Construction of environmental DNA libraries in Escherichia coli and screening for the presence of genes conferring utilization of 4-hydroxybutyrate.
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Environmental DNA libraries from three different soil samples were constructed. The average insert size was 5 to 8 kb and the percentage of plasmids with inserts was approximately 80%. The recombinant Escherichia coli strains (approximately 930,000) were screened for 4-hydroxybutyrate utilization. Thirty-six positive E. coli clones were obtained during the initial screen, and five of them contained a recombinant plasmid (pAH1 to pAH5) which conferred a stable 4-hydroxybutyrate-positive phenotype. These E. coli clones were studied further. All five were able to grow with 4-hydroxybutyrate as sole carbon and energy source and exhibited 4-hydroxybutyrate dehydrogenase activity in crude extracts. Sequencing of pAH5 revealed a gene homologous to the gbd gene of Ralstonia eutropha, which encodes a 4-hydroxybutyrate dehydrogenase. Two other genes (orf1 and orf6) conferring utilization of 4-hydroxybutyrate were identified during subcloning and sequencing of the inserts of pAH1 and pAH3. The deduced orf1 gene product showed similarities to members of the DedA family of proteins. The sequence of the deduced orf6 gene product harbors the fingerprint pattern of enoyl-coenzyme A hydratases/isomerases. The other sequenced inserts of the plasmids recovered from the positive clones revealed no significant similarity to any other gene or gene product whose sequence is available in the National Center for Biotechnology Information databases. (+info)
Cloning and expression of succinic semialdehyde reductase from human brain. Identity with aflatoxin B1 aldehyde reductase.
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The neuromodulator gamma-hydroxybutyrate is synthesized in vivo from gamma-aminobutyrate by transamination to succinic semialdehyde and subsequent reduction of the aldehyde group. In human brain, succinic semialdehyde reductase is thought to be responsible for the conversion of succinic semialdehyde to gamma-hydroxybutyrate. In the present work, we cloned the cDNA coding for succinic semialdehyde reductase and expressed it in Escherichia coli. A data bank search indicated that the enzyme is identical with aflatoxin B1-aldehyde reductase, an enzyme implicated in the detoxification of xenobiotic carbonyl compounds. Structurally, succinic semialdehyde reductase thus belongs to the aldo-keto reductase superfamily. The recombinant protein was indistinguishable from native human brain succinic semialdehyde reductase by SDS/PAGE. In addition to succinic semialdehyde, it readily catalyzed the reduction 9,10-phenanthrene quinone, phenylglyoxal and 4-nitrobenzaldehyde, typical substrates of aflatoxin B1 aldehyde reductase. The results suggest multiple functions of succinic semialdehyde reductase/aflatoxin B1 aldehyde reductase in the biosynthesis of gamma-hydroxybutyrate and the detoxification of xenobiotic carbonyl compounds, respectively. (+info)
Development and evaluation of a new canine myocardial infarction model using a closed-chest injection of thrombogenic material.
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A new canine myocardial infarction model using thrombi induced by closed-chest injection of thrombin and autogenous blood with fibrinogen into coronary arteries was developed. Occlusive thrombi were formed in all treated animals. Occluded vessels did not spontaneously reperfuse 1 day after occlusion, but did so within 3 days. Infarction was confirmed by increased levels of creatine kinase-MB, glutamate-oxaloacetate transaminase and a-hydroxybutyrate dehydrogenase. Additionally, the left ventricular ejection fraction (LVEF) decreased within 0.5 h after occlusion and had not improved 4 weeks later. After 1 week, extensive transmural anteroinferior myocardial infarction was observed and heart mass had increased. By 4 weeks after occlusion, pulmonary capillary wedge pressure and central venous pressure were increased, and oxygen pressure was decreased. Dropout of nuclei in cardiomyocytes and increased amount of collagen fiber were observed in myocardial infarct regions of hearts excised 4 weeks after occlusion. This canine model may be useful and convenient in evaluating treatment efficacy and the long-term outcome of acute myocardial infarction. (+info)
Measurement of myocardial infarct size from plasma fatty acid-binding protein or myoglobin, using individually estimated clearance rates.
