Characterization Of The Specific Pyruvate Transport System Of Escherichia Coli K12

A mutant of Escherichia coli K12 lacking pyruvate dehydrogenase and phosphoenolpyruvate synthase was used to study transport of pyruvate by whole cells. Uptake of pyruvate is maximal with mid-log phase cells and the Michaelis constant for transport is 20{dollar}\mu{dollar}M. Pretreatment of the cells with respiratory chain poisons or uncouplers, with the exception of arsenate, inhibits transport of pyruvate by up to 95%. Lactate and alanine, natural analogs of pyruvate, competitively inhibit transport only at very high concentrations. The synthetic analogues 3-bromopyruvate and pyruvic acid methyl ester are good competitive inhibitors.;In order to further minimize metabolism of pyruvate, membrane vesicles of a wild type E. coli K12 were prepared. Transport is dependent on an artificial electron donor system, phenazine methosulfate and sodium ascorbate, added to the vesicles. Pyruvate is concentrated 7-15 times in these energized vesicles and the Michaelis constant is 15{dollar}\mu{dollar}M. Uptake of pyruvate can also be energized by a phenazine methosulfate and NADH system, but not by the metabolic intermediates lactate, glucose or ATP. Energy poisons, with the exception of arsenate, inhibit the transport of pyruvate. Synthetic analogues such as 3-bromopyruvate are good competitive inhibitors of transport. Lactate initially appeared to be a good competitive inhibitor of pyruvate transport in vesicles, but under conditions in which the oxidation of lactate to pyruvate is minimized, this apparent inhibition disappears.;Transport of pyruvate in whole cells was also found to be sensitive to osmotic shock, indicating that a periplasmic binding protein was involved in the transport system. Column chromatography techniques were employed in an attempt to isolate this binding protein from osmotic shock fluid. Partial purification of pyruvate binding activity was achieved through the use of an affinity column, but purification to homogeneity was not accomplished with subsequent column steps. The binding protein has been tentatively identified as a 36,000 molecular weight moiety by SDS-polyacrylamide gel electrophoresis.;A brief summary of these results indicates that pyruvate is transported in Escherichia coli by a unique and specific active transport system. This transport system utilizes the respiratory chain to provide the driving force for uptake and appears to have a periplasmic component capable of binding pyruvate

Health-status outcomes with invasive or conservative care in coronary disease

BACKGROUND In the ISCHEMIA trial, an invasive strategy with angiographic assessment and revascularization did not reduce clinical events among patients with stable ischemic heart disease and moderate or severe ischemia. A secondary objective of the trial was to assess angina-related health status among these patients. METHODS We assessed angina-related symptoms, function, and quality of life with the Seattle Angina Questionnaire (SAQ) at randomization, at months 1.5, 3, and 6, and every 6 months thereafter in participants who had been randomly assigned to an invasive treatment strategy (2295 participants) or a conservative strategy (2322). Mixed-effects cumulative probability models within a Bayesian framework were used to estimate differences between the treatment groups. The primary outcome of this health-status analysis was the SAQ summary score (scores range from 0 to 100, with higher scores indicating better health status). All analyses were performed in the overall population and according to baseline angina frequency. RESULTS At baseline, 35% of patients reported having no angina in the previous month. SAQ summary scores increased in both treatment groups, with increases at 3, 12, and 36 months that were 4.1 points (95% credible interval, 3.2 to 5.0), 4.2 points (95% credible interval, 3.3 to 5.1), and 2.9 points (95% credible interval, 2.2 to 3.7) higher with the invasive strategy than with the conservative strategy. Differences were larger among participants who had more frequent angina at baseline (8.5 vs. 0.1 points at 3 months and 5.3 vs. 1.2 points at 36 months among participants with daily or weekly angina as compared with no angina). CONCLUSIONS In the overall trial population with moderate or severe ischemia, which included 35% of participants without angina at baseline, patients randomly assigned to the invasive strategy had greater improvement in angina-related health status than those assigned to the conservative strategy. The modest mean differences favoring the invasive strategy in the overall group reflected minimal differences among asymptomatic patients and larger differences among patients who had had angina at baseline

Initial invasive or conservative strategy for stable coronary disease

BACKGROUND Among patients with stable coronary disease and moderate or severe ischemia, whether clinical outcomes are better in those who receive an invasive intervention plus medical therapy than in those who receive medical therapy alone is uncertain. METHODS We randomly assigned 5179 patients with moderate or severe ischemia to an initial invasive strategy (angiography and revascularization when feasible) and medical therapy or to an initial conservative strategy of medical therapy alone and angiography if medical therapy failed. The primary outcome was a composite of death from cardiovascular causes, myocardial infarction, or hospitalization for unstable angina, heart failure, or resuscitated cardiac arrest. A key secondary outcome was death from cardiovascular causes or myocardial infarction. RESULTS Over a median of 3.2 years, 318 primary outcome events occurred in the invasive-strategy group and 352 occurred in the conservative-strategy group. At 6 months, the cumulative event rate was 5.3% in the invasive-strategy group and 3.4% in the conservative-strategy group (difference, 1.9 percentage points; 95% confidence interval [CI], 0.8 to 3.0); at 5 years, the cumulative event rate was 16.4% and 18.2%, respectively (difference, 121.8 percentage points; 95% CI, 124.7 to 1.0). Results were similar with respect to the key secondary outcome. The incidence of the primary outcome was sensitive to the definition of myocardial infarction; a secondary analysis yielded more procedural myocardial infarctions of uncertain clinical importance. There were 145 deaths in the invasive-strategy group and 144 deaths in the conservative-strategy group (hazard ratio, 1.05; 95% CI, 0.83 to 1.32). CONCLUSIONS Among patients with stable coronary disease and moderate or severe ischemia, we did not find evidence that an initial invasive strategy, as compared with an initial conservative strategy, reduced the risk of ischemic cardiovascular events or death from any cause over a median of 3.2 years. The trial findings were sensitive to the definition of myocardial infarction that was used