[Effect of curcumin on nitric oxide induced structural and functional modifications of high molecular weight cystatin from the goat brain].

Cystatins are thiol proteinase inhibitors ubiquitously present in the mammalian body. In brain, they prevent unwanted proteolysis and are involved in several neurodegenerative diseases. Under physiological conditions nitric oxide can be found in almost all the tissues, but under pathological conditions NO has damaging effects. Its increased concentration, under various neural diseases leads to cell damage through formation of highly reactive peroxynitrite. Our present study was designed to investigate the protective effect of curcumin against NO induced damage of HM-GBC. NO caused intensive structural and functional damage of HM-GBC, resulting in 89% loss of its antiproteolytic activity after 2 h of incubation. Structural damage occurs in the form of protein degradation. Curcumin significantly protected HM-GBC against this damage. This suggests that curcumin has a significant potential in the treatment of diseases caused by nitrogen free radicals and this potential must be further explored for the development of novel drugs.

Beneficial effects of Annona squamosa extract in streptozotocin-induced diabetic rats.

INTRODUCTION: The present study investigated the possible therapeutic effects of Annona squamosa (A. squamosa) extract on certain biochemical markers in streptozotocin (STZ)-induced diabetes mellitus in rats. METHODS: The effects of an aqueous extract of A. squamosa leaves on blood glucose, insulin, C-peptide, albumin, albumin/globulin ratio, urea, uric acid and creatinine and the activities of diagnostic marker enzymes aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase and gamma-glutamyl transpeptidase were examined in the plasma, liver and kidney tissues of control and experimental groups. RESULTS: Oral administration of A. squamosa (300 mg/kg) aqueous extract to diabetic rats for 30 days significantly reduced blood glucose, urea, uric acid and creatinine, but increased the activities of insulin, C-peptide, albumin, albumin/globulin ratio and restored all marker enzymes to near control levels. CONCLUSION: The present results shown that A. squamosa extract has an antihyperglycaemic effect and consequently may alleviate liver and renal damage associated with STZ-induced diabetes mellitus in rats.

Effect of polyols and salts on the acid-induced state of human placental cystatin.

Polyols (glycerol and sorbitol) and salts (magnesium sulfate, sodium sulfate, and magnesium chloride) have been used to study the refolding of the acid-induced state of human placental cystatin (HPC), which is a low molecular weight (12,500 daltons) thiol proteinase inhibitor, in terms of CD spectroscopy, binding of hydrophobic dye 1-anilinonaphthalene-8-sulfonic acid (ANS), and intrinsic fluorescence measurements. The helical content of acid-denatured HPC increased with increase in glycerol concentration (0-80%). At 80% glycerol concentration, the secondary structural features observed in the far UV-CD region are similar to those of the native state (pH 6.0). The intrinsic fluorescence and near UV-CD studies showed that this 80% glycerol-induced state has a significant amount of tertiary structure with decreased ANS binding compared to the acid-denatured state. It was found that glycerol is more effective in stabilizing the acid-denatured state of HPC as compared to sorbitol. Among salts the stability effect was more for MgCl(2) (used up to concentration of 3 M) compared to MgSO(4) and Na(2)SO(4) (used up to the concentration of 1.5 M due to restricted solubility of HPC at higher sulfate salt concentrations) as determined by CD studies and fluorescence measurements, which showed secondary and tertiary structural resemblance of this MgCl(2)-induced state close to native state and showed overall spectral features in between the native state and the acid-denatured state. This MgCl(2) (3 M)-induced state showed decreased ANS fluorescence as compared to the acid-denatured state but more than that of the native state. The results taken together suggest that the acid-denatured state of HPC in the presence of 80% glycerol or 3 M MgCl(2) has a conformation in between that of the native state (pH 6.0) and the acid-induced state at pH 2.0.

Antidiabetic and antioxidant activity of Annona squamosa extract in streptozotocin-induced diabetic rats.

