Nitish R Mahapatra

Chromogranin A (CHGA) is ubiquitously expressed in secretory cells of the endocrine, neuroendocrine, and neuronal tissues. Although this protein has long been known as a marker for neuroendocrine tumors, its role in cardiovascular disease states including essential hypertension (EH) has only recently been recognized. It acts as a prohormone giving rise to bioactive peptides such as vasostatin-I (human CHGA1-76) and catestatin (human CHGA352-372) that exhibit several cardiovascular regulatory functions. CHGA is over-expressed but catestatin is diminished in EH. Moreover, genetic variants in the promoter, catestatin, and 3'-untranslated regions of the human CHGA gene alter autonomic activity and blood pressure. Consistent with these findings, targeted ablation of this gene causes severe arterial hypertension and ventricular hypertrophy in mice. Transgenic expression of the human CHGA gene or exogenous administration of catestatin restores blood pressure in these mice. Thus, the accumulated evidence establishes CHGA as a novel susceptibility gene for EH.

Antagonistic bacteria are common soil inhabitants with potential to control several soil-borne diseases of various crops. In this study, two methomyl degrading Bacillus sp. were screened for their antagonistic potential against soil borne pathogen identified as Alternaria sp. which causes leaf spot and blight disease in lentil. Both the strains produced non-volatile and volatile organic compounds, extracellular enzymes, siderophore, indole acetic acid and solubilized phosphate which ascribed to the mechanism of bio-control and plant growth promotion. These bacterial strains produced indole acetic acid, chitinase and solubilized phosphate even in presence of pesticides (namely methomyl, carbendazim and imidacloprid). The production of chitinase increased by 51–140% in presence of different tested pesticides by the bacterial strains. However, phosphate solublization was only increased up to 79% in B. cereus and 87% in B. safensis in presence of methomyl. Both strains promoted plant growth and suppressed leaf spot and the incidence of blight in lentil plants under controlled conditions in green house. Application of B. cereus and B. safensis isolates to sterile rhizospheric soil increased the dry weight of plants by 40.8% and 43.2%, respectively as compared to control. In similar set of experiments the disease incidence was reduced by 67.7–81.6% in B. cereus and 57.2–78.8% in B. safensis in sterile condition and by 51.4–76.5% and 48.6–63.4%, respectively in non-sterile condition. The present investigation shows both B. cereus and B. safensis as potential plant growth promoting rhizobacteria that can be exploited as efficient bio-control organisms against soil borne plant pathogens as well as can be applied in plant growth enhancement even in pesticide infested soil.

Catestatin (CST), a chromogranin A (CHGA)-derived peptide, is a potent inhibitor of catecholamine release from adrenal chromaffin cells and postganglionic sympathetic axons. We resequenced the CST region of CHGA in an Indian population (n = 1010) and detected two amino acid substitution variants: G364S and G367V. Synthesized CST variant peptides (viz. CST-Ser-364 and CST-Val-367) were significantly less potent than the wild type peptide (CST-WT) to inhibit nicotine-stimulated catecholamine secretion from PC12 cells. Consistently, the rank-order of blockade of nicotinic acetylcholine receptor (nAChR)-stimulated inward current and intracellular Ca2+ rise by these peptides in PC12 cells was: CST-WT > CST-Ser-364 > CST-Val-367. Structural analysis by CD spectroscopy coupled with molecular dynamics simulations revealed the following order of α-helical content: CST-WT > CST-Ser-364 > CST-Val-367; docking of CST peptides onto a major human nAChR subtype and molecular dynamics simulations also predicted the above rank order for their binding affinity with nAChR and the extent of occlusion of the receptor pore, providing a mechanistic basis for differential potencies. The G364S polymorphism was in strong linkage disequilibrium with several common CHGA genetic variations. Interestingly, the Ser-364 allele (detected in ∼15% subjects) was strongly associated with profound reduction (up to ∼2.1-fold) in plasma norepinephrine/epinephrine levels consistent with the diminished nAChR desensitization-blocking effect of CST-Ser-364 as compared with CST-WT. Additionally, the Ser-364 allele showed strong associations with elevated levels of plasma triglyceride and glucose levels. In conclusion, a common CHGA variant in an Indian population influences several biochemical parameters relevant to cardiovascular/metabolic disorders. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.

