Ajaz Ahmad Waza

Protein kinase C (PKC) mediated interaction between conexin43 (Cx43) and K(+)(ATP) channel subunit (Kir6.1) in cardiomyocyte mitochondria: Implications in cytoprotection against hypoxia induced cell apoptosis.

PURPOSE OF RESEARCHnWe have recently shown that adenosine-triphosphate-sensitive potassium [K(+)(ATP)] channel protein subunit, Kir6.1 is a phosphospecific interaction partner of the gap-junction protein connexin43 (Cx43). Since, both Cx43 and K(+)(ATP) are known to be involved in cell survival during hypoxia, we addressed the question, whether the interaction between Cx43 and K(+)(ATP) has a role in protecting cell against hypoxia-induced cell death.nnnPRINCIPLE RESULTSnWe report here that the Kir6.1 protein interacts, in a phosphospecific manner with Cx43 in the mitochondria of cardiomyocytic cell line H9C2. The hypoxia for 12-h resulted in the appreciable increase in the phosphorylation at the serine 262 (S262) of the Cx43 with the concomitant increase in the Cx43 and Kir6.1 interaction. Moreover, the increased interaction was mediated by a signaling pathway involving PKC and more specifically by PKC epsilon. Functional implications of the association between the Cx43 and Kir6.1 were found to prevent mitochondria mediated hypoxia induced cell apoptosis.nnnMAJOR CONCLUSIONSnOur results demonstrate that PKC epsilon regulates the interaction between Cx43 and Kir6.1 in the cardiomyocyte mitochondria and this interaction prevents hypoxia induced cell death. Our results provide an interesting lead in developing effective strategies to protect cardiomyocytes from hypoxia/ischemia induced cell death.

Adenosine-triphosphate-sensitive K+ channel (Kir6.1): a novel phosphospecific interaction partner of connexin 43 (Cx43).

Connexin 43 (Cx43) is a phosphoprotein expressed in a wide variety of cells. Cx43 and adenosine-triphosphate-sensitive K(+)channels [K(+)(ATP)] are part of same signaling pathway that regulates cell survival during stress and ischemia preconditioning. Molecular mechanism for their coordinated role in ischemia/hypoxia preconditioning is not well known. Using pull down, co-immunoprecipitation assays and co-localization studies we provide evidence, for the first time that Kir6.1, a K(+)(ATP) channel protein component, can interact with Cx43. Further we show that the interaction was phospho-specific such that Cx43 phosphorylated at serine 262 (S262) interacted with Kir6.1 in preference to the unphosphorylated form of Cx43. Introduction of phospho-deficient mutation at serine 262 (S262A) in Cx43 completely abolished the interaction. Our data provide an interesting lead about a possible partnership between Cx43 and Kir6.1, which will help in better understanding their role in ischemia/hypoxia preconditioning.

A review on heme oxygenase-1 induction: is it a necessary evil

Heme oxygenase-1 (HO-1) is considered to be the main protein in diseases arising as a result of oxidative and inflammatory insults. Tremendous research has been carried out on HO-1 since years, pertaining its cytoprotective effect against oxidative injury and other cellular stresses. HO-1, by regulating intracellular levels of pro-oxidant heme, or by other benefits of its by-products such as carbon monoxide (CO) and biliverdin (BV) had become an important candidate protein to be up-regulated to combat diverse stressful events. Although the beneficial effects of HO-1 induction have been reported in a number of cells and tissues, a growing body of evidence indicates that this increased HO-1 expression may lead to the progression of several diseases such as neurodegeneration, carcinogenesis. But it is not clear, what accounts for the increased expression of HO-1 in cells and tissues. The observed friendly role of HO-1 in a wide range of stress conditions since times is now doubtful. Therefore, more studies are needed to elucidate the exact role of HO-1 in various stressful events. Being more concise, elucidating the effect of HO-1 up-regulation on critical genes involved in particular diseases such as cancer will help to a larger extent to comprehend the exact role of HO-1. This review will assist in understanding the dual role (protective and detrimental) of HO-1 and the signaling pathway involved and will help in unraveling the doubtful role of HO-1 induction.