INTRODUCTION: The aim of the study is to analyse the antioxidant effect of oral administration of aqueous extract of Annona squamosa (A. squamosa) leaf on blood glucose, haemoglobin, glycosylated haemoglobin, plasma insulin, antioxidant enzymes and lipid peroxidation in liver and kidney to streptozotocin (STZ)-induced diabetic rats. METHODS: Aqueous extract of A. squamosa on blood glucose, haemoglobin, glycosylated haemoglobin, plasma insulin, serum lipid and the levels of lipid peroxides and antioxidant enzymes, such as catalase, superoxide dismutase, glutathione peroxidase and reduced glutathione, were examined in the liver and kidney tissues of control and experimental groups. RESULTS: Oral administration of A. squamosa aqueous extract to diabetic rats for 30 days significantly reduced the levels of blood glucose, lipids and lipid peroxidation, but increased the activities of plasma insulin and antioxidant enzymes, like catalase, superoxide dismutase, reduced glutathione and glutathione peroxidase. CONCLUSION: The A. squamosa aqueous extract supplementation is useful in controlling the blood glucose level, improves the plasma insulin, lipid metabolism and is beneficial in preventing diabetic complications from lipid peroxidation and antioxidant systems in experimental diabetic rats; therefore, it could be useful for prevention or early treatment of diabetes mellitus.

Studies on low molecular mass phytocystatins purified from Phaseolus mungo (Urd).

In the present study two phytocystatins (thiol protease inhibitors) have been isolated and purified to homogeneity from Phaseolus mungo by a simple two-step procedure using ammonium sulfate fractionation and gel filtration on Sephacryl-100 HR. The latter procedure yielded two peaks of the inhibitors (PMC I and PMC II). The pH optimum of both phytocystatins was pH 7.0; the temperature optima for PMC I and PMC II were 65 and 70 degrees C, respectively. The molecular masses of the purified phytocystatins were 19 and 17 kD, respectively, as determined by SDS-PAGE and mass spectrometry. Antibodies raised against the purified cystatins gave a single precipitin line in Ouchterlony double immunodiffusion. Kinetics of inhibition showed that PMC I and PMC II strongly inhibit papain and ficin but not trypsin and chymotrypsin. Binding stoichiometry of PMC I and PMC II with both papain and ficin was 1 : 2. The effect of urea on PMC I and PMC II was analyzed by fluorescence and circular dichroism spectroscopy. The CD results suggest an unfolding of PMC I and PMC II accompanying a decrease in the amount of extended (hydrated) coil structure and an increase in sheet-like structure. FTIR results show that PMC I is structurally similar to PMC II. Hydrophobic interactions are observed over a long time scale (5-150 min). Furthermore, fluorescence spectroscopy results were found to be in accordance with CD results, by showing quenching of fluorescence intensity of PMC I and PMC II, although to different extents, due to perturbations of the environment of aromatic residues in the protein. Both cystatins showed strong inhibitory activity against Escherichia coli and Staphylococcus aureus.

Detailed biochemical characterization of human placental cystatin (HPC).

A low molecular weight thiol protease inhibitor (12,500) purified from human placenta has been characterized in detail. Human placental cystatin (HPC) was found to be stable in the pH range 3.0-9.0 and temperature stability was between 40 and 100 degrees C. It does not have any disulphide groups and carbohydrate content. There was no cross-reaction between anti-HPC serum and other purified cystatins like HMW kininogens isolated from sheep plasma and phytocystatins isolated from Phaseolus mungo. The kinetics of inhibition of HPC was studied with ficin and bromelain and the comparison was made with our already reported results with papain. The respective K(i) values obtained for ficin and bromelain are 8.4 x 10(-8) M and 9.5 x 10(-8) M, respectively, whereas the value for papain was 5.5 x 10(-8) M. The values of association constants (K(ass)) for ficin and bromelain were 2.9 x 10(3) and 8.6 x 10(2) M(-1) s(-1), respectively, however, the value for papain was 3.4 x 10(4) M(-1) s(-1), the respective dissociation constant values for ficin and bromelain were 2.6 x 10(-5) and 2.1 x 10(-5) s(-1), respectively, and the value obtained for papain was 2.3 x 10(-5) s(-1). These kinetic parameters taken together along with t(1/2) values and IC(50) values imply that HPC binds more effectively to papain, then ficin and least with bromelain. Far-UV-CD analysis shows that HPC has 21.08% alpha-helical structure and significant amount of beta structure. Near-UV-CD spectra of HPC show positive peak at 280 nm indicating significant amount of tertiary interactions. The partial amino acid sequence analysis shows that HPC has highest sequence homology with chicken cystatin and Gly residue is present at position 11 rather than at conserved position 9, which has also been reported for human stefin A structure. The hydropathy plot of 1-30 amino acid residues shows that most amino acids of this stretch are present in the hydrophobic core of the protein. Owing to low molecular weight, absence of disulphide bonds and carbohydrate content HPC can be placed in type I cystatin family with some resemblance to chicken cystatin as shown by CD studies and amino acid sequence analysis.

