Integrated morphometric and molecular approaches to screen hybrid from wild Labeo rohita and Labeo catla parent populations.

BACKGROUND: Interspecific hybrids of rohu (Labeo rohita) and catla (Labeo catla) are common, especially in India due to constrained breeding. These hybrids must segregate from their wild parents as part of conservational strategies. This study intended to screen the hybrids from wild rohu and catla parents using both morphometric and molecular approaches. METHODS & RESULTS: The carp samples were collected from Jharkhand and West Bengal, India. The correlation and regression analysis of morphometric features are considered superficial but could be protracted statistically by clustering analysis and further consolidated by nucleotide variations of one mitochondrial and one nuclear gene to differentiate hybrids from their parents. Out of 21 morphometric features, 6 were used for clustering analysis that exhibited discrete separation among rohu, catla, and their hybrids when the data points were plotted in a low-dimensional 2-D plane using the first 2 principal components. Out of 40 selected single nucleotide polymorphism (SNP) positions of the COX1 gene, hybrid showed 100% similarity with catla. Concerning SNP similarity of the 18S rRNA nuclear gene, the hybrid showed 100% similarity with rohu but not with catla; exhibiting its probable parental inheritance. CONCLUSIONS: Along with morphometric analysis, the SNP comparison study together points towards strong evidence of interspecific hybridization between rohu and catla, as these hybrids share both morphological and molecular differences with either parent. However, this study will help screen the hybrids from their wild parents, as a strategy for conservational management.

Prevalence of familial hypercholesterolemia in patients with confirmed premature coronary artery disease in Ranchi, Jharkhand.

BACKGROUND: Familial hypercholesterolemia (FH) is an under-diagnosed autosomal co-dominant genetic disorder characterized by very high plasma levels of low-density lipoprotein cholesterol (LDL-C), premature coronary artery disease (CAD) with arcus cornealis, and xanthomas. Among patients with CAD, the frequency of FH is significantly higher than that of the general population, but little data are available in India in this regard. This study aimed to assess the prevalence of FH in patients with premature coronary artery disease for the first time in the Jharkhand population. RESULTS: The study was conducted on 200 premature CAD patients at RIMS hospital, Ranchi, from January 2020 to June 2021 with CAG-confirmed acute coronary syndrome. The study, without taking the aid of genetic profiling of the patients and using the Dutch Lipid Clinic Network Criteria, revealed quite a high (23.5%) prevalence of potential FH in patients with premature CAD apart from the conventional risk factors. Mean LDL-C levels among patients with definite, probable, possible, and no FH were recorded as 250.39, 184.32, 136.11, and 108.09 mg/dl, respectively. Arcus cornealis was seen in 55.31% of patients with potential FH, 90% in definite FH, and 44.40% with probable FH. Patients with potential FH were more likely to be younger (age < 40 years) males, having a history of CAD and a family history of premature CAD as compared to patients without FH. CONCLUSIONS: There was no previous report of large studies on FH or its epidemiology and its natural history from India. The present study is the first one to show a high prevalence of potential FH in premature CAD (about 23.5%). This preliminary study revealed that the prevalence of FH in patients with premature CAD who came to the tertiary care hospital of Ranchi, Jharkhand, was high, apart from the conventional risk factors.

Prevalence of CVD risk factors among some tribal and nontribal populations of Jharkhand - A comparative survey.

