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.     

Enhancement of HLA class II-restricted CD4+ T cell recognition of human melanoma cells following treatment with bryostatin-1

The majority of melanoma cells express detectable levels of HLA class II proteins, and an increased threshold of cell surface class II is crucial for the stimulation of CD4+ T cells. Bryostatin-1, a protein kinase C (PKC) activator, has been considered as a potent chemotherapeutic agent in a variety of in vitro tumor models. Little is known about the role of bryostatin-1 in HLA class II Ag presentation and immune activation in malignant tumors, especially in melanoma. In this study, we show that bryostatin-1 treatment enhances CD4+ T cell recognition of melanoma cells in the context of HLA class II molecules. We also show that bryostatin-1 treatment of melanoma cells increases class II protein levels by upregulating the class II transactivator (CIITA) gene. Flow cytometry and confocal microscopic analyses revealed that bryostatin-1 treatment upregulated the expression of costimulatory molecules (CD80 and CD86) in melanoma cells, which could prolong the interaction of immune cells and tumors. Bryostatin-1 also induced cellular differentiation in melanoma cells, and reduced tumorigenic factors such as pro-cathepsins and matrix-metalloproteinase-9. These data suggest that bryostatin-1 could be used as a chemo-immunotherapeutic agent for reducing tumorigenic potential of melanoma cells while enhancing CD4+ T cell recognition to prevent tumor recurrence.     

Dissociation between Dorsal and Ventral Posterior Parietal Cortical Responses to Incidental Changes in Natural Scenes

Background

The posterior parietal cortex (PPC) is thought to interact with the medial temporal lobe (MTL) to support spatial cognition and topographical memory. While the response of medial temporal lobe regions to topographical stimuli has been intensively studied, much less research has focused on the role of PPC and its functional connectivity with the medial temporal lobe.

Methodology/Principle Findings

Here we report a dissociation between dorsal and ventral regions of PPC in response to different types of change in natural scenes using an fMRI adaptation paradigm. During scanning subjects performed an incidental target detection task whilst viewing trial unique sequentially presented pairs of natural scenes, each containing a single prominent object. We observed a dissociation between the superior parietal gyrus and the angular gyrus, with the former showing greater sensitivity to spatial change, and the latter showing greater sensitivity to scene novelty. In addition, we observed that the parahippocampal cortex has increased functional connectivity with the angular gyrus, but not superior parietal gyrus, when subjects view change to the scene content.

Conclusions/Significance

Our findings provide support for proposed dissociations between dorsal and ventral regions of PPC and suggest that the dorsal PPC may support the spatial coding of the visual environment even when this information is incidental to the task at hand. Further, through revealing the differential functional interactions of the SPG and AG with the MTL our results help advance our understanding of how the MTL and PPC cooperate to update representations of the world around us.     

Responses to oleic,linoleic and α-linolenic acids in high-carbohydrate,high-fat diet-induced metabolic syndrome in rats

We investigated the changes in adiposity, cardiovascular and liver structure and function, and tissue fatty acid compositions in response to oleic acid-rich macadamia oil, linoleic acid-rich safflower oil and α-linolenic acid-rich flaxseed oil (C18 unsaturated fatty acids) in rats fed either a diet high in simple sugars and mainly saturated fats or a diet high in polysaccharides (cornstarch) and low in fat. The fatty acids induced lipid redistribution away from the abdomen, more pronounced with increasing unsaturation; only oleic acid increased whole-body adiposity. Oleic acid decreased plasma total cholesterol without changing triglycerides and nonesterified fatty acids, whereas linoleic and α-linolenic acids decreased plasma triglycerides and nonesterified fatty acids but not cholesterol. α-Linolenic acid improved left ventricular structure and function, diastolic stiffness and systolic blood pressure. Neither oleic nor linoleic acid changed the left ventricular remodeling induced by high-carbohydrate, high-fat diet, but both induced dilation of the left ventricle and functional deterioration in low fat-diet-fed rats. α-Linolenic acid improved glucose tolerance, while oleic and linoleic acids increased basal plasma glucose concentrations. Oleic and α-linolenic acids, but not linoleic acid, normalized systolic blood pressure. Only oleic acid reduced plasma markers of liver damage. The C18 unsaturated fatty acids reduced trans fatty acids in the heart, liver and skeletal muscle with lowered stearoyl-CoA desaturase-1 activity index; linoleic and α-linolenic acids increased accumulation of their C22 elongated products. These results demonstrate different physiological and biochemical responses to primary C18 unsaturated fatty acids in a rat model of human metabolic syndrome.     

The catalase activity of diiron adenine deaminase

Adenine deaminase (ADE) from the amidohydrolase superfamily (AHS) of enzymes catalyzes the conversion of adenine to hypoxanthine and ammonia. Enzyme isolated from Escherichia coli was largely inactive toward the deamination of adenine. Molecular weight determinations by mass spectrometry provided evidence that multiple histidine and methionine residues were oxygenated. When iron was sequestered with a metal chelator and the growth medium supplemented with Mn²⁺ before induction, the post-translational modifications disappeared. Enzyme expressed and purified under these conditions was substantially more active for adenine deamination. Apo-enzyme was prepared and reconstituted with two equivalents of FeSO₄. Inductively coupled plasma mass spectrometry and Mössbauer spectroscopy demonstrated that this protein contained two high-spin ferrous ions per monomer of ADE. In addition to the adenine deaminase activity, [Fe^II/Fe^II]-ADE catalyzed the conversion of H₂O₂ to O₂ and H₂O. The values of k_cat and k_cat/K_m for the catalase activity are 200 s⁻¹ and 2.4 × 10⁴ M⁻¹ s⁻¹, respectively. [Fe^II/Fe^II]-ADE underwent more than 100 turnovers with H₂O₂ before the enzyme was inactivated due to oxygenation of histidine residues critical for metal binding. The iron in the inactive enzyme was high-spin ferric with g_ave = 4.3 EPR signal and no evidence of anti-ferromagnetic spin-coupling. A model is proposed for the disproportionation of H₂O₂ by [Fe^II/Fe^II]-ADE that involves the cycling of the binuclear metal center between the di-ferric and di-ferrous oxidation states. Oxygenation of active site residues occurs via release of hydroxyl radicals. These findings represent the first report of redox reaction catalysis by any member of the AHS.     

