Purification of the cardiac sarcoplasmic reticulum membrane protein phospholamban from recombinant Escherichia coli

Phospholamban (PLN) was expressed in Escherichia coli as a protein fusion with glutathione S-transferase (GST). GST-PLN was mostly present in the insoluble protein fraction and accounted for approximately 50% of total insoluble protein. Attempts to suppress inclusion body formation or to use GST as an affinity-purification tag failed. A successful purification method is based on preparative SDS/PAGE and electrodialysis. From 1 g cells we typically purified 13.5 mg fusion protein with a PLN content of 2.8 mg. We genetically inserted an enterokinase (EK) protease site just in front of the PLN sequence and demonstrated the proteolytical liberation of PLN from the carrier protein. The approach described represents a substantial advancement in PLN expression and purification.     

The effect of topical corticosteroids on refractive outcome and corneal haze after photorefractive keratectomy

BACKGROUND: The effect of topical corticosteroids after excimer laser photorefractive keratectomy (PRK) remains a matter of some controversy. Refractive effects may be different according to the amount of myopia and timing of instillation. METHODS: Two groups of patients were studied: Study A consisted of 215 eyes (128 patients) with PRK (mean baseline myopia, -6.53 +/- 2.22 D) that received no corticosteroids (No Corticosteroid Group) unless significant regression or corneal haze appeared (Delayed Corticosteroid Group), and in Study B, we randomly assigned eyes to the Initial Corticosteroid Group (mean baseline myopia, -6.39 +/- 1.84 D) or the No/delayed Corticosteroid Group (mean baseline myopia -5.78 +/- 2.02 D). Clinical results after PRK for low-to-moderate and high myopia were compared. RESULTS: In the first group, 70.9% (73 eyes) of moderately myopic eyes (mean, -4.56 +/- 1.10 D) belonged to the No Corticosteroid Group that had a mean refraction of -5.39 +/- 1.77 D. Delayed Corticosteroid Group eyes were more myopic (mean, -7.52 +/- 2.10 D), and showed more severe haze than those in the No Corticosteroid Group. In study B, only in high myopes with more than -6.00 D (mean, -7.76 +/- 1.15 D) did refraction and corneal haze outcomes show significant difference between the Initial Corticosteroid Group and the No/delayed Corticosteroid Group. CONCLUSIONS: The effects of topical corticosteroids after PRK were less in moderate myopes compared to high myopes. Delayed instillation of corticosteroids did not reverse the regression or haze whereas initial instillation showed a beneficial effect on high myopes but not on moderate myopes.     

Glucose-induced positive cooperativity of fructose phosphorylation by human B-cell glucokinase

Human B-cell glucokinase displays sigmoidal kinetics towards D-glucose or D-mannose, but fails to do so towards D-fructose. Yet, D-glucose, D-mannose and 2-deoxy-D-glucose confer to the enzyme positive cooperativity towards D-fructose. For instance, in the presence of 5 mM D-[U-14C]fructose, its rate of phosphorylation is increased to 214.3 +/- 11.0%, 134.0 +/- 4.3% and 116.5 +/- 3.0% of paired control value by D-glucose, D-mannose and 2-deoxy-D-glucose (each 6 mM), respectively. D-glucose and, to a lesser extent, D-mannose also display reciprocal kinetic cooperativity. D-fructose, however, fails to affect D-glucose or D-mannose phosphorylation under conditions in which positive cooperativity is otherwise observed. These findings are relevant to the reciprocal effects of distinct hexoses upon their phosphorylation by B-cell glucokinase and, as such, to the metabolic and functional response evoked in pancreatic islet B-cells by these sugars, when tested either separately or in combination.     

Association of angiotensin-converting enzyme gene I/D polymorphism with change in left ventricular mass in response to physical training