BACKGROUND: World especially India had been witnessing a sharp increase of death and disability due to cardiovascular disease (CVD). Prevention, cure and management of cardiovascular diseases (CVD) necessitate true picture of the spread of CVD risk factors throughout the country. Among few surveys in India, very little reports were from state Jharkhand. This study aimed to report the status of CVD risk factors among tribal and nontribal population of Jharkhand. METHODS: Comparative study was conducted on rural tribals and urban nontribals with no apparent CVD related symptoms. Informed consents, filled up CVD risk questionnaire were collected. Anthropometric and behavioral data with measured Blood pressure (BP), blood sugar and body mass index (BMI) were analyzed by multivariate logistic regression to find out the association of inappropriate features related to CVD, if any with age, gender and ethnicity. RESULTS: Almost 2 fold more susceptibility to develop hypertension, pre-hypertension and obesity was found in nontribals over 8.0%, 15.6% and 22.1% of respective affected tribals. This trend increased up to 3 fold in > 40 years age group. This >40 when compared with < 40 years age group nearly 16 fold and 11 fold significantly increased hypertension risk was reported in nontribals and tribals respectively We report 14.8% diabetic in tribals, highest so far in India. CONCLUSION: With age above 40 years, susceptibility to different CVD risk factors like hypertension, pre-hypertension, obesity, high MAP reported to increase severely in urban nontribals than rural tribals.

Catecholamine biosynthesis and secretion: physiological and pharmacological effects of secretin.

Pituitary adenylyl cyclase activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) augment the biosynthesis of tyrosine hydroxylase (TH). We tested whether secretin belonging to the glucagon/PACAP/VIP superfamily would increase transcription of the tyrosine hydroxylase (Th) gene and modulate catecholamine secretion. Secretin activated transcription of the endogenous Th gene and its transfected promoter (EC(50) ∼4.6 nM) in pheochromocytoma (PC12) cells. This was abolished by pre-treatment with a secretin receptor (SCTR) antagonist and by inhibition of protein kinase A (PKA), mitogen-activated protein kinase, or CREB (cAMP response element-binding protein). In agreement, secretin increased PKA activity and induced phosphorylation of CREB and binding to Th CRE, suggesting secretin signaling to transcription via a PKA-CREB pathway. Secretin stimulated catecholamine secretion (EC(50) ∼3.5 µM) from PC12 cells, but this was inhibited by pre-treatment with VIP-preferring receptor (VPAC1)/PACAP-preferring receptor (PAC1) antagonists. Secretin-evoked secretion occurred without extracellular Ca(2+) and was abolished by intracellular Ca(2+) chelation. Secretin augmented phospholipase C (PLC) activity and increased inositol-1,4,5-triphosphate (IP(3)) levels in PC12 cells; PLC-ß inhibition blocked secretin-induced catecholamine secretion, indicating the participation of intracellular Ca(2+) from a phospholipase pathway in secretion. Like PACAP, secretin evoked long-lasting catecholamine secretion, even after only a transient exposure. Thus, transcription is triggered by nanomolar concentrations of the peptide through SCTR, with signaling along the cAMP-PKA and extracellular-signal-regulated kinase 1/2 pathways and through CREB. By contrast, secretion is triggered only by micromolar concentrations of peptide through PAC1/VPAC receptors and by utilizing a PLC/intracellular Ca(2+) pathway.

Biochanin-A, an isoflavon, showed anti-proliferative and anti-inflammatory activities through the inhibition of iNOS expression, p38-MAPK and ATF-2 phosphorylation and blocking NFκB nuclear translocation.

Biochanin-A, an isoflavone, existing in red clover, cabbage and alfalfa, has an inhibitory and apoptogenic effect on certain cancer cells. However, the actual mechanism by which this compound inhibits proliferation and induces apoptosis in cancer cells and the mechanism of its anti-inflammatory activities have not been well characterized. In this study, we have investigated the anti-inflammatory and anti-proliferative activity of Biochanin-A. The effects of Biochanin-A on RAW 264.7, HT-29 cell lines and mouse peritoneal macrophages have been investigated in vitro. Cell proliferation and anti-inflammatory effects were analyzed by 3-(4-5-dimethylthiozol-2-yl)2-5-diphenyl-tetrazolium bromide (MTT) assay, (3)H-thymidine incorporation assay, Western blot, cytokines estimation, Luciferase assay, Electrophoretic mobility shift assay (EMSA) and Kinase assay. Present investigation demonstrated that, Biochanin-A inhibited lipopolysacharide (LPS)-induced nitric oxide(NO) production in macrophage and showed dose dependent inhibition of inducible nitric oxide synthase (iNOS) expression. The induction of NF-κB binding activity by LPS was inhibited markedly by co-incubation with different doses of Biochanin-A. Biochanin-A inhibited the LPS-induced IkB kinase (IKK) activity and nuclear factor kappa beta (NF-κB) activation associated with the inhibition of iNOS expression. LPS-induced phosphorylation of IκBα and p38 MAPK was blocked by Biochanin-A and it inhibited IL-6, IL-1ß and TNF-α production in RAW264.7 cells indicating its anti-inflammatory activity in association with anti-proliferation. Biochanin-A is important for the prevention of phosphorylation and degradation of IκBα, thereby blocking NF-κB activation, which in turn leads to decreased expression of the iNOS, thus preventing proliferation and inflammation.

