Glutathione transferase-derived compounds as potential therapeutic agents

Glutathione transferases are generally recognized for their role as antioxidant enzymes in phase II detoxification reactions. Recent studies identified a diverse range of other functions which are unrelated to detoxification. One such action is the specific inhibition of cardiac ryanodine receptor Ca{u00B2}{u207A} release channel. It is well documented that excessively active RyR2 channels are partly responsible for low store Ca{u00B2}{u207A} levels and defective Ca{u00B2}{u207A} release in heart failure. Therefore, inhibition of RyR2 is a potential strategy for the treatment of heart failure as it would help to maintain low RyR2 activity during diastole. Previous studies which examined the effects of truncating GSTM2 on its ability to alter Ca{u00B2}{u207A} release from SR and RyR2 channel activity identified the GSTM2 C terminal domain (GSTM2C) as the region responsible for the inhibitory effect. Although it supported the inhibition of RyR2, it did not support the activation of RyR1 channels which was shown by the full length protein. Activity of smaller fragments from the GSTM2C indicated that the helix 6 in the C terminal domain is critical for the inhibitory effect. Although fragments containing the helix 6 sequences inhibited Ca{u00B2}{u207A} release from cardiac SR and RyR2 channel activity, helix 6 sequence alone was not effective. Structural analysis on circular dichroism spectroscopy revealed the helix 6 sequence to be unstructured which may have explained the lack of activity, if the helical nature of the fragment was essential for its efficacy. On the other hand, the active fragment of GSTM2 responsible for the inhibition of RyR2 activity may involve not only helix 6 but also the flanking helices. Therefore, the main aims of the thesis were to identify the minimum fragment of GSTM2 which is capable of inhibiting RyR2, to determine the effect of mutations on the inhibitory effect, to confirm the region of RyR2 that interacts with GSTM2 and finally, to determine the effects of GSTM2 fragments on the contractility of ventricular cardiomyocytes. First, I examined the activity of RyR2 in the presence of two GSTM2C constructs with potentially destabilizing mutations in helix 6. The mutants, Fl57A and Y160A failed to inhibit cardiac RyR2 activity in single channel lipid biayer experiments and Ca{u00B2}{u207A} release from cardiac SR. Although they retained the helical structure as indicated in circular dichroism spectroscopy, tryptophan fluorescence indicated changes in folding. Interestingly, wild type GSTM2C inhibited cardiac RyR2 only at positive potentials, which may develop during Ca{u00B2}{u207A} efflux, but not at negative potentials. This further suggested that therapeutics mimicking the structure of GSTM2C may reduce excess Ca{u00B2}{u207A} release during diastole, which can lead to fatal arrhythmias. Previous experiments carried out in our laboratory also showed that H5678 fragment of GSTM2C (containing helices 5,6,7 and 8) is capable of binding to the DR3 region of RyR2 which explains the isoform specific activity of GSTM2. Tryptophan fluorescence quenching experiments that I performed confirmed the binding between H5678 and 22 amino acid fragment (1869-1890) of DR3 region, which further narrowed down the GSTM2 binding site in cardiac RyR2. Consistent with previous results, binding between the two proteins was weak with dissociation constants in micro molar range. Since DR3 is located in the clamp region of the 3D structure of RyR2, specific binding of GSTM2C to RyR2 could dramatically influence the gating of the channel. I investigated the effect of GSTM2C and the mutants, Fl57A and Yl60A on the contractility of neonatal cardiomyocytes. Consistent with the results obtained with single channel lipid bilyer experiments and Ca{u00B2}{u207A} release assays, the mutants failed to reduce the contractility of cardiomyocytes. Ability of GSTM2C to inhibit RyR2 activity hence Ca{u00B2}{u207A} release from SR was further indicated when the contractility of cardiomyocytes was reduced significantly in the presence of GSTM2C. A new property of GSTM2C was identified when the GSTM2C was shown to be capable of entering into cardiomyoctytes without a carrier cell penetrating peptide. The overall results presented in this study identify GSTM2C as a potential therapeutic for the treatment of heart failure. The helix 6 sequence in the GSTM2C and its flanking helices played a major role in stabilizing the molecule and in binding to the DR3 region of RyR2. Due to the voltage dependent activity, GSTM2C could be used during diastole to reduce the abnormal Ca{u00B2}{u207A} leak through ryanodine receptors. Thus, GSTM2C could be the only isoform specific, endogenous inhibitor of cardiac ryanodine receptor activity reported so far