Protective effects of Piper nigrum and Vinca rosea in alloxan induced diabetic rats.

In the present study aqueous extract of Piper nigrum seeds and Vinca rosea flowers were administered orally to alloxan induced diabetic rats once a day for 4 weeks. These treatments lead to significant lowering of blood sugar level and reduction in serum lipids. The levels of antioxidant enzymes, catalase and glutathione peroxidase decreased in alloxan induced diabetic rats however these levels returned to normal in insulin, P. nigrum and V. rosea treated rats. There was no significant difference in superoxide dismutase activity in all groups compared to controls. Lipid peroxidation levels were significantly higher in diabetic rats and it was slightly increased in insulin, P. nigrum and V. rosea treated rats as compared to control rat. These results suggest that oxidative stress plays a key role in diabetes, and treatment with P. nigrum and V. rosea are useful in controlling not only the glucose and lipid levels but these components may also be helpful in strengthening the antioxidants potential.

Comparative effect of olive oil and fish oil supplementation in combating gentamicin induced nephrotoxicity in rats.

The present study is related to the comparative effects of fish oil and olive oil supplementation on gentamicin induced nephrotoxicity in rats. Three treatment groups (Pretrement, Co-treatment and post treatment) were chosen for the study. Nephrotoxicity in rats was induced by intraperitonial administration of gentamicin (80 mg/kg/d) for 3,5,7,10,& 12 consecutive days. The animals were sacrificed 12 hrs after last treatment in each group. The maximum nephrotoxicity was developed on 10 days treatment of gentamicin. For each group a control group was taken without any oil or gentamicin treatment. Beneficial effects of oils were evidenced by reduced serum urea and creatinine concentrations in the group receiving oils compared to the non oil treatment animals receiving gentamicin only. Further, the changed values of alkaline phosphatase and acid phosphatase activity retumed to normal in kidney and liver tissue homogenates after fish and olive oil treatment. In this study, it was found that co-treatment of fish and olive oil is more effective antagonist of gentamicin induced nephrotoxicity. However fish oil was found to be more effective. Hypercholesteromia associated with gentamicin induced nephrotoxicity is also lowered by oil supplementations. The beneficial effects of these oils are due to counteracting effect of the biochemical alterations induced by the drug.

Studies on the interaction of papain with human placental cystatin by UV, fluorescence and CD spectroscopy.

The interaction of activated papain with low molecular weight cystatin (Mr 12500) purified from human placenta has been studied. Analysis of inhibition of caesinolytic activity of papain by cystatin showed stoichiometry of 1:1. Kinetic studies gave an inhibition constant (K(i)) value of 5.5 x 10(-8) M and association rate constant (K(+1)) value of 3.4 x 10(4) (M(-1) s(-1)). All spectroscopic studies showed conformational changes in both papain and cystatin on formation of complex. The data suggest perturbation of environment of aromatic residues and change of their native structure and conformation thereby shedding light on the behaviour of cystatins, especially interaction of placental cystatin with thiol protease inhibitors.