Morphological and Molecular Characterization of Diplozoon kashmirensis;D. aegyptensis and D. guptai Collected from Fishes of Kashmir Valley-India

The study reports the results of molecular characterization of the Internal Transcribed Spacer (ITS) of ribosomal DNA of 3 Monogenean species using polymerase chain reaction (PCR), nucleotide sequencing and construction of phylogenetic trees from different fish hosts of Kashmir. The present study shows that the size of the amplified product is 873bp long for D. kashmirensis, 1120bp long in D. aegyptensis and 687bp long in D. guptai revealing that there are intraspecific differences in their base pair lengths. Guanine and Cytocine (G+C) content of three Diplozoon species was found nearly constant for three species i.e., 47% (D. kashmirensis); 47% (D. aegyptensis) and 48% (D. guptai), this GC richness contributes to physical attributes of RNA structures, as there is correlation between GC content and optimal growth temperature. An important observation during the present study has been noticed that Schizothorax niger is infected by all the three species of Diplozoidae; D. kashmirensis; D. aegyptensis and D. guptai, but when all six fishes were collected simultaneously, parasitism by all the parasite species was never observed. Phylogenetic trees Maximum Parsimony (MP), Maximum Likelihood (ML) and Neighbor Joining (NJ) showed that D. kashmirensis and D. aegyptensis share a common host Carassius carassius and S. niger.

Role of SCN1A and SCN2A Gene Polymorphisms in Epilepsy Syndromes-A Study from India

Introduction: Epilepsy is the most common heterogeneous neurological disorder affecting approximately 42 million people worldwide. Juvenile myoclonic epilepsy (JME) is a common form of idiopathic generalized epilepsy representing 5-10% of all epilepsy cases. Lennox-Gastaut syndrome (LGS) is one of the most severe epileptic encephalopathies of childhood onset, the cause of which may be symptomatic, i.e., secondary to an underlying brain disorder or cryptogenic, i.e., with no known cause. Sodium channels are integral membrane proteins which play a central role in neuronal membrane excitability and action potential generation. Alpha subunit of voltage gated sodium channels encoded by SCN1A, SCN2A and other genes is pivotal for neuronal signalling. It was planned to analyse the roles of SCN1A Thr1067Ala and SCN2A Arg19Lys polymorphisms in the pathophysiology and risk JME and LGS in the Indian population. nMethods: A total of 50 JME patients, 50 LGS Patients and 100 age and sex matched healthy volunteers were recruited in this study. The genotyping of SCN1A Thr1067Ala i.e., 3184 A>G (rs2298771) and SCN2A Arg19Lys i.e., 56 G>A (rs17183814) polymorphism was performed by polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP) analysis. nResults: The SCN1A Thr1067Ala polymorphism genotypic distribution in LGS was significantly different from the normal population (P=0.008), with mutant homozygous (GG) plus heterozygous (AG) genotypes’ percentage in LGS patients (16%) being lower than in healthy controls (24%). Frequency of the mutant ‘G’ allele of this SNP in LGS patients was 0.1, while it was 0.2 in control subjects (P=0.04). These observations which suggest a protective role of SCN1A Thr1067Ala polymorphism in LGS, were in sync with computation of an odds ratio of 0.21 (95% CI 0.07 to 0.66, p=0.005) for the GG genotype in LGS patients. Though no correlation of SCN1A Thr1067Ala SNP with the severity of disease phenotype in LGS viz. frequency/duration of seizures etc. was noted, a conflicting finding was the significant association of its mutant genotypes with an early age of onset of the syndrome (p=0.007). Contrary to the findings in SCN1A Thr1067Ala, in case of SCN2A Arg19Lys polymorphism, though a significantly different genotypic distribution was present in LGS, in comparison to normal population (p=0.03), the mutant homozygous (AA) and heterozygous (GA) combined percentage in LGS patients (16%) was greater than in healthy controls (11%). This was complemented by observation of an odds ratio of 4.24 (95% CI 1.15 to 15.55, p=0.029, in case of LGS patients with heterozygous (GA) genotype, indicative of an increased disease susceptibility. Unlike LGS, in JME patients no significant differences in genotypic/allelic frequencies of SCN1A Thr1067Ala and SCN2A Arg19Lys polymorphisms were noted and the associated odds ratios for mutant genotypes were also non-significant. nConclusion: The SCN1A Thr1067Ala and SCN2A Arg19Lys polymorphisms may play contrary roles in the pathophysiology of LGS. Inheritance of SCN1A Thr1067Ala mutant allele decreases the susceptibility for LGS occurrence, and may hamper Na+ channels opening and neuronal excitability. On the other hand, the mutant allele of SCN2A Arg19Lys polymorphism confers an increased risk for development of LGS, consequent to a likely facilitatory effect on action potential generation and misfiring in neurons. Neither of these two SNPs appears to influence the pathogenesis/susceptibility to JME.