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OBJECTIVE: In patients with acute myocardial infarction (AMI), estimation of infarct size from the early markers, fatty acid-binding protein (FABP) and myoglobin (MYO), usually assumes average (fixed) rate constants (FCR) for protein clearance from plasma. However, individual variation in FCR is large. Renal dysfunction causes slower clearance of FABP and MYO from plasma and, hence, overestimation of infarct size in 20-25% of patients. We investigated whether or not more accurate values of infarct size could be obtained with individually estimated clearance rates. METHODS: Concentrations of FABP and MYO and, for comparison, activities of the established cardiac markers, creatine kinase (CK) and alpha-hydroxybutyrate dehydrogenase (HBDH), were assayed in serial plasma samples from 138 patients with AMI. Individual FCR values of FABP and MYO were estimated from plasma creatinine concentrations, sex and age. RESULTS: Individual FCR values varied from 0.4 to 2.4 h-1. Use of these individual FCR values significantly improved the correlation between infarct size, as estimated from FABP or MYO on the one hand, and from CK and HBDH on the other. Approximately equal estimates of infarct size were obtained for all four marker proteins. CONCLUSIONS: Using individually estimated clearance rates, renal insufficiency no longer hampers calculation of infarct size from FABP and MYO, and reliable estimates of total myocardial damage can be obtained within 24 h after first symptoms. (+info)
Chronic disturbances in NO production results in histochemical and subcellular alterations of the rat heart.
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The mechanisms and myocardial alterations associated with NO-deficient hypertension are still far from clear. The aim of the present study was to focus on the enzyme histochemical and subcellular changes in the heart of L-NAME treated rats, as well as to examine the influence of captopril treatment. Wistar rats were administered either L-NAME (40 mg/kg/day) alone or together with captopril (100 mg/kg/day) for a period of 4 weeks. A significant increase of blood pressure confirmed the reliability of the model. The results showed that long-lasting L-NAME administration was accompanied by a decrease of endothelial NO-synthase activity and by a significant local decrease of the following enzyme activities: capillary-related alkaline phosphatase, 5'-nucleotidase and ATPase (but not dipeptidyl peptidase IV) and cardiomyocyte-related glycogen phosphorylase, succinic dehydrogenase, beta-hydroxybutyrate dehydrogenase and ATPases. No activity of these enzymes was found in the scar, whereas a marked increase of alkaline phosphatase and dipeptidyl peptidase IV activities was found in the foci of fibrotization. Histochemical changes correlated with subcellular changes, which were characterized by 1) apparent fibroblast activation associated with interstitial/perivascular fibrosis, 2) heterogeneous population of the normal, hypertrophic and injured cardiomyocytes, 3) enhancement of the atrial granules and their translocation into the sarcolemma, and 4) impairment of capillaries as well as by induction of angiogenesis. Similar alterations were also found in the heart of captopril co-treated rats, despite of the significant suppression of blood pressure. The results indicate that NO-deficient hypertension is accompanied by metabolic disturbances and ultrastructural alterations of the heart and these changes are probably not induced by the renin-angiotension system only. (+info)
Elevated homocysteine levels are associated with increased ischemic myocardial injury in acute coronary syndromes.
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OBJECTIVES: This study was conducted to determine whether the amount of myocardial damage during acute coronary syndromes (ACS) is related to the admission plasma homocysteine concentration. BACKGROUND: Elevated homocysteine levels are associated with increased thrombosis in patients presenting with ACS. It is not known whether this association is reflected in the degree of myocardial injury in those patients. METHODS: We studied consecutive patients presenting with acute myocardial infarction (MI) (n = 205) and unstable angina pectoris (UAP) (n = 185). Plasma samples were collected on admission and prior to clinical intervention and were assayed for homocysteine by high performance liquid chromatography (HPLC). Myocardial necrosis was assessed by measurements of cardiac troponin T (cTnT) on admission and 12 h after admission (peak cTnT). The patients were studied by quintiles of homocysteine concentration. RESULTS: There was a significant increase in peak cTnT in the 5th homocysteine quintile in MI (analysis of variance [ANOVA], p = 0.005), the levels being 4.10, 3.86, 4.13, 6.20 and 7.85 microg/liter for quintiles 1 to 5, respectively (p < 0.0001, for top vs. bottom quintile). Similarly, there was a step-up in peak cTnT levels in the top homocysteine quintile in UAP (ANOVA, p < 0.0001), the levels being 0.03, 0.03, 0.02, 0.04 and 0.15 microg/liter, (p < 0.0001 for top vs. bottom quintile). In a multivariate regression model, the association between peak cTnT and the top homocysteine quintile remained strong after adjustment of other confounders including age, gender, final diagnosis and thrombolysis treatment (odds ratio [OR]: 2.92 (1.75-4.87) p < 0.0001). The patients with UAP were further examined according to peak cTnT levels below (cTnT negative) or above (cTnT positive) 0.1 microg/liter. Homocysteine levels were significantly higher in cTnT positive than cTnT negative patients; 13.8 (11.7-15.3) vs. 10.3 (9.4-11.3) micromol/liter, respectively, p = 0.002. CONCLUSIONS: Elevated homocysteine levels are associated with a higher risk of ischemic myocardial injury in patients presenting with ACS. (+info)
Expression of a glutamate decarboxylase homologue is required for normal oxidative stress tolerance in Saccharomyces cerevisiae.