Flexibility and viscometric studies of globular proteins ovalbumin and ovotransferrin.

The ultrasonic velocity, density and viscosity of two egg proteins, ovalbumin and ovotransferrin in phosphate buffer have been studied at the physiological pH values. The thermodynamic functions for unfolding, ellipticity, surface amino acid residues and compressibility have been obtained for thermal and chemical denaturation in these food proteins. The computed values of Huggin's constant and shape factor, at a fixed ionic strength 0.16 M are found to be in agreement with the reported values for globular proteins. The slow increase in free-energy of unfolding with temperature at a fixed pH 7 suggests uncoiling and in turn, disappearance of biological activity. It has been observed that the effects of temperature and chemical denaturant on the native protein may give rise to different conformational states. In the presence of urea and sodium dodecyl sulphate (SDS), the proteins gave the excessively denatured states at 25 degrees C and pH 7, in comparison to the thermal denatured state. The positive values of partial adiabatic compressibility (see symbol in text) beta s over the temperature range 45-75 degrees C suggest the possibility of large internal flexibility in ovotransferrin than in ovalbumin.

Purification and physicochemical studies of human plasma thiol proteinase inhibitors.

The physicochemical properties of thiol proteinase inhibitors (TPI) isolated from outdated human blood have been studied. A simple technique which includes ammonium sulphate precipitation, Sephadex G-200 gel filtration and ion-exchange chromatography led to the isolation of 4 isolates namely TPI-1, TPI-2, TPI-3 and TPI-4 having molecular mass of 70, 155, 195 and 497 kDa respectively. The latter two forms are the new isolates unreported previously. They exhibit similar pH stability, inhibition spectra with papain, cathepsin B and trypsin, antigenic properties and glycoprotein nature. The TPI-4, however, was found to be most heat stable showing no decrease in inhibitory activity when heated upto 70 degrees C for 30 min. Our work suggests that TPI-3 and TPI-4 are the oligomers of TPI-1.

Characterisaton of human plasma thiol proteinase inhibitors.

Earlier, we had reported purification of three thiol proteinase inhibitors (TPI-1 of 70 kDa, TPI-3 of 195 kDa and TPI-4 of 497 kDa) from human plasma. In the present study we report that TPI-1 binds to papain in the stoichiometry ratio (E/I) of 1:1 while TPI-3 and TPI-4 bind in the ratio of 1.5:1 and 3.2:1 respectively. The K(m) for papain with BAPNA as substrate and Kcat/K(m) values for TPI-1, TPI-3 and TPI-4 were 2.7 x 10(-6) M, 0.84 nM/sec; 3.2 x 10(-6) M, 0.75 nM/sec; and 3.6 x 10(-6) M, 0.72 nM/sec respectively. The Ki values were found to be 1.48 nM for TPI-1, 0.133 nM for TPI-3 and 0.117 nM for TPI-4. The UV absorption and fluorescence emission spectra study suggest involvement of aromatic residues in the binding process. This study suggests that TPI-4 is the most potent inhibitor of thiol proteinases.

Structural requirements for cathepsin B and cathepsin H inhibition by kininogens.

Domain 3 (D3) of human kininogens, the major cysteine proteinase inhibitors in plasma, has been shown to be the tightest binding inhibitory domain for cathepsins B and H. D3 was expressed in three fragments as its exon products as follows: exon 7 (Gly235-Gln292), exon 8 (Gln292-Gly328), and exon 9 (Gln329-Met357). Exon products 7, 8, and 9 alone as well as exon product 7 + 9 each exhibited an 1C50 value 5- to 30-fold higher (5-30 microM) than exon products 7 + 8 and 8 + 9 (0.9-1.3 microM) for cathepsins B and H, respectively. However, in turn, the exon products 7 + 8 and 8 + 9 seemed to be less potent inhibitors than the intact D3 (10, 200 nM) or HK (200, 500 nM) molecule. These results clearly indicate that an intact molecule of HK or its domain 3 as a whole is required for optimal inhibition of cathepsins B and H.