Govindan Sadasivam Selvam

Potential risk modifications of GSTT1, GSTM1 and GSTP1 (glutathione-S-transferases) variants and their association to CAD in patients with type-2 diabetes.

BACKGROUND Type-2 diabetes mellitus (T2DM) is a major risk factor for coronary artery disease (CAD) resulting in high morbidity and mortality. Glutathione S-transferases (GSTM1, GSTT1 and GSTP1) are known for their broad range of detoxification and in the metabolism of xenobiotics. Recent studies revealed the relationship of GSTs variants with T2DM and CAD. In this case-control study we ascertained the association of GSTs variants in association with the development of CAD in patients with T2DM. METHODS From the Southern part of India, we enrolled 222 T2DM patients, 290 T2DM patients with CAD and 270 healthy controls matched for age, sex and origin. Serum lipid profiles were measured and DNA was extracted from the blood samples. Multiplex PCR for GSTM1/T1 (null polymorphism) and PCR-RFLP for GSTP1 (105 A>G), were performed for genotyping of study participants. Gene frequency and lipid profiles were statistically analyzed for disease association. RESULTS Regression analysis showed that, GSTM1-null genotype is associated with a 2-fold increase (OR=2.925; 95% CI=2.078-4.119; P<0.0001) and GSTT1-null genotype is associated with a 3-fold increase (OR=3.114; 95% CI=2.176-4.456; P<0.0001) to T2DM development. Ile/Val and Val/Val genotypes of GSTP1 also showed a significant risk for T2DM (OR=1.423, CI=1.041-1.946; P=0.027 and OR=1.829, CI=1.064-3.142; P=0.029). Increased odds ratio showed that GSTT1-null genotype had a moderately higher occurrence in T2DM-CAD patients (OR=1.918, 95% CI=1.144-3.214; P=0.014) than T2DM patients without CAD. The level of HDL has significantly decreased in GSTT1-present than in GSTT1-null genotype (43.50±4.10 vs. 45.20±3.90; P=0.004) when compared with control and T2DM patients. However, LDL level showed a significant increase in GSTT1-null than GSTT1-present genotype (108.70±16.90 vs. 102.20±12.60; P=0.005). Although the GSTM1-null polymorphism showed no correlation with lipid profiles among T2DM and T2DM with CAD patients, GSTT1-null polymorphism attained a statistical significance for the level of LDL (127±28.20 vs. 134±29.10; P=0.039) and triglycerides in T2DM with CAD patients (182.10±21.10 vs. 191.20±24.10; P=0.018). CONCLUSION Our work concludes that GSTM1, GSTT1 and GSTP1 variants might contribute to the development of T2DM and GSTT1 variant alone is involved in the development of T2DM associated CAD complications in the South Indian population.

Plasmid profile and curing analysis of Pseudomonas aeruginosa as metal resistant

The isolate Pseudomonas aeruginosa exhibited resistance to heavy metals such as cadmium, chromium, nickel and lead. Plasmid DNA was isolated from P. aeruginosa and designated as pBC15. The size of the plasmid DNA was approximately 23 kb. Escherichia coli DH5α was transformed with plasmid pBC15 subsequired resistance to nickel and ampicillin. The same size of the plasmid was isolated from E. coli transformant and separated on 0.7 % agarose gel electrophoresis. The restriction analysis of pBC15 showed that the plasmid DNA has single site for Bam HI and Eco RI and three sites for Xho I which were compared with 1 Kb DNA and λ Hind III digest molecular markers. Therefore, the size of the plasmid DNA of pBC15 was confirmed to be 23 kb. Curing was carried out by ethidium bromide, acridine orange, novobiocin, sodium dodechyl sulphate and elevated temperature (40 °C). Transformation and curing results suggest that nickel and ampicillin resistance gene was conferred by plasmid DNA. Cadmium resistant gene was present on chromosomal DNA along with the gene for chromium resistance. Lead resistance gene was shown to be present on the chromosomal DNA rather than the plasmid DNA as the cured and uncured cultures remained similar in lead resistance. Therefore, the ability of P. aeruginosa resistant to nickel and ampicillin is plasmid mediated and transferable to other strains whereas cadmium, chromium and lead could be chromosomal encoded. The heavy metal and antibiotic resistances of P. aeruginosa can be used to exploit for clean up industrial wastewater and bioremediation of heavy metal contaminated soil.

