N- and C-terminal cooperation in rotavirus enterotoxin: novel mechanism of modulation of the properties of a multifunctional protein by a structurally and functionally overlapping conformational domain.

Rotavirus NSP4 is a multifunctional endoplasmic reticulum (ER)-resident nonstructural protein with the N terminus anchored in the ER and about 131 amino acids (aa) of the C-terminal tail (CT) oriented in the cytoplasm. Previous studies showed a peptide spanning aa 114 to 135 to induce diarrhea in newborn mouse pups with the 50% diarrheal dose approximately 100-fold higher than that for the full-length protein, suggesting a role for other regions in the protein in potentiating its diarrhea-inducing ability. In this report, employing a large number of methods and deletion and amino acid substitution mutants, we provide evidence for the cooperation between the extreme C terminus and a putative amphipathic alpha-helix located between aa 73 and 85 (AAH73-85) at the N terminus of DeltaN72, a mutant that lacked the N-terminal 72 aa of nonstructural protein 4 (NSP4) from Hg18 and SA11. Cooperation between the two termini appears to generate a unique conformational state, specifically recognized by thioflavin T, that promoted efficient multimerization of the oligomer into high-molecular-mass soluble complexes and dramatically enhanced resistance against trypsin digestion, enterotoxin activity of the diarrhea-inducing region (DIR), and double-layered particle-binding activity of the protein. Mutations in either the C terminus, AAH73-85, or the DIR resulted in severely compromised biological functions, suggesting that the properties of NSP4 are subject to modulation by a single and/or overlapping highly sensitive conformational domain that appears to encompass the entire CT. Our results provide for the first time, in the absence of a three-dimensional structure, a unique conformation-dependent mechanism for understanding the NSP4-mediated pleiotropic properties including virus virulence and morphogenesis.

Plasmepsin inhibitors: design, synthesis, inhibitory studies and crystal structure analysis.

Plasmepsin group of enzymes are key enzymes in the life cycle of malarial parasites. As inhibition of plasmepsins leads to the parasite's death, these enzymes can be utilized as potential drug targets. Although many drugs are available, it has been observed that Plasmodium falciparum, the species that causes most of the malarial infections and subsequent death, has developed resistance against most of the drugs. Based on the cleavage sites of hemglobin, the substrate for plasmepsins, we have designed two compounds (p-nitrobenzoyl-leucine-beta-alanine and p-nitrobenzoyl-leucine-isonipecotic acid), synthesized them, solved their crystal structures and studied their inhibitory effect using experimental and theoretical (docking) methods. In this paper, we discuss the synthesis, crystal structures and inhibitory nature of these two compounds which have a potential to inhibit plasmepsins.

Expression, purification, crystallization and preliminary crystallographic analysis of the diarrhoea-causing and virulence-determining region of rotaviral nonstructural protein NSP4.

The region spanning the tetrameric coiled-coil domain and the interspecies-variable virulence-determining region of the cytoplasmic tail of rotaviral nonstructural protein NSP4 has been crystallized. The crystals belong to space group I222, with unit-cell parameters a = 30.70, b = 38.07, c = 181.62 A, and contain two molecules in the asymmetric unit. Diffraction data have been collected utilizing a MAR imaging plate to a resolution of 2.2 A. The tetramer is generated by the crystallographic dyad along the c axis.

Evaluation of antidiabetic effect of Momordica cymbalaria fruit in alloxan-diabetic rats.

The oral treatment with the aqueous extract of Momordica cymbalaria fruit (MC) (0.5 g/kg) for 6 weeks showed a significant antihyperglycemic as well as antihyperlipidemic effects in the alloxan-induced diabetic rats.

Effect of omega-3 fatty acids on lipid peroxidation and antioxidant enzyme status in type 2 diabetic patients.