Common functional genetic variants in catecholamine storage vesicle protein promoter motifs interact to trigger systemic hypertension.

OBJECTIVES: The purpose of this study is to understand whether naturally occurring genetic variation in the promoter of chromogranin B (CHGB), a major constituent of catecholamine storage vesicles, is functional and confers risk for cardiovascular disease. BACKGROUND: CHGB plays a necessary (catalytic) role in catecholamine storage vesicle biogenesis. Previously, we found that genetic variation at CHGB influenced autonomic function, with association maximal toward the 5' region. METHODS: Here we explored transcriptional mechanisms of such effects, characterizing 2 common variants in the proximal promoter, A-296C and A-261T, using transfection/cotransfection, electrophoretic mobility shift assay (EMSA), and chromatin immunoprecipitation (ChIP). We then tested the effects of promoter variation on cardiovascular traits. RESULTS: The A-296C disrupted a c-FOS motif, exhibiting differential mobility shifting to chromaffin cell nuclear proteins during EMSA, binding of endogenous c-FOS on ChIP, and differential response to exogenous c-FOS. The A-261T disrupted motifs for SRY and YY1, with similar consequences for EMSA, endogenous factor binding, and responses to exogenous factors. The 2-SNP CHGB promoter haplotypes had a profound (p=3.16E-20) effect on blood pressure (BP) in the European ancestry population, with a rank order of CT

Conserved regulatory motifs at phenylethanolamine N-methyltransferase (PNMT) are disrupted by common functional genetic variation: an integrated computational/experimental approach.

The adrenomedullary hormone epinephrine transduces environmental stressors into cardiovascular events (tachycardia and hypertension). Although the epinephrine biosynthetic enzyme PNMT genetic locus displays both linkage and association to such traits, genetic variation underlying these quantitative phenotypes is not established. Using an integrated suite of computational and experimental approaches, we elucidate a functional mechanism for common (minor allele frequencies > 30%) genetic variants at PNMT. Transcription factor binding motif prediction on mammalian PNMT promoter alignments identified two variant regulatory motifs, SP1 and EGR1, disrupted by G-367A (rs3764351), and SOX17 motif created by G-161A (rs876493). Electrophoretic mobility shifts of approximately 30-bp oligonucleotides containing ancestral versus variant alleles validated the computational hypothesis. Queried against chromaffin cell nuclear protein extracts, only the G-367 and -161A alleles shifted. Specific antibodies applied in electrophoretic gel shift experiments confirmed binding of SP1 and EGR1 to G-367 and SOX17 to -161A. The in vitro allele-specific binding was verified in cella through promoter reporter assays: lower activity for -367A haplotypes cotransfected by SP1 (p = 0.002) and EGR1 (p = 0.034); and enhanced inhibition of -161A haplotypes (p = 0.0003) cotransfected with SP1 + SOX17. Finally, we probed cis/trans regulation with endogenous factors by chromatin immunoprecipitation using SP1/EGR1/SOX17 antibodies. We describe the systematic application of complementary computational and experimental techniques to detect and document functional genetic variation in a trait-associated regulatory region. The results provide insight into cis and trans transcriptional mechanisms whereby common variation at PNMT can give rise to quantitative changes in human physiological and disease traits. Thus, PNMT variants in cis may interact with nuclear factors in trans to govern adrenergic activity.