Naringenin confers protection against oxidative stress through upregulation of Nrf2 target genes in cardiomyoblast cells

Cardiovascular diseases are the major health concern and the leading cause of death. Numerous studies have shown that oxidative stress stimuli have been incriminated in the pathogenesis of both acute and chronic heart disease. Though it is well known that bioflavonoids protect cells against reactive oxygen species (ROS)-induced damage, the molecular mechanisms involved are uncertain. Understanding the possible intracellular signaling pathways triggered by flavonoids will help to overcome the cardiac diseases resulting from oxidative stress. In the present study, we investigated whether naringenin (NGN) supplementation would improve the antioxidant defence under oxidative stress through the activation of Nrf2 signaling in cultured cardiomyoblast. NGN pretreatment significantly reduced stress-mediated apoptotic cell death and lipid peroxidation and showed increased level of reduced glutathione in H2O2-treated cardiomyoblast. In addition, NGN inhibited the production of NO and trigged the synthesis of antioxidant marker enzymes. Gene expression studies revealed that NGN upregulated the transcription of Akt and downregulated NF-κB and Caspase 3 genes. Notably, transcription of Nrf2 and its target genes was also upregulated. Taken together, the present study revealed that NGN elicits potent cytoprotective effect against oxidative stress by regulating Nrf2 and its target genes. In conclusion, the present work suggests that improving Nrf2 signaling by NGN supplementation would be a rational approach to facilitate ROS detoxification by augmenting both expression and activity of phase II detoxification enzymes for the alleviation of cardiac complications.

L-Arginine ameliorates cardiac left ventricular oxidative stress by upregulating eNOS and Nrf2 target genes in alloxan-induced hyperglycemic rats

Hyperglycemia is independently related with excessive morbidity and mortality in cardiovascular disorders. L-Arginine-nitric oxide (NO) pathway and the involvement of NO in modulating nuclear factor-E2-related factor-2 (Nrf2) signaling were well established. In the present study we investigated, whether L-arginine supplementation would improve the myocardial antioxidant defense under hyperglycemia through activation of Nrf2 signaling. Diabetes was induced by alloxan monohydrate (90 mg kg(-1) body weight) in rats. Both non-diabetic and diabetic group of rats were divided into three subgroups and they were administered either with L-arginine (2.25%) or L-NAME (0.01%) in drinking water for 12 days. Results showed that L-arginine treatment reduced the metabolic disturbances in diabetic rats. Antioxidant enzymes and glutathione levels were found to be increased in heart left ventricles, thereby reduction of lipid peroxidation by L-arginine treatment. Heart histopathological analysis further validates the reversal of typical diabetic characteristics consisting of alterations in myofibers and myofibrillary degeneration. qRT-PCR studies revealed that L-arginine treatment upregulated the transcription of Akt and downregulated NF-κB. Notably, transcription of eNOS and Nrf2 target genes was also upregulated, which were accompanied by enhanced expression of Nrf2 in left ventricular tissue from diabetic and control rats. Under these findings, we suggest that targeting of eNOS and Nrf2 signaling by L-arginine supplementation could be used as a potential treatment method to alleviate the late diabetic complications.

In vitro degradation of oxalate by recombinant Lactobacillus plantarum expressing heterologous oxalate decarboxylase

The aim of the present study is to constitutively express heterologous oxalate decarboxylase (OxdC) in Lactobacillus plantarum and to examine its ability to degrade oxalate in vitro for their future therapy against enteric hyperoxaluria.