BACKGROUND: This study was conducted to investigate the effect of omega-3 fatty acids on lipid peroxidation and antioxidant enzyme activities in non-insulin dependent diabetic patients. METHODS: Thirty-four non-insulin dependent diabetic patients were selected for this study and they were initially treated with antidiabetic drugs alone for one month. This was followed by supplementation with omega-3 fatty acids (1,080 mg of EPA and 720 mg of DHA per day) along with the antidiabetic drugs for a period of two months. RESULTS: No change in glycaemic control was observed in diabetic patients at the end of two months of omega-3 fatty acids therapy along with antidiabetic drugs. The combined treatment significantly reduced serum triglycerides (2.07 +/- 0.94 mmol/l, before combined therapy vs 1.54 +/- 0.49 mmol/l after combined therapy, P<0.05) and increased HDL-cholesterol levels (0.93 +/- 0.099 mmol/l, before combined therapy vs 1.04 +/- 0.098 mmol/l after therapy, P<0.01). The raised lipid peroxide levels (5.14 +/- 0.61 micromol MDA/l in controls vs 6.36 +/- 1.56 micromol MDA/l in diabetic patients, P<0.001) were significantly decreased in these patients after the combined therapy (6.36 +/- 1.56 micromol MDA/l, before combined therapy vs 5.16 +/- 0.7 micromol MDA/l, after combined therapy, P<0.01). Among the erythrocyte antioxidant enzymes, the Glutathione peroxidase activity was increased (32.5 +/- 9.9 U/g Hb/min, before combined therapy vs 42.25 +/- 4.6 U/g Hb/min, after combined therapy, P<0.01) while no change was observed in Catalase (99.7 +/- 30.4 KU/g Hb before combined therapy vs 85.35 +/- 23.41 KU/g Hb, after combined therapy) and Superoxide dismutase activities (2.6 +/- 1.04 U/mg Hb/min, before therapy vs 3.01 +/- 1.08 U/mg Hb/min, after combined therapy) after the 2 months of combined treatment with antidiabetic agents and omega-3 fatty acids. CONCLUSION: Supplementation with omega-3 fatty acids has beneficial effects on serum triglycerides, HDL-cholesterol, lipid peroxidation and antioxidant enzymes, which may lead to decreased rate of occurrence of vascular complications in diabetes.

Antihyperglycemic activity of Momordica cymbalaria in alloxan diabetic rats.

Aqueous, ethanolic and hexane fractions of Momordica cymbalaria fruits were prepared and given individually at different doses to different batches of rats (both normal and alloxan diabetic rats) after an overnight fast. The blood glucose levels were measured at 0, 1, 3, 5 and 7 h after the treatment. The aqueous extract of Momordica cymbalaria at a dosage of 0.5 g/kg b.w. is showing maximal blood glucose lowering effect in diabetic rats. The same dosage did not produce any hypoglycemic activity in normal rats. The antihyperglycemic activity of Momordica cymbalaria fruit was compared with the treatment of Glibenclamide, an oral hypoglycemic agent.

Lipid peroxidation and antioxidant enzyme status in Type 2 diabetics with coronary heart disease.

Lipid peroxides are thought to be formed by free radicals and may play an important role in the development of atheromatous vascular diseases. The relationship between serum lipids, lipoproteins, lipid peroxides [thiobarbituric acid reactive substances (TBARS)] and erythrocyte antioxidant enzymes [catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD)] was investigated in non-insulin-dependent diabetic patients with and without coronary heart disease (CHD), and a comparison was made for all the above parameters with non-diabetic patients with CHD. Lipid peroxide concentrations were significantly increased in both groups of diabetic patients and also in non-diabetic patients with CHD, compared to those in control subjects. Diabetic patients with CHD had higher levels of TBARS compared to those diabetics without CHD. Hyperlipidaemia and abnormal lipoprotein levels were observed in all three groups of patients. Increased total cholesterol and LDL-cholesterol were observed in diabetics with CHD compared to those without CHD. Among the erythrocyte antioxidant enzymes, CAT activity was increased, GPx activity was decreased and no change was observed in SOD activity in both groups of diabetic patients and non-diabetic patients with CHD compared to those in controls. A clear correlation was observed between the CAT activity and lipid peroxide concentrations in all the diabetic patients. These observations suggest that there are similar abnormalities in lipid metabolism and erythrocyte antioxidant enzymes in diabetic patients and non-diabetic patients with CHD.

Effect of oral administration of bark extracts of Pterocarpus santalinus L. on blood glucose level in experimental animals.

The effect of administration of different doses of Pterocarpus santalinus L. bark extracts in normal and diabetic rats, on blood glucose levels was evaluated in this study. Among the three fractions (aqueous, ethanol and hexane), ethanolic fraction at the dose of 0.25 g/kg body weight showed maximum antihyperglycemic activity. The same dose did not cause any hypoglycemic activity in normal rats. The results were compared with the diabetic rats treated with glibenclamide and the antihyperglycemic activity of ethanolic extract of PS bark at the dose of 0.25 g/kg b.w. was found to be more effective than that of glibenclamide.

Lipid peroxidation and antioxidant enzyme levels in type 2 diabetics with microvascular complications.