Concordance between Body Composition Indices Measured with Dual-Energy X-Ray Absorptiometry and Bioelectrical Impedance Analysis in Obese Children in Sri Lanka

Dual-energy X-ray absorptiometry (DXA) is the reference standard in the measurement of body composition indices. But, its utility is limited due to the high cost, expertise required, lack of portability, and restricted availability. Therefore, bioelectrical impedance analysis (BIA) has gained recognition in resource-limited settings for the measurement of body composition indices in the screening of children for childhood obesity. To determine whether BIA represents a viable alternative to DXA in the assessment of body composition in obese children in the community setting in Sri Lanka, the concordance between BIA and DXA was determined. Fat mass (FM), percentage fat mass (%FM), and fat-free mass (FFM) were measured in 97 obese children using DXA and BIA, and the concordance between the methods was analyzed using independent sample t-test, regression analysis, and Bland-Altman plots. Significant mean differences were observed between DXA and BIA in measuring FM and FFM. However, high correlations were seen in DXA- and BMI-derived FM and FFM measurements (FM r=0.92 and FFM 0.83, P<0.001 for both). Compared to DXA, BIA overestimated FM and %FM and underestimated FFM. When compared with DXA-derived measurements, the accuracy errors (SEE) of BIA for FM, FFM, and %FM were relatively higher in boys (3.56 kg, 4.49 kg, and 5.46%, respectively) than in girls (2.44 kg, 3.72 kg, and 3.5%), respectively. BA plots showed a systematic error in the measurements of FM, FFM, and %FM in both sexes. Despite the limitations inherited, BIA is a viable alternative to DXA for the measurement of body composition in obese children of 5-15 yrs. The accuracy errors observed, however, need to be taken into consideration when interpreting results at the individual level

Ameliorative effects of <em>Asparagus falcatus</em> L. and <em>Vetiveria</em> <em>zizanioides</em> (L.) Nash on carbon tetrachloride induced oxidative stress in mice

417-424The hepatoprotective and antioxidative effects of two aqueous plant extracts, Asparagus falcatus and Vetiveria zizanioides, were determined against carbon tetrachloride (CCl4) induced oxidative stress in mice. Hepatotoxicity was induced by the administration of a single intraperitonial dose of CCl4 (0.5 mL kg-1 CCl4 in olive oil) after a 16 hrs fast. Aqueous extracts of the tubers of Asparagus and root of Vetiveria (0.9 gm kg-1) were used on pre and post-treatment basis. The ability of plant extracts to protect the liver against changes mediated by carbon tetrachloride confirms that plants possess anti-hepatotoxic properties against CCl4 induced liver damage. Both pre and post-treatment decreased the CCl4 mediated increase in serum enzyme activities (ALT, AST, ALP) and increased the reduced glutathione concentration in the liver. Administration of Vetiveria alone improved the GSH status significantly. Glutathione reductase and glutathione S-transferase activities were increased significantly (P 4 control group. Histopathological studies provided supportive evidence for the biochemical analysis. The magnitude of hepatoprotective properties varied between the two plant extracts but the hepatoprotection mediated by Vetiveria was more potent than that of Asparagus