Molecular basis of neuroendocrine cell type-specific expression of the chromogranin B gene: Crucial role of the transcription factors CREB, AP-2, Egr-1 and Sp1.

The molecular basis of neuroendocrine-specific expression of chromogranin B gene (Chgb) has remained elusive. Utilizing wild-type and mutant Chgb promoter/luciferase reporter constructs, this study established a crucial role for the cAMP response element (CRE) box at -102/-95 bp in endocrine [rat pheochromocytoma (chromaffin) cell line (PC12) and rat pituitary somatotrope cell line (GC)] and neuronal [rat dorsal root ganglion/mouse neuroblastoma hybrid cell line (F-11), cortical and hippocampal primary neurons] cells. Additionally, G/C-rich domains at -134/-127, -125/-117 and -115/-110 bp played especially important roles for endocrine-specific expression of the Chgb gene. Co-transfection of expression plasmids for CREB, activator protein-2 (transcription factor) (AP-2), early growth response protein (transcription factor) (Egr-1) or specificity protein 1 (transcription factor) (Sp1) with the Chgb promoter constructs trans-activated expression of the Chgb gene. Nuclear extracts from either PC12 or F-11 cells formed specific complexes with the Chgb (-110/-87 bp) (CRE) oligonucleotide, which were either supershifted or disrupted by anti-CREB antibodies. In addition PC12 nuclear extracts also formed a specific complex with a Chgb (-140/-104-bp) oligonucleotide containing three G/C-rich regions, which was dose-dependently disrupted by anti-AP-2, anti-Egr-1 or anti-Sp1 antibodies; indeed, any one of these three antibodies completely abolished the complex, suggesting that all three factors bind the region simultaneously, at least in vitro. Chromatin immunoprecipitation assays documented the binding of the transcription factors CREB, AP-2, Egr-1 and Sp1 to the chromosomal Chgb gene promoter in vivo in PC12 cells within the context of chromatin. We conclude that the neuroendocrine-specific expression of Chgb is mediated by the CRE and G/C boxes in cis and the transcription factors CREB, AP-2, Egr-1 and Sp1 in trans.

Integration of metal-resistant determinants from the plasmid of an Acidocella strain into the chromosome of Escherichia coli DH5alpha.

Acidophilic bacteria of mine origin are ideal systems for studying microbial metal resistance because of their ability to grow in the presence of high concentrations of metal salts. We have previously shown that the metal-resistant transformants obtained after transformation of Escherichia coli DH5alpha with plasmid DNA preparation from Acidocella sp. strain GS19h did not contain any plasmid suggesting chromosomal integration of the plasmid(s) (Appl Environ Microbiol 1997; 63: 4523-4527). The present study provides evidence in support of this suggestion. The pulsed field gel electrophoresis (PFGE) pattern of genomic DNA of the plasmidless metal-resistant transformants differed markedly from that of the untransformed DH5alpha strain. Moreover, when the recombinant plasmids constructed by cloning plasmid DNA fragments of the Acidocella strain GS19h in the vector pBluescript II KS+ were used to transform E. coli DH5alpha strain, no plasmid DNA was detected in some of the zinc- and ampicillin-resistant (ZnrAmpr) clones. The PFGE pattern of genomic DNA of such a transformed clone also differed markedly from that of the parent strain, suggesting chromosomal integration of the recombinant plasmid(s) containing both ampicillin- and zinc-resistance determinants. This observation was further supported by hybridization of chromosomal DNA of the plasmidless ZnrAmpr E. coli DH5alpha clone with the probes made from the plasmid DNA of strain GS19h and the vector DNA. Thus, this study corroborates our previous finding and documents the phenomenon of integration of metal-resistant determinants from the Acidocella GS19h plasmid(s) into the chromosome of E. coli DH5alpha.