Serum levels of total cholesterol, triglycerides, lipoproteins, lipid peroxides (TBARS) and erythrocyte antioxidant enzyme activities were measured in 105 non insulin dependent diabetic patients, among whom 38 had microvascular complications (MVC) of diabetes. All the diabetic patients had higher concentrations of glycated hemoglobin (HbA1) compared to controls (10.51 +/- 2.42% vs 6.31 +/- 0.85% P <0.001). Significant increase of serum triglycerides (TG), total cholesterol (TC), low density lipoprotein cholesterol (LDL-C) and very low density lipoprotein cholesterol (VLDL-C) and a significant decrease of high density lipoprotein cholesterol (HDL-C) were observed in the diabetic patients compared to controls (TG: 2.31 +/- 0.9 mmol/l vs 1.53 +/- 0.48 mmol/l P <0. 001; TC: 5.94 +/- 1.4 mmol/l vs 4.3 +/- 0.85 mmol/l P <0.001; LDL-C: 3.96 +/- 1.33 mmol/l vs 2.39 +/- 0.8 mmol/l P <0.001; VLDL-C: 0.46 +/- 0.2 mmol/l vs 0.3 +/- 0.09 mmol/l P <0.001; HDL-C: 0.81 +/- 0.24 mmol/l vs 1.04 +/- 0.18 mmol/l P <0.001). Significantly increased levels of serum TBARS were observed in diabetic patients compared to those in controls (TBARS: 6.7 +/- 1.5 mmol/l vs 5.14 +/- 0.61 mmol/l P <0.001). Erythrocyte catalase (CAT) activity was increased and Glutathione peroxidase (GPx) activity was decreased in diabetic patients compared to controls, but no significant change in Superoxide dismutase (SOD) activity was observed in diabetic patients (CAT: 104.94 +/- 37.1 KU/g Hb vs 85.8 +/- 23.6 KU/g Hb, P <0.01; GPx: 30 +/- 9.7 U/g Hb/min vs 40.84 +/- 12.3 U/g Hb/min, P <0. 001; SOD: 2.4 +/- 1.2 U/mg Hb/min vs 2.55 +/- 0.84 U/mg Hb/min, P=NS). In comparison with the diabetic group without MVC, the diabetic group with MVC had decreased GPx and SOD activities, while no difference was observed between these two groups regarding CAT activity (GPx: 25.32 +/- 8.4 U/g Hb/min vs 34.5 +/- 8.8 U/g Hb/min, P <0.001; SOD: 1.83 +/- 0.53 U/mg Hb/min vs 2.84 +/- 1.4 U/mg Hb/min, P<0.001; CAT: 106.3 +/- 39.9 KU/g Hb vs 103 +/- 34.9 KU/g Hb, P =NS). TBARS concentrations were significantly increased in the group with MVC compared to the group without these complications, indicating a positive relationship between TBARS and MVC of diabetes (7.05 +/- 1.23 mmol/l vs 6.3 +/- 1.02 mmol/l, P <0.001). Serum triglycerides, LDL and VLDL cholesterol concentration were significantly higher in diabetics with MVC than in diabetics without the complications (TG: 2.7 +/- 0.98 mmol/l vs 2.13 +/- 0.82 mmol/l, P<0.01; LDL - C: 4.45 +/- 1.3 mmol/l vs 3.67 +/- 1.3 mmol/l, P <0. 02; VLDL-C: 0.53 +/- 0.19 mmol/l vs 0.43 +/- 0.16 mmol/l, P <0.01), and the serum levels of TC in the group with MVC showed a positive correlation with their lipid peroxide levels (r =0.368, P <0.001). The increase in TBARS and the decreased GPx and SOD activities in diabetics with MVC in this study indicate that these factors may contribute to the occurrence of micro vascular complications in NIDDM patients.

Antidiabetic and hypolipidemic effects of Momordica cymbalaria Hook. fruit powder in alloxan-diabetic rats.

This study was undertaken to investigate the effect of Momordica cymbalaria fruit powder on blood glucose and other biochemical parameters in alloxan-induced diabetic rats. The treatment was given for 15 days. After the treatment, a significant reduction was observed in fasting blood glucose levels in the treated diabetic rats, but no hypoglycaemic activity in the treated normal rats. M. cymbalaria treatment showed considerable lowering of serum cholesterol and triglycerides in the treated diabetic group. There was a significant improvement in hepatic glycogen level in treated diabetic rats close to normal level after the treatment with M. cymbalaria. These results suggest that the M. cymbalaria fruit powder possesses antidiabetic and hypolipidemic effects in alloxan-induced diabetic rats.

Hyperlipidemia, increased lipid peroxidation and changes in antioxidant enzymes, Na(+)-K(+)-ATPase in erythrocytes of type 2 diabetic patients in andhra pradesh.

Plasma levels of lipids, lipoproteins and lipid peroxides and erythrocyte Na(+)-K(+) ATPase, Mg(2+)ATPase and antioxidant enzymes were measured in type-2 diabetic patients. A significant decrease in Na(+)-K(+)-ATPase activity was observed in diabetic patients which was negatively correlated with blood glucose and lipid peroxides, while the Mg(2+)-ATPase activity was increased. In the diabetic subjects the plasma concentrations of Na(+) and K(+) were increased where as erythrocyte levels of Na(+) were increased and K(+) were decreased. Hyperlipidaemia and increased levels of lipid peroxides were observed in the diabetic subjects. There was a significant increase in erythrocyte catalase activity in diabetics which positively correlated with their lipid peroxides. There was no change in GPx activities between controls and diabetics.