Comparison of In Vitro Antibacterial Activity of Epaltes divaricata and Vetiveria zizanioides against Methicillin-Resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of hospital- and community-acquired infections worldwide. Therefore, this study was conducted to explore the antibacterial activity of the two medicinal plants Epaltes divaricata and Vetiveria zizanioides against strains of MRSA which were isolated from patients with skin and soft tissue infections. Hexane, ethanol, and water extracts of E. divaricata (whole plant) and V. zizanioides (roots) were prepared. Clinical isolates of MRSA strains (n = 20) were used for the study. Bacterial susceptibility was tested using a disc diffusion assay. Minimum inhibitory concentration (MIC) was determined by a broth microdilution method. Vancomycin was used as the positive control. Hexane, ethanol, and water extracts of E. divaricata showed inhibitory zones against MRSA. Except for water extract, both hexane and ethanol extracts of V. zizanioides showed inhibitory zones. MIC ranges of hexane, ethanol, and water extracts in E. divaricata were 0.012–0.32 mg/mL, 0.019–2.4 mg/mL, and 0.019–0.48 mg/mL, respectively. Respective MIC ranges of hexane and ethanol extracts of V. zizanioides were 0.003–0.032 mg/mL and 0.019–2.4 mg/mL. The hexane extract of V. zizanioides inhibited 55% of the selected MRSA strains at a relatively low MIC value of 0.012 mg/mL. The hexane extract of both plants demonstrated inhibition of 75% of MRSA strains at a MIC value of 0.064 mg/mL. Ethanol extract of V. zizanioides and E. divaricata, respectively, inhibited 70% and 45% of MRSA strains at the MIC of 0.096 mg/mL, whereas water extract of E. divaricata inhibited 80% of MRSA strains at the same MIC. Both E. divaricata and V. zizanioides were equally effective against MRSA at a MIC of 0.064 mg/mL. But V. zizanioides was more effective since the hexane extract inhibited more than 50% of MRSA strains at significantly a lower MIC value of 0.012 mg/mL. Fractionation, purification, and identification of active compounds will warrant further evaluation of the therapeutic potential of both plant extracts

Chemical Composition and Antimicrobial Activity of Two Sri Lankan Lichens, Parmotrema rampoddense, and Parmotrema tinctorum against Methicillin-Sensitive and Methicillin-Resistant Staphylococcus aureus

Introduction. Medicinal utility of lichens is ascribed to the presence of various secondary metabolites of low molecular weight and they have been used in traditional medicine including Ayurveda in the treatment of wounds and skin disorders. Despite the urgent need to effectively address the antibiotic resistance worldwide, the discovery of new antibacterial drugs has declined in the recent past. This emphasizes the increasing importance of investigating and developing new classes of antibiotics that can withstand antibiotic resistance. Aims of the study. The present study was conducted to investigate the chemical composition and the antibacterial activity of hexane, ethanol, and aqueous extracts of Parmotrema rampoddense and Parmotrema tinctorum, two lichens collected from Belihuloya, Sri Lanka, against Gram-negative and Gram-positive bacteria including twenty clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA). Materials and methods. Phytochemical analysis, thin layer chromatography (TLC), and Gas Chromatography Mass Spectrometry (GC-MS) were performed to determine the chemical composition of the two lichens. Hexane, ethanol, and aqueous extracts of both lichens were tested against clinical isolate of Gram-negative and Gram-positive bacteria including twenty clinical isolates of MRSA. Bacterial susceptibility was tested using a disc diffusion assay. Minimum inhibitory concentration (MIC) was determined by a broth microdilution method. Vancomycin was used as the positive control. Results. Alectorialic acid, atranorin, atraric acid, orcinol, and O-orsellinaldehyde were among the secondary metabolites identified by the TLC and GC-MS analysis. None of the lichen extracts were active against Gram-negative bacteria but both lichens showed a concentration-dependent activity against methicillin-sensitive Staphylococcus aureus (MSSA) and MRSA. Ethanol extract of P. rampoddense showed the highest activity against MSSA with the MIC, 0.0192 mg/ml, but all MRSA isolates investigated showed MIC between 0.096 and 2.4 mg/ml for the same extract. Conclusion. Both lichens, P. rampoddense and P. tinctorum, represent potentially important sources of future antimicrobial drugs. Further investigation on the ethanol extract of P. rampoddense will enable us to determine the most active phytoconstituents responsible for the activity, their mechanism of action against bacterial pathogens, and also their cytotoxicity against normal cells