Renalase, a recently identified oxidoreductase, is emerging as a novel regulator of cardiovascular and metabolic disease states. The mechanism of regulation of renalase gene, especially at the post-transcriptional level, is completely unknown. We set out to investigate the possible role of microRNAs in regulation of renalase gene in this study. Computational predictions using multiple algorithms coupled with systematic functional analysis revealed specific interactions of miR-29a/b/c and miR-146a/b with mouse and human renalase 3′-UTR (untranslated region) in cultured cells. Next, we estimated miR-29b and miR-146a, as well as renalase expression, in genetically hypertensive blood pressure high and genetically hypotensive blood pressure low mice. Kidney tissues from blood pressure high mice showed diminished (∼ 1.6- to 1.8-fold) renalase mRNA/protein levels and elevated (∼ 2.2-fold) miR-29b levels as compared to blood pressure low mice. A common single nucleotide polymorphism in human renalase 3′-UTR (C/T; rs10749571) creates a binding site for miR-146a; consistently, miR-146a down-regulated human renalase 3′-UTR/luciferase activity in case of the T allele suggesting its potential role in regulation of renalase in humans. Indeed, genome-wide association studies revealed directionally concordant association of rs10749571 with diastolic blood pressure, glucose and triglyceride levels in large human populations (n ≈ 58,000-96,000 subjects). This study provides evidence for post-transcriptional regulation of renalase gene by miR-29 and miR-146 and has implications for inter-individual variations on cardiometabolic traits.

Although the transcript level of inducible heat shock protein 70.3 (Hsp70.3, also known as Hspa1a) is altered in various disease states, its transcriptional regulation remains incompletely understood. Here, we systematically analyzed the Hspa1a promoter to identify major cis elements and transcription factors that may govern the constitutive/inducible gene expression. Computational analyses coupled with extensive in vitro (promoter-reporter activity and electrophoretic mobility shift assays) and in vivo (chromatin immunoprecipitation assays) revealed interaction of several transcription factors with Hspa1a promoter motifs: HSF-1 (heat shock factor 1) at - 114/- 97 bp and - 788/- 777 bp, NF-Y (nuclear transcription factor Y) at - 73/- 58 bp, NF-κB (nuclear factor kappa B) at - 133/- 124 bp, and CREB (cAMP response element binding protein) at - 483/- 476 bp. Consistently, siRNA (small interfering RNA)-mediated down-regulation of each of these transcription factors caused substantial reduction of endogenous Hspa1a expression. Heat-shock-induced activation of Hspa1a was coordinately regulated by HSF-1 and NF-Y/NF-κB. The Hspa1a expression was augmented by TNF-α (tumor necrosis factor-alpha) and forskolin in NF-κB and CREB-dependent manners, respectively. NF-κB and CREB also activated Hspa1a transcription in cardiac myoblasts upon exposure to ischemia-like conditions. Taken together, this study discovered previously unknown roles for NF-κB and CREB to regulate Hspa1a expression and a coordinated action by several transcription factors for Hspa1a transactivation under heat-shock/ischemia-like conditions and thereby provided new insights into the mechanism of Hspa1a regulation. © 2013 Elsevier Ltd.

Despite the well-known role of cystathionine γ-lyase (Cth) in cardiovascular pathophysiology, transcriptional regulation of Cth remains incompletely understood. Sequencing of the Cth promoter region in mouse models of genetic/essential hypertension (viz. Blood Pressure High [BPH], Blood Pressure Low [BPL] and Blood Pressure Normal [BPN] mice) identified several genetic variations. Transient transfections of BPH/BPL-Cth promoter-reporter plasmids into various cell types revealed higher promoter activity of BPL-Cth than that of BPH-Cth. Corroboratively, endogenous Cth mRNA levels in kidney and liver tissues were also elevated in BPL mice. Computational analysis of the polymorphic Cth promoter region predicted differential binding affinity of c-Rel, HOXA3 and IRF1 with BPL/BPH-Cth promoter domains. Over-expression of c-Rel/HOXA3/IRF1 modulated BPL/BPH-Cth promoter activities in a consistent manner. Gel shift assays using BPH/BPL-Cth-promoter oligonucleotides with/without binding sites for c-Rel/HOXA3/IRF1 displayed formation of specific complexes with c-Rel/HOXA3/IRF1; addition of antibodies to reaction mixtures resulted in supershifts/inhibition of Cth promoter-transcription factor complexes. Furthermore, chromatin immunoprecipitation (ChIP) assays proved differential binding of c-Rel, HOXA3 and IRF1 with the polymorphic promoter region of BPL/BPH-Cth. Tumor necrosis factor-α (TNF-α) reduced the activities of BPL/BPH-Cth promoters to different extents that were further declined by ectopic expression of IRF1; on the other hand, siRNA-mediated down-regulation of IRF1 rescued the TNF-α-mediated suppression of the BPL/BPH-Cth promoter activities. In corroboration, ChIP analysis revealed enhanced binding of IRF1 with BPH/BPL-Cth promoter following TNF-α treatment. BPL/BPH-Cth promoter activity was diminished upon exposure of hepatocytes and cardiomyoblasts to ischemia-like pathological condition due to reduced binding of c-Rel with BPL/BPH-Cth-promoter. Taken together, this study reveals the molecular basis for the differential expression of Cth in mouse models of essential hypertension under basal and pathophysiological conditions.