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The action of gamma-aminobutyrate (GABA) as an intercellular signaling molecule has been intensively studied, but the role of this amino acid metabolite in intracellular metabolism is poorly understood. In this work, we identify a Saccharomyces cerevisiae homologue of the GABA-producing enzyme glutamate decarboxylase (GAD) that is required for normal oxidative stress tolerance. A high copy number plasmid bearing the glutamate decarboxylase gene (GAD1) increases resistance to two different oxidants, H(2)O(2) and diamide, in cells that contain an intact glutamate catabolic pathway. Structural similarity of the S. cerevisiae GAD to previously studied plant enzymes was demonstrated by the cross-reaction of the yeast enzyme to a antiserum directed against the plant GAD. The yeast GAD also bound to calmodulin as did the plant enzyme, suggesting a conservation of calcium regulation of this protein. Loss of either gene encoding the downstream steps in the conversion of glutamate to succinate reduced oxidative stress tolerance in normal cells and was epistatic to high copy number GAD1. The gene encoding succinate semialdehyde dehydrogenase (UGA5) was identified and found to be induced by H(2)O(2) exposure. Together, these data strongly suggest that increases in activity of the glutamate catabolic pathway can act to buffer redox changes in the cell. (+info)
Assessment of coronary reperfusion in patients with myocardial infarction using fatty acid binding protein concentrations in plasma.
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OBJECTIVE: To examine whether successful coronary reperfusion after thrombolytic treatment in patients with confirmed acute myocardial infarction can be diagnosed from the plasma marker fatty acid binding protein (FABP), for either acute clinical decision making or retrospective purposes. DESIGN: Retrospective substudy of the GUSTO trial. SETTING: 10 hospitals in four European countries. PATIENTS: 115 patients were treated with thrombolytic agents within six hours after the onset of acute myocardial infarction. Patency of the infarct related artery was determined by angiography within 120 minutes of the start of thrombolysis. MAIN OUTCOME MEASURES: First hour rate of increase in plasma FABP concentration after thrombolytic treatment, compared with increase in plasma myoglobin concentration and creatine kinase isoenzyme MB (CK-MB) activity. Infarct size was estimated from the cumulative release of the enzyme alpha hydroxybutyrate dehydrogenase in plasma during 72 hours, or from the sum of ST segment elevations on admission. Logistic regression analyses were performed to construct predictive models for patency. RESULTS: Complete reperfusion (TIMI 3) occurred in 50 patients, partial reperfusion (TIMI 2) in 36, and no reperfusion (TIMI 0+1) in 29. Receiver operating characteristic (ROC) curve analyses showed that the best performance of FABP was obtained when TIMI scores 2 and 3 were grouped and compared with TIMI score 0+1. The performance of FABP as a reperfusion marker was improved by combining it with alpha hydroxybutyrate dehydrogenase infarct size, but not with an early surrogate of infarct size (ST segment elevation on admission). In combination with infarct size FABP performed as well as myoglobin (areas under the ROC curve 0.868 and 0.857, respectively) and better than CK-MB (area = 0.796). At optimum cut off levels, positive predictive values were 97% for FABP, 95% for myoglobin, and 89% for CK-MB (without infarct size, 87%, 88%, and 87%, respectively), and negative predictive values were 55%, 52%, and 50%, respectively (without infarct size, 44%, 42%, and 34%). CONCLUSIONS: FABP and myoglobin perform equally well as reperfusion markers, and successful reperfusion can be assessed, with positive predictive values of 87% and 88%, or even 97% and 95% when infarct size is also taken into account. However, identification of non-reperfused patients remains a problem, as negative predictive values will generally remain below 70%. (+info)