PURPOSE: To determine the radiologic characteristics of cystic dystrophy of the duodenal wall. MATERIALS AND METHODS: Ten patients with cystic dystrophy of the duodenal wall and chronic pancreatitis underwent ultrasonography (US) (n = 10), computed tomography (CT) (n = 10), endoscopic US (n = 5), and endoscopic retrograde cholangiopancreatography (ERCP) (n = 9). Cystic dystrophy of the duodenal wall was classified as either cystic or solid. The imaging findings were retrospectively analyzed and compared with findings at pancreatoduodenectomy (n = 10). RESULTS: The more frequent cystic type (n = 7) of cystic dystrophy of the duodenal wall was characterized by the presence of easily recognizable cystic lesions (diameter, more than 1 cm), located within the thickened wall of the second portion of the duodenum. The solid type (n = 3) of cystic dystrophy of the duodenal wall demonstrated fibrous thickening of the duodenal wall within which small cysts (diameter, less than 1 cm) were present. The intraduodenal cysts were usually elongated or bilobate with a thick wall. The thickening of the duodenal wall appeared as a solid layer between the duodenal lumen and the pancreas, hypoechoic at US, isoattenuating at unenhanced CT, and hypoattenuating in the early phase (after initiation of infusion of contrast material) and isoattenuating in the late phase (after completion of infusion) at contrast material-enhanced CT. Findings at retrospective analysis of CT and endoscopic US images were characteristic. CONCLUSION: Imaging modalities, notably CT and endoscopic US, helped establish the diagnosis of cystic dystrophy of the duodenal wall.     

The distribution of HBV, HCV and HGV among livers with fulminant hepatic failure of different aetiology

BACKGROUND/AIMS: The aim of the study was to assess the impact factor of HCV and HGV in fulminant hepatic failure. METHODS: The 5'-untranslated regions of HCV RNA and HGV RNA and a segment of the core antigen sequence of HBV were amplified after extracting the nucleic acids from snap-frozen tissue aliquots from explanted livers of 26 consecutive patients undergoing orthotopic liver transplantation for fulminant hepatic failure preoperatively diagnosed as either autoimmune (n=2), HAV/HBV (n=8), toxic (n=4) or aetiologically unknown (n=12). RESULTS: HCV RNA was detected in five of 26 (19.2%) livers with fulminant hepatic failure. All five HCV RNA-positive livers belonged to the group of non-toxic, non-autoimmune liver failure (n=20), three of them were found in the group of liver failure with unknown aetiology (n=12) and two in the group of HBV-associated liver failure (n=7), making an HCV incidence of 25%, 25% and 28.6%, in the different groups, respectively. HGV RNA was detected in 10 of 17 (58.8%) explants and in all four groups of fulminant hepatic failure as defined preoperatively. HBV DNA was identified in six livers of 26 patients (23.1%) with fulminant hepatic failure. Neither HCV RNA nor HBV DNA was detected in the livers of patients with toxic or autoimmune fulminant hepatic failure. CONCLUSIONS: These results indicate that HBV and HCV, but not HGV, play an aetiologic role in fulminant hepatic failure. HCV-positive cases were concentrated either in the group of otherwise unexplained fulminant hepatic failure or in the group of HBV fulminant hepatic failure. HGV-positive cases, on the other hand, were found within all four preoperatively defined groups, indicating a role as cofactor rather than as single aetiologic agent.     

Dissociation between the potency and reversibility of the insulinotropic action of two meglitinide analogues

The non-sulphonylurea hypoglycaemic agent repaglinide is about one order of magnitude less potent, in terms of insulinotropic efficiency, than S3075, another meglitinide analogue. In the present study, the effects of these two drugs upon 86Rb outflow, 45Ca efflux and insulin release from prelabelled rat pancreatic islets were investigated in a perifusion system. At a concentration of 10 microM, which is sufficient to evoke a maximal secretory response in incubated islets, both agents inhibited 86Rb efflux from islets perifused in the absence of D-glucose, and stimulated both 45Ca efflux and insulin release from islets perifused in the presence of 6 mM D-glucose. The stimulation of 45Ca efflux from prelabelled islets was suppressed in the absence of extracellular Ca2+. The cationic and secretory response to repaglinide differed, however, from that evoked by S3075, in persisting for at least 20 min after removal of the drug from the perifusion medium, whilst the changes in 86Rb outflow, 45Ca efflux and insulin release caused by S3075 were rapidly reversible. These findings indicate that there is no parallel between the insulinotropic efficiency of distinct meglitinide analogues, and the reversibility of their functional effects. Since a comparable dissociation was recently documented in the case of hypoglycaemic sulphonylureas, the present observations reinforce the view that distinct molecular determinants may rule the relative insulinotropic potency of sulphonylureas and structurally related meglitinide analogues, on one hand, and the reversibility of their biological action, on the other hand.