Biophysical evaluation of two red-shifted hypocrellin B derivatives as novel PDT agents

Two novel cyclohexane-1,2-diamino and N,N dimethyl amino-propyl substituted hypocrellin B derivatives, abbreviated as CHA2HB and DMAHB, respectively were synthesized. These derivatives exhibited enhanced absorption in phototherapeutic window. Photodynamic action of these derivatives, investigated using optical and electron spin resonance methods, depended on both Type I and Type II mechanisms. Gel electrophoresis indicated 1O2/O2(.-) mediated DNA damage. CHA2HB displayed 20 fold increase in light dependent cytotoxicity on colon cancer cell line (HCT 116) than the well-known hypocrellin B (HB). The light induced, LD(50) values for CHA2HB and DMAHB were found to be 0.1 microM and 1.5 microM, respectively. The singlet oxygen generating efficiency followed the order HB>CHA(2)HB>DMAHB. But, the enhanced red absorption as well as the hydrophilicity renders the CHA2HB a better photodynamic therapeutic agent.

Recombinant Lactobacillus plantarum expressing and secreting heterologous oxalate decarboxylase prevents renal calcium oxalate stone deposition in experimental rats

BackgroundCalcium oxalate (CaOx) is the major constituent of about 75% of all urinary stone and the secondary hyperoxaluria is a primary risk factor. Current treatment options for the patients with hyperoxaluria and CaOx stone diseases are limited. Oxalate degrading bacteria might have beneficial effects on urinary oxalate excretion resulting from decreased intestinal oxalate concentration and absorption. Thus, the aim of the present study is to examine the in vivo oxalate degrading ability of genetically engineered Lactobacillus plantarum (L. plantarum) that constitutively expressing and secreting heterologous oxalate decarboxylase (OxdC) for prevention of CaOx stone formation in rats. The recombinants strain of L. plantarum that constitutively secreting (WCFS1OxdC) and non-secreting (NC8OxdC) OxdC has been developed by using expression vector pSIP401. The in vivo oxalate degradation ability for this recombinants strain was carried out in a male wistar albino rats. The group I control; groups II, III, IV and V rats were fed with 5% potassium oxalate diet and 14th day onwards group II, III, IV and V were received esophageal gavage of L. plantarum WCFS1, WCFS1OxdC and NC8OxdC respectively for 2-week period. The urinary and serum biochemistry and histopathology of the kidney were carried out. The experimental data were analyzed using one-way ANOVA followed by Duncan’s multiple-range test.ResultsRecombinants L. plantarum constitutively express and secretes the functional OxdC and could degrade the oxalate up to 70–77% under in vitro. The recombinant bacterial treated rats in groups IV and V showed significant reduction of urinary oxalate, calcium, uric acid, creatinine and serum uric acid, BUN/creatinine ratio compared to group II and III rats (P < 0.05). Oxalate levels in kidney homogenate of groups IV and V were showed significant reduction than group II and III rats (P < 0.05). Microscopic observations revealed a high score (4+) of CaOx crystal in kidneys of groups II and III, whereas no crystal in group IV and a lower score (1+) in group V.ConclusionThe present results indicate that artificial colonization of recombinant strain, WCFS1OxdC and NC8OxdC, capable of reduce urinary oxalate excretion and CaOx crystal deposition by increased intestinal oxalate degradation.