Connexin 43 and ATP-sensitive potassium channels crosstalk: a missing link in hypoxia/ischemia stress

Connexin 43 (Cx43) is a gap junction protein expressed in various tissues and organs of vertebrates. Besides functioning as a gap junction, Cx43 also regulates diverse cellular processes like cell growth and differentiation, cell migration, cell survival, etc. Cx43 is critical for normal cardiac functioning and is therefore abundantly expressed in cardiomyocytes. On the other hand, ATP-sensitive potassium (KATP) channels are metabolic sensors converting metabolic changes into electrical activity. These channels are important in maintaining the neurotransmitter release, smooth muscle relaxation, cardiac action potential repolarization, normal physiology of cellular repolarization, insulin secretion and immune function. Cx43 and KATP channels are part of the same signaling pathway, regulating cell survival during stress conditions and ischemia/hypoxia preconditioning. However, the underlying molecular mechanism for their combined role in ischemia/hypoxia preconditioning is largely unknown. The current review focuses on understanding the molecular mechanism responsible for the coordinated role of Cx43 and KATP channel protein in protecting cardiomyocytes against ischemia/hypoxia stress.

Promoter Hypermethylation and Its Impact on Expression of MGMT Gene in the GIT Malignant Patients of Kashmiri Origin

ABSTRACT Epigenetic alterations, in addition to multiple gene abnormalities, are involved in the genesis and progression of human cancers. Gastrointestinal tract (GIT) cancer is a major medical and economic burden worldwide. Aberrant methylation of CpG islands within promoter regions is associated with transcriptional inactivation of various tumor suppressor genes. Although a number of cancer-associated genes have been found to be hypermethylated in GIT cancer, valuable methylation markers for early diagnosis and prognostic evaluation of this cancer remain largely unknown. O6-methyguanine DNA methyltransferase (MGMT) is a DNA-repair gene that removes mutagenic and cytotoxic adducts from the O6 position of guanine induced by alkylating agents. MGMT promoter hypermethylation and reduced expression have been found in some primary human carcinomas. We studied DNA methylation of CpG islands of the MGMT gene and its relation with MGMT protein expression in human GIT carcinomas. A total of 210 GIT tumor samples and 90 adjacent normal tissues were analyzed for MGMT promoter methylation by methylation-specific polymerase chain reaction after bisulfite modification of DNA and same samples were analyzed for MGMT protein expression by Western blotting. The methylation frequencies of MGMT gene promoter were 41.4%, 34.2%, and 44.2% in stomach, esophageal, and colorectal cancer cases while as 16.6, 13.3, and 13.3 in respective controls. MGMT protein was found downregulated in controls of all GIT. The results suggest that methylation at CpG islands of MGMT may be responsible for the downregulation of MGMT protein expression in GIT cancers.