Renalase, a novel monoamine oxidase, is emerging as an important regulator of cardiovascular, metabolic, and renal diseases. However, the mechanism of transcriptional regulation of this enzyme remains largely unknown. We undertook a systematic analysis of the renalase gene to identify regulatory promoter elements and transcription factors. Computational analysis coupled with transfection of human renalase promoter/luciferase reporter plasmids (5′-promoter-deletion constructs) into various cell types (HEK-293, IMR32, and HepG2) identified two crucial promoter domains at base pairs -485 to -399 and -252 to -150. Electrophoretic mobility shift assays using renalase promoter oligonucleotides with and without potential binding sites for transcription factors Sp1, STAT3, and ZBP89 displayed formation of specific complexes with HEK-293 nuclear proteins. Consistently, overexpression of Sp1, STAT3, and ZBP89 augmented renalase promoter activity; additionally, siRNA-mediated downregulation of Sp1, STAT3, and ZBP89 reduced the level of endogenous renalase transcription as well as the transfected renalase promoter activity. In addition, chromatin immunoprecipitation assays showed in vivo interactions of these transcription factors with renalase promoter. Interestingly, renalase promoter activity was augmented by nicotine and catecholamines; while Sp1 and STAT3 synergistically activated the nicotine-induced effect, Sp1 appeared to enhance epinephrine-evoked renalase transcription. Moreover, renalase transcript levels in mouse models of human essential hypertension were concomitantly associated with endogenous STAT3 and ZBP89 levels, suggesting crucial roles for these transcription factors in regulating renalase gene expression in cardiovascular pathological conditions.

The authors regret typing errors on page 132 in the DNA sequence of mHsp-2686-RT-RP primer in the second sentence of the subsection entitled “Extraction of RNA and real-time PCR” under the Materials and Methods section of the article. The sentence should be read as follows: Real-time PCR was carried out using the DyNAmo™ HS SYBR® Green qPCR kit (Finnzymes, USA) and mHspa1a gene-specific primers (forward, mHsp-2406-RT-FP: 5′-TGCCCCGCTGATGTGATTTG-3′ and reverse, mHsp-2686-RT-RP: 5′-CACCAACCTGGAAACAAGTCCTAC-3′). The authors would like to apologize for any inconvenience caused.

3-Hydroxy-3-methylglutaryl-coenzyme A [HMG-CoA] reductase gene (Hmgcr) is a susceptibility gene for essential hypertension. Sequencing of the Hmgcr locus in genetically hypertensive BPH (blood pressure high), genetically hypotensive BPL (blood pressure low) and genetically normotensive BPN (blood pressure normal) mice yielded a number of single nucleotide polymorphisms (SNPs). BPH/BPL/BPN Hmgcr promoter-luciferase reporter constructs were generated and transfected into liver HepG2, ovarian CHO, kidney HEK-293 and neuronal N2A cells for functional characterization of the promoter SNPs. The BPH-Hmgcr promoter showed significantly less activity than the BPL-Hmgcr promoter under basal as well as nicotine/cholesterol-treated conditions. This finding was consistent with lower endogenous Hmgcr expression in liver and lower plasma cholesterol in BPH mice. Transfection experiments using 5′-promoter deletion constructs (strategically made to assess the functional significance of each promoter SNP) and computational analysis predicted lower binding affinities of transcription factors c-Fos, n-Myc and Max with the BPH-promoter as compared to the BPL-promoter. Corroboratively, the BPH promoter-luciferase reporter construct co-transfected with expression plasmids of these transcription factors displayed less pronounced augmentation of luciferase activity than the BPL construct, particularly at lower amounts of transcription factor plasmids. Electrophoretic mobility shift assays also showed diminished interactions of the BPH promoter with HepG2 nuclear proteins. Taken together, this study provides mechanistic basis for the differential Hmgcr expression in these mouse models of human essential hypertension and have implications for better understanding the role of this gene in regulation of blood pressure. © 2011 Sonawane et al.