Solution structure of the guanine nucleotide-binding STAS domain of SLC26-related SulP protein Rv1739c from Mycobacterium tuberculosis

The structure and intrinsic activities of conserved STAS domains of the ubiquitous SulP/SLC26 anion transporter superfamily have until recently remained unknown. Here we report the heteronuclear, multidimensional NMR spectroscopy solution structure of the STAS domain from the SulP/SLC26 putative anion transporter Rv1739c of Mycobacterium tuberculosis. The 0.87-Å root mean square deviation structure revealed a four-stranded β-sheet with five interspersed α-helices, resembling the anti-σ factor antagonist fold. Rv1739c STAS was shown to be a guanine nucleotide-binding protein, as revealed by nucleotide-dependent quench of intrinsic STAS fluorescence and photoaffinity labeling. NMR chemical shift perturbation analysis partnered with in silico docking calculations identified solvent-exposed STAS residues involved in nucleotide binding. Rv1739c STAS was not an in vitro substrate of mycobacterial kinases or anti-σ factors. These results demonstrate that Rv1739c STAS binds guanine nucleotides at physiological concentrations and undergoes a ligand-induced conformational change but, unlike anti-σ factor antagonists, may not mediate signals via phosphorylation.     

Association of CYP3A5*3 polymorphism with development of acute leukemia

BACKGROUND:

CYP3A5 was observed to be an important genetic contributor to inter individual differences in CYP3A-dependent drug metabolism in acute leukemic patients. Loss of CYP3A5 expression was mainly conferred by a single nucleotide polymorphism at 6986A>G (CYP3A5*3). We investigated the association between CYP3A5*3 polymorphism and acute leukemia.

MATERIALS AND METHODS:

Two hundred and eighty nine acute leukemia cases comprising of 145 acute lymphocytic leukemia (ALL), 144 acute myeloid leukemia and 241 control samples were analyzed for CYP3A5*3 polymorphism using PCR-RFLP method. Statistical analysis was performed with SPSS version (15.0) to detect the association between CYP3A5*3 polymorphism and acute leukemia.

RESULTS:

The CYP3A5*3 polymorphism 3/3 genotype was significantly associated with acute leukemia development (χ²- 133.53; df-2, P 0.000). When the data was analyzed with respect to clinical variables, mean WBC, blast % and LDH levels were increased in both ALL and AML cases with 3/3 genotype. The epidemiological variables did not contribute to the genotype risk to develop either AML or ALL.

CONCLUSION:

The results suggest that the CYP3A5*3 polymorphism might confer the risk to develop ALL or AML emphasizing the significance of effective phase I detoxification in carcinogenesis. Association of the polymorphism with clinical variables indicate that the 3/3 genotype might also contribute to poorer survival of the patients.     

Role of MAPK phosphatase-1 in sustained activation of JNK during ethanol-induced apoptosis in hepatocyte-like VL-17A cells

Ethanol metabolism plays a central role in activating the mitogen-activated protein kinase (MAPK) cascade leading to inflammation and apoptosis. Sustained activation of c-Jun N-terminal kinase (JNK), one of the MAPKs, has been shown to induce apoptosis in hepatocytes. MAPK phosphatase-1 (MKP-1) has been shown to dephosphorylate MAPKs in several cells. The aim of the study is to evaluate the role of MKP-1 in sustained JNK activation as a mechanism to explain ethanol-induced hepatocyte apoptosis. VL-17A cells (HepG2 cells overexpressing alcohol dehydrogenase and cytochrome P450-2E1) were exposed to ethanol for different time periods. Western blots were performed for MKP-1, phospho-JNK, phosphotyrosine, and protein kinase Cdelta (PKCdelta). Electrophoretic mobility shift assays for AP-1 were performed. Apoptosis was measured by caspase-3 activity assay, TUNEL, and 4',6-diamidino-2-phenylindole staining. Reactive oxygen species were neutralized by overexpressing both superoxide dismutase-3 and catalase genes using lentiviral vectors in VL-17A cells. Ethanol incubation markedly decreased the MKP-1 protein levels to 15% of control levels and was associated with sustained phosphorylation of p46 JNK and p54 JNK, as well as increased apoptosis. VL-17A cells overexpressing superoxide dismutase-3 and catalase, treatment with a tyrosine kinase inhibitor, or incubation of the cells with PKCdelta small interference RNAs significantly inhibited the ethanol-induced MKP-1 degradation and apoptosis. Ethanol-induced oxidative stress enhanced the tyrosine phosphorylation of PKCdelta, which in turn caused the proteasomal degradation of MKP-1, leading to sustained JNK activation and increased apoptosis in VL-17A cells.