Kamna Srivastava

Direct Detection and Identification of Mycobacterium tuberculosis and Mycobacterium bovis in Bovine Samples by a Novel Nested PCR Assay: Correlation with Conventional Techniques

ABSTRACT Mycobacterium tuberculosis and M. bovis infect animals and humans. Their epidemiologies in developed and developing countries differ, owing to differences in the implementation of preventive measures (World Health Organization, 1999). Identification and differentiation of these closely related mycobacterial species would help to determine the source, reservoirs of infection, and disease burden due to diverse mycobacterial pathogens. The utility of the hupB gene (Rv2986c in M. tuberculosis, or Mb3010c in M. bovis) to differentiate M. tuberculosis and M. bovis was evaluated by a PCR-restriction fragment length polymorphism (RFLP) assay with 56 characterized bovine isolates (S. Prabhakar et al., J. Clin. Microbiol. 42:2724-2732, 2004). The degree of concordance between the PCR-RFLP assay and the microbiological characterization was 99.0% (P < 0.001). A nested PCR (N-PCR) assay was developed, replacing the PCR-RFLP assay for direct detection of M. tuberculosis and M. bovis in bovine samples. The N-PCR products of M. tuberculosis and M. bovis corresponded to 116 and 89 bp, respectively. The detection limit of mycobacterial DNA by N-PCR was 50 fg, equivalent to five tubercle bacilli. M. tuberculosis and/or M. bovis was detected in 55.5% (105/189) of the samples by N-PCR, compared to 9.4% (18/189) by culture. The sensitivities of N-PCR and culture were 97.3 and 29.7, respectively, and their specificities were 22.2 and 77.7%, respectively. The percentages of animals or samples identified as infected with M. tuberculosis or M. bovis by N-PCR and culture reflected the clinical categorizations of the cattle (P of <0.05 to <0.01). Mixed infection by N-PCR was detected in 22 animals, whereas by culture mixed infection was detected in 1 animal.

Prevalence of eNOS Glu298Asp Polymorphism in Healthy Volunteers from a Region of Northern India

Objective: Endothelial nitric oxide synthase (eNOS) Glu298Asp polymorphisms are under extensive study worldwide due to their suggested role in cardiovascular disorders. This polymorphism had gained more attention since several reports suggest its association with hypertension and coronary artery disease (CAD). Asian Indians are highly susceptible to ischemic heart dis eases. We determined the prevalence of eNOS Glu298 Asp polymorphism in 139 healthy volunteers from Delhi and the surrounding areas. The subjects were recruited from those who willingly participated in this study in response to a publicized call and a standard questionnaire. Male to female ratio was 2.7:1 due to the larger number of male participants in this investigation. This, however, does not represent normal male to female distribution in the area. Despite the male bias, this investigation was justified. The prevalence of CAD in males is about 3 times higher in this region and no data had so far been available on the distribution of this polymorphism from India. Method: The eNOS Glu298Asp polymorphism was studied by PCR-RFLP. Results: Distribution of genotype GG, GT and TT in the study subjects was found to be 71.22, 28.06 and 0.72%, respectively, and allele frequency was G,0.853;T,0.147. Conclusion: T allele had been described as susceptibility allele for CAD in several population studies. The frequency of the T allele was found to be two times higher in our subjects than that reported for Japanese and Korean populations. This study does not provide any direct evidence for eNOS gene disease associations but is the first report on the prevalence of eNOS Glu298Asp gene polymorphism in Indian subjects. Whether the observed pattern of eNOS Glu298Asp polymorphism contributes to the greater susceptibility of Asian Indians to CAD as compared to the other population groups, needs to be investigated.

Association of Angiotensin II Type 1 Receptor (A1166C) Gene Polymorphism and Its Increased Expression in Essential Hypertension: A Case-Control Study

Objectives Hypertension is one of the major cardiovascular diseases. It affects nearly 1.56 billion people worldwide. The present study is about a particular genetic polymorphism (A1166C), gene expression and protein expression of the angiotensin II type I receptor (AT1R) (SNP ID: rs5186) and its association with essential hypertension in a Northern Indian population. Methods We analyzed the A1166C polymorphism and expression of AT1R gene in 250 patients with essential hypertension and 250 normal healthy controls. Results A significant association was found in the AT1R genotypes (AC+CC) with essential hypertension (χ2 = 22.48, p = 0.0001). Individuals with CC genotypes were at 2.4 times higher odds (p = 0.0001) to develop essential hypertension than individuals with AC and AA genotypes. The statistically significant intergenotypic variation in the systolic blood pressure was found higher in the patients with CC (169.4±36.3 mmHg) as compared to that of AA (143.5±28.1 mmHg) and AC (153.9±30.5 mmHg) genotypes (p = 0.0001). We found a significant difference in the average delta-CT value (p = 0.0001) wherein an upregulated gene expression (approximately 16 fold) was observed in case of patients as compared to controls. Furthermore, higher expression of AT1R gene was observed in patients with CC genotype than with AC and AA genotypes. A significant difference (p = 0.0001) in the protein expression of angiotensin II Type 1 receptor was also observed in the plasma of patients (1.49±0.27) as compared to controls (0.80±0.24). Conclusion Our findings suggest that C allele of A1166C polymorphism in the angiotensin II type 1 receptor gene is associated with essential hypertension and its upregulation could play an important role in essential hypertension.

Design, Synthesis and Evaluation of Anticonvulsant Activity of Pyridinyl-Pyrrolidones: A Pharmacophore Hybrid Approach

Various 1‐[6‐(4‐substituted phenyl)‐3‐cyano‐4‐(substituted phenyl)‐pyridin‐2‐yl]‐5‐oxopyrrolidine‐3‐carboxylic acids (3a–t) were designed and synthesized by clubbing pyrrolidinones and pyridines, the two active anticonvulsant pharmacophores. All the synthesized compounds fulfilled the requirements of suggested pharmacophoric model for anticonvulsant activity. Their in vivo anticonvulsant evaluation was performed by maximal electroshock seizure (MES) and subcutaneous pentylenetetrazole (scPTZ) tests. The minimal motor impairment was assessed by rotorod test and the estimation of various liver enzymes was performed to check the magnitude of liver toxicity posed by the synthesized compounds. Compounds 3d and 3k displayed comparable anticonvulsant activity to the standard drugs with ED50 values of 13.4 and 18.6 mg/kg in electroshock screen, repectively. The compounds 3d and 3k were also found to have encouraging anticonvulsant activity (ED50 = 86.1 and 271.6 mg/kg, respectively) in scPTZ screen. Interestingly, they did not show any sign of motor impairment at the maximum dose administered and were not toxic to the liver.

Association of Angiotensin Converting Enzyme (Insertion/Deletion) Gene Polymorphism with Essential Hypertension in Northern Indian Subjects

OBJECTIVE Essential hypertension is a multifactorial disease in which genetic and environmental factors play an important role. The renin-angiotensin system (RAS) is known to play a critical role in the homeostasis of blood pressure. Angiotensin-I converting enzyme (ACE) is a significant component of RAS, and an insertion/deletion (I/D) polymorphism in its gene has been implicated in predisposition to hypertension. The purpose of the current study is to investigate the association of I/D polymorphism of the ACE gene with essential hypertension in northern Indians. METHOD Two hundred twenty-two patients with essential hypertension and 252 controls were recruited for the study. DNA samples were isolated from peripheral blood by using a kit. Polymerase chain reaction was used