Both rare and common polymorphisms contribute functional variation at CHGA, a regulator of catecholamine physiology.

The chromogranin/secretogranin proteins are costored and coreleased with catecholamines from secretory vesicles in chromaffin cells and noradrenergic neurons. Chromogranin A (CHGA) regulates catecholamine storage and release through intracellular (vesiculogenic) and extracellular (catecholamine release-inhibitory) mechanisms. CHGA is a candidate gene for autonomic dysfunction syndromes, including intermediate phenotypes that contribute to human hypertension. Here, we show a surprising pattern of CHGA variants that alter the expression and function of this gene, both in vivo and in vitro. Functional variants include both common alleles that quantitatively alter gene expression and rare alleles that qualitatively change the encoded product to alter the signaling potency of CHGA-derived catecholamine release-inhibitory catestatin peptides.

Generation of novel plasmids in Escherichia coli S17-1(pSUP106).

When the highly metal-resistant acidophilic heterotrophic strain, Acidiphilium symbioticum KM2, was incubated with two Escherichia coli strains, viz. S17-1 (pSUP106) and K12, on a medium that supported growth of these two divergent species of different habitats, E. coli transconjugants were isolated that contained novel plasmids and were resistant to Zn(2+) (48 m M), Cu(2+) (12 m M), Ni(2+) (12 m M), chloramphenicol (50 microg/ml), and tetracycline (25 microg/ml). The transconjugant plasmids did not hybridize with any of the A. symbioticum KM2 plasmids. After curing of the plasmids, the transconjugants became sensitive to 12 m M Zn(2+), 12 m M Cu(2+), and 12 m M Ni(2+), but remained chloramphenicol and tetracycline resistant-the phenotypic markers that were originally present in pSUP106. That a part of pSUP106 was integrated into the chromosome of the transconjugants was evident from the hybridization of pSUP106 with chromosomal DNA of the cured derivatives of the transconjugants. Further, the transconjugant plasmids hybridized only with the chromosomal DNA of E. coli S17-1 and not with the chromosomal DNA of A. symbioticum KM2 or E. coli K12, suggesting their host chromosomal origin. Thus, the present study describes a unique event of genetic rearrangements in the E. coli strain S17-1 (pSUP106), resulting in the formation of novel plasmids conferring metal-resistance phenotypes in the cell.

Resistance to cadmium and zinc in Acidiphilium symbioticum KM2 is plasmid mediated.

The acidophilic heterotroph, Acidiphilium symbioticum KM2, is highly resistant to several metals and harbors three plasmids of 3.8, 7.1, and 56 kb in size. The bacterium becomes extremely sensitive to metals when it is cured of its plasmids. A mini-plasmid library was constructed by ligating the plasmid DNA fragments generated by MboI partial digestion into the BamHI site of pBluescriptII KS+. The Lac(-)Amp(r) transformants of Escherichia coli DH5alpha, isolated after transformation with the library, were counter-selected on Cu(2+), Cd(2+), Ni(2+), and Zn(2+)-containing plates. Only Cd(2+)- and Zn(2+)-resistant colonies were developed, and, after screening, four types of recombinant plasmids designated as pNM201 (7.2 kb), pNM206 (3.4 kb), pNM208 (4.5 kb), and pNM215 (4.9 kb) were obtained. The DNA insert in pNM206 hybridized strongly with the 3.8-kb plasmid and weakly with the 7.1-kb plasmid of Acidiphilium symbioticum KM2. The DNA insert in pNM215 hybridized only with the 7.1-kb plasmid. These results strongly suggested that resistance to cadmium and zinc in A. symbioticum KM2 is mediated by these plasmids. The smallest insert of 422 bp in pNM206 conferring metal resistance in E. coli has no sequence similarity with the reported metal-resistant genes. All the putative ORFs are significantly rich (up to 37%) in basic amino acids, mainly arginine.