monitored for ECG and intraarterial pressure. Two volumes (0.5 or 0.8 mL) and three durations (30, 60, and 90 seconds) of superselective infusion of DMSO were used to evaluate the effect of a single-dose rate within an ipsilateral rete. Contralateral control infusions of normal saline were also administered. Acute hemodynamic and angiographic outcomes were assessed. After recovery, follow-up angiography and sacrifice were performed at either 10 or 28 days. Brains and retia were harvested for gross and microscopic histopathologic evaluation. RESULTS: No significant hemodynamic alterations occurred acutely. Twenty-three of the 24 infused retia showed variable acute vasospasm that typically was mild to moderate in severity and transient (10 to 20 minutes). Follow-up angiography at sacrifice always showed normal retial arterial anatomy. No adverse clinical sequelae were noted. Gross inspection of brains showed no evidence of infarction or subarachnoid hemorrhage. Microscopic histopathologic examination of retia showed mostly nonspecific changes in both exposed and control samples. Possible causal histotoxicity was seen in four retia (three of four exposed to higher dose rates), in which involvement was limited to one to three retial arteries. CONCLUSION: Lower total dose and dose rates of superselective infusion of DMSO into the retial microarterial network resulted in substantially less angiotoxicity than that found in a previous study, as defined by clinical, angiographic, gross, and histopathologic criteria. (+info)
Identification and disruption of BetL, a secondary glycine betaine transport system linked to the salt tolerance of Listeria monocytogenes LO28.
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The trimethylammonium compound glycine betaine (N,N, N-trimethylglycine) can be accumulated to high intracellular concentrations, conferring enhanced osmo- and cryotolerance upon Listeria monocytogenes. We report the identification of betL, a gene encoding a glycine betaine uptake system in L. monocytogenes, isolated by functional complementation of the betaine uptake mutant Escherichia coli MKH13. The betL gene is preceded by a consensus sigmaB-dependent promoter and is predicted to encode a 55-kDa protein (507 amino acid residues) with 12 transmembrane regions. BetL exhibits significant sequence homologies to other glycine betaine transporters, including OpuD from Bacillus subtilis (57% identity) and BetP from Corynebacterium glutamicum (41% identity). These high-affinity secondary transporters form a subset of the trimethylammonium transporter family specific for glycine betaine, whose substrates possess a fully methylated quaternary ammonium group. The observed Km value of 7.9 microM for glycine betaine uptake after heterologous expression of betL in E. coli MKH13 is consistent with values obtained for L. monocytogenes in other studies. In addition, a betL knockout mutant which is significantly affected in its ability to accumulate glycine betaine in the presence or absence of NaCl has been constructed in L. monocytogenes. This mutant is also unable to withstand concentrations of salt as high as can the BetL+ parent, signifying the role of the transporter in Listeria osmotolerance. (+info)