ArgO145, a Stx2a prophage of a bovine O145:H- STEC strain, is closely related to phages of virulent human strains.

Shiga toxins (Stx) are the main virulence factor of a pathogroup of Escherichia coli strains that cause severe human diseases. These toxins are encoded in prophages (Stx prophages), and generally their expression depends on prophage induction. Several studies have reported high diversity among both Stx prophages and Stx. In particular, the toxin subtype Stx2a is associated with high virulence and HUS. Here, we report the genome of ArgO145, an inducible Stx2a prophage identified in a bovine O145:H- strain which produced high levels of Shiga toxin and Stx phage particles. The ArgO145 genome shared lambda phage organization, with recombination, regulation, replication, lysis, and head and tail structural gene regions, although some lambda genes encoding regulatory proteins could not be identified. Remarkably, some Stx2a phages of strains isolated from patients in other countries showed high similarity to ArgO145.

Glycolytic pathway, redox state of NAD(P)-couples and energy metabolism in lens in galactose-fed rats: effect of an aldose reductase inhibitor.

PURPOSE: The present study was aimed at evaluating early changes in glycolysis, the redox state of free cytosolic NAD(P)-couples, and the adenine nucleotide system in lens in both control and 50% galactose-fed rats, with the possibility of preventing these with an aldose reductase inhibitor (ARI). METHODS: Experiments were performed on male Sprague-Dawley rats fed the galactose diet for 2-14 days. The levels of glucose, galactose, glycolytic intermediates, alpha-glycerophosphate, malate, NAD, ATP, ADP, AMP were assayed spectrofluorometrically in individual lenses by enzymatic procedures, while galactitol and myo-inositol were quantified by GC-MS. Free cytosolic NAD+/NADH, NADP+/NADPH, and ATP/ADP x P(i) (phosphate potential) were estimated from lactate dehydrogenase, malic enzyme, and triose phosphate isomerase-glyceraldehyde 3-phosphate dehydrogenase-3-phosphoglycerate kinase systems. Lactate and pyruvate production by lenses of both control and galactose-fed rats was measured in a set of in vitro incubation studies (2 hr, 37 degrees C, Krebs bicarbonate-Hepes buffer, pH 7.45, with 5mM glucose or 5mM glucose + 30 mM galactose, respectively). RESULTS: Lens galactitol levels in 2, 4, 6, 8, 10, and 14-day galactose-fed rats were 48 +/- 8, 58 +/- 9, 68 +/- 8, 73 +/- 5, 81 +/- 20, and 75 +/- 11 mmol/g wet weight (mean +/- SD), respectively. NAD+/NADH ratios were indistinguishable from controls after 2-6 days on the galactose diet, but fell dramatically between 8 and 10 days, and did not correlate with polyol accumulation per se. The pattern of glycolytic intermediates (no change in G6P, F6P, and 3-PG, increase in GA3P, decrease in FDP, PEP, pyruvate, and lactate), as well as reduced in vitro lactate and pyruvate production, suggest inhibition of glycolysis at the sites of phosphofructokinase, glyceraldehyde 3-phosphate dehydrogenase, enolase, and pyruvate kinase. ATP levels as well as total ATP/ADP, ATP/ADP x Pi, adenylate charge, and cytosolic phosphate potential were decreased in galactose-fed rats, while galactose 1-phosphate and a-glycerophosphate levels as well as NADP+/NADPH ratio were increased. Lens galactitol levels were reduced approximately 57% in 10-day galactose-fed rats treated with the ARI (tolrestat, 100 mg/kg bwt/day, 6-day pretreatment); the changes in the lower segment of glycolysis, alpha-glycerophosphate levels, redox state of NAD-couples, and energy metabolism were partially prevented while NADP+/ NADPH ratios were unchanged and galactose 1-phosphate levels were further increased. CONCLUSIONS: Depressed glycolysis in lens in galactose-fed rats is consistent with decreased NAD+/NADPH and adenine nucleotide phosphorylation. Early changes in lens glucose utilization, redox state of NAD-couples, and energy metabolism in this model of galactosemia are similar to those in diabetes, are at least in part mediated by aldose reductase involved mechanisms, and can be partially prevented by an aldose reductase inhibitor.

Elevated glucose levels increase retinal glycolysis and sorbitol pathway metabolism. Implications for diabetic retinopathy.

PURPOSE: To assess effects of elevated glucose levels on retinal glycolysis and sorbitol pathway metabolism. METHODS: Freshly isolated retinas from normal male Sprague-Dawley rats were incubated for 2 hours at 37 degrees C, pH 7.45, in Krebs bicarbonate-Hepes buffer containing 5, 10, 20, or 30 mM glucose. Glycolytic metabolites, sorbitol, and fructose were measured in extracts of retina and medium. RESULTS: Elevated glucose levels increased retinal levels of sorbitol and triose phosphates, decreased sn-glycerol-3-phosphate levels, increased lactate and fructose production, and increased the retinal lactate-pyruvate ratio (indicative of an increased cytosolic ratio of free NADH-NAD+ like that induced by hypoxia). An inhibitor of aldose reductase (AL 4114) normalized sorbitol, fructose, triose phosphates, and the lactate-pyruvate ratio without affecting lactate production or sn-glycerol 3-phosphate levels. CONCLUSIONS: Elevation of retinal glucose levels causes a hypoxia-like redox imbalance "pseudohypoxia" that results from increased oxidation of sorbitol to fructose in the second step of the sorbitol pathway. This redox imbalance provides a plausible explanation for impaired regulation of retinal blood flow (in the absence of vascular structural changes) in humans with diabetes and in nondiabetic acutely hyperglycemic animals. These findings, together with other observations, suggest that this redox imbalance precedes, and may contribute to, hypoxic and ischemic retinopathy associated with diabetes.

Dietary myo-inositol supplementation does not prevent retinal and glomerular vascular structural changes in chronically diabetic rats.

To assess effects of dietary myo-inositol supplementation on diabetes-induced vascular structural lesions, diabetes was induced in Sprague-Dawley rats with streptozotocin; one-third of these rats was fed a 2% myo-inositol diet for 9 months, one-third was left untreated for 5 months then treated with myo-inositol for the last 4 months, and one-third was untreated for the entire 9 months. Controls included untreated and myo-inositol-treated groups. Weight gain was impaired and plasma glucose, glycosylated hemoglobin, food consumption, urine volume, and albuminuria were increased significantly in diabetic versus age-matched control rats. Plasma myo-inositol levels were increased approximately fivefold in controls and approximately six- to eightfold in diabetic rats treated with myo-inositol. In general, myo-inositol did not affect any of the above parameters in control or diabetic rats. Retinal capillary basement membrane width (CBMW) was increased significantly (approximately 50% versus controls) after 9 months of diabetes. In the control group myo-inositol increased CBMW to the level of untreated diabetic rats; myo-inositol had no effect on CBMW in each diabetic group. The number of retinal capillaries containing pericyte nuclei and pericyte capillary coverage were increased in untreated as well as myo-inositol-treated diabetic rats and in the myo-inositol-treated control group. Glomerular CBMW was increased after 5 and 9 months of diabetes versus age-matched controls, and was increased even more by myo-inositol. Mesangial fractional volume of the glomerulus was increased 36% by diabetes and was decreased slightly but significantly by myo-inositol. These results indicate that diets supplemented with 2% myo-inositol (1) cause capillary basement membrane (CBM) thickening and pericyte changes in retinal capillaries of normal rats, (2) are ineffective in preventing or reversing diabetes-induced retinal CBM thickening, and (3) cause further thickening of glomerular CBM in diabetic rats.