Diminished expression of MGMT & RASSF1A genes in gastric cancer in ethnic population of Kashmir

BACKGROUNDnCancer initiation and progression are accompanied by profound changes in DNA. DNA methylation that was the first epigenetic alterations identified in cancer. DNA hypermethylation at promoter sites is closely associated with down regulation of protein and as major participant in the development and progression of series of human tumors. Therefore we hypothesized that promoter hypermethylation of RASSF1A & MGMT gene could influence susceptibility to gastric cancer (GC) as well, and we conducted this study to test the hypothesis in Kashmiri population.nnnMETHODSnA hospital based case-control study; including 200 GC cases and 200 matched controls from patients who went surgical resection. Promoter hypermethylation was determined by Methylation Specific Polymerase chain reaction. The expression of MGMT & RASSF1A protein was examined by Western blotting technique.nnnRESULTSnFrequency of promoter region hypermethylation of MGMT gene were 46.5% in cases and 5.5% in controls (P<0.05) while as in case of RASSF1A frequency was 44% in cases and 4.5% in controls (P<0.05). Further, frequency of hypermethylation of both genes was found predominant in males, aged and advanced pathological stage subjects. Loss of MGMT expression was found in 46.5% cases (P<0.05) while as loss of RASSF1A expression was found in 40.5% cases (P<0.05). In both genes a positive correlation was observed between promoter CpG island hypermethylation and down regulation of respective proteins.nnnCONCLUSIONSnThese findings indicate that promoter hypermethylation at CpG island may be responsible for reduction of expression at protein level which may be an initial event in carcinogenesis and the progression of GC.

Relaxin protects cardiomyocytes against hypoxia-induced damage in in-vitro conditions: Involvement of Nrf2/HO-1 signaling pathway

&NA; Relaxin, a peptide hormone has emerged as a cardioprotective agent against the heart failure and has been found to protect cardiac muscle cells against hypoxia/reoxygenation injury under in vitro conditions. The present study was conducted to study its possible role in activating the Nrf2/HO‐1 signaling pathway in cardiomyocytes, as a means to counter hypoxia associated oxidative damage and cell death. H9C2 cell line was induced with chemical hypoxia alone or together with relaxin. Hypoxia associated cellular damage and reactive oxygen species (ROS) production was accessed by Lactate dehydrogenase (LDH) release and DCFDA activity respectively. The anti‐oxidative property of RLXH2 was measures by assessing the activities of different enzymes like Superoxide dismutase (SOD), Catalase (CAT) and Glutathione peroxidase (GSX). Expression levels of Nrf2 and HO‐1 was studied by immunoblotting and quantitative real time PCR (qRT‐PCR). Translocation of Nrf2 to nucleus was studied by immunoblotting. Our results found that hypoxia associated lactate dehydrogenase leakage and ROS production is countered by RLXH2 treatment. Similarly, RLXH2 was able to counter hypoxia induced oxidative damage as evident by increased activities of SOD, CAT and GSX. Furthermore, it was found that RLXH2 treatment induces translocation of Nrf2 from cytosol to nucleus and in turn enhances expression level of HO‐1. Our results suggest that RLXH2 exerts cytoprotective action in cardiomyocytes against the hypoxia induced damage and activates Nrf2/HO‐1 signaling pathway.

Association of GABAA Receptor Gene with Epilepsy Syndromes

GABA has always been an inviting target in the etiology and treatment of epilepsy. The GABRA1, GABRG2, and GABRD genes provide instructions for making α1, ϒ2, and δ subunits of GABAA receptor protein respectively. GABAA is considered as one of the most important proteins and has found to play an important role in many neurological disorders. We explored the association of GABAA receptor gene mutation/SNPs in JME and LGS patients in Indian population. A total of 100 epilepsy syndrome patients (50 JME and 50 LGS) and 100 healthy control subjects were recruited and analyzed by AS-PCR and RFLP-PCR techniques. In our study, GABRA1 965 Cu2009>u2009A mutation and 15 Au2009>u2009G polymorphism gene may play an important role in modulating the drug efficacy in LGS patients. The GABRA1 15 Au2009>u2009G polymorphism may also play an important role in the susceptibility of LGS and the inheritance of GG genotype of this polymorphism may provide an increased risk of development of LGS. The GABRG2 588 Cu2009>u2009T polymorphism may decrease the duration of seizures in JME patients. The GABRD 659xa0Gu2009>u2009A polymorphism may play an important role in the susceptibility of JME and LGS and this polymorphism may also increase the duration of postictal period in JME patients but may decrease the duration of seizure in LGS patients.