CONSTRUCTION OF GREEN FLUORESCENT PROTEIN BASED BACTERIAL BIOSENSOR FOR HEAVY METAL REMEDIATION

Environmental contamination by heavy metals is a worldwide problem. Therefore, it is necessary to develop sensitive, effective and inexpensive methods, which can efficiently monitor and determine the level of hazardous metals in the environment. Conventional techniques to analyze metals, suffer from the disadvantages of high cost. Alternatively, development of simple system for monitoring heavy metals pollution is therefore needed. The present approach is based on the use of bacteria that are genetically engineered so that a measurable signal is produced when the bacteria are in contact with the bioavailable metal ions. Reporter genes are widely used as genetic tools for quantification and detection of specific cell population, gene expression and constructing whole cell biosensors as specific and sensitive devices for measuring biologically relevant concentrations of pollutants. An attempt has been made to construct the reporter gene enhanced green fluorescent protein and was expressed under the control of cadR gene, responsible for cadmium resistance. Recombinant strain Escherichia colicadR30 was used, that carried cadR gene in pET30b expression vector and cloned. Clones confirmed by the expression of enhanced green fluorescent protein was detected under ultraviolet illumination and separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The construction of green fluorescent protein based Escherichia coli bacterial biosensor was developed based on green fluorescent protein expression under the control cadR gene of Pseudomonas aeruginosa BC15. The constructed bacterial biosensor is useful and applicable in determining the availability of heavy metals in soil and wastewater.

Secretion of Biologically Active Heterologous Oxalate Decarboxylase (OxdC) in Lactobacillus plantarum WCFS1 Using Homologous Signal Peptides

Current treatment options for patients with hyperoxaluria and calcium oxalate stone diseases are limited and do not always lead to sufficient reduction in urinary oxalate excretion. Oxalate degrading bacteria have been suggested for degrading intestinal oxalate for the prevention of calcium oxalate stone. Here, we reported a recombinant Lactobacillus plantarum WCFS1 (L. plantarum) secreting heterologous oxalate decarboxylase (OxdC) that may provide possible therapeutic approach by degrading intestinal oxalate. The results showed secretion and functional expression of OxdC protein in L. plantarum driven by signal peptides Lp_0373 and Lp_3050. Supernatant of the recombinant strain containing pLp_0373sOxdC and pLp_3050sOxdC showed OxdC activity of 0.05 U/mg and 0.02 U/mg protein, while the purified OxdC from the supernatant showed specific activity of 18.3 U/mg and 17.5 U/mg protein, respectively. The concentration of OxdC protein in the supernatant was 8–12 μg/mL. The recombinant strain showed up to 50% oxalate reduction in medium containing 10 mM oxalate. In conclusion, the recombinant L. plantarum harboring pLp_0373sOxdC and pLp_3050sOxdC can express and secrete functional OxdC and degrade oxalate up to 50% and 30%, respectively.

Analysis of polymorphism in Renin Angiotensin System and other related genes in South Indian chronic kidney disease patients

BACKGROUND Several Renin Angiotensin System (RAS) polymorphisms alter the homeostasis to an abnormal state. Similarly, other genes such as Nephrin (NPHS1) and Podocin (NPHS2) contribute to the loss of renal function during renal diseases. In Indian population, studies in RAS and other renal specific gene polymorphisms in Chronic Kidney Disease (CKD) patients are scanty. METHODS We examined 118 CKD patients and 98 control subjects for the occurrence of common polymorphisms in angiotensin converting enzyme insertion/deletion (ACE; I/D), angiotensinogen (AGT; M235T), chymase (CMA; -1903G>A), angiotensin receptor type-1 (AGTR1-1166A>C), methylene tetrahydrofolate reductase (MTHFR; 677C>T), nephrin (NPHS1; R1160X) and podocin (NPHS2; R291W and R229Q). RESULT Significant association was observed in AGT-M235T polymorphism between CKD patients and controls. The frequency of TT genotype was higher in CKD patients when compared with controls (0.39 vs. 0.14; chi(2)=20.3, P<0.001). ACE-DD genotype showed a higher level of systolic pressure with a median of 166 mmHg (P<0.05) when compared to II and ID genotypes. Two heterozygous conditions of NPHS2-R229Q polymorphism were found among 105 CKD patients. No significant associations were found in genotype frequencies in other above polymorphisms between CKD patients and controls. CONCLUSION Asian Indian population with AGT-TT genotypes may have a higher relative risk towards CKD with odds ratio (OR) 3.98 (95% CI=1.92-8.25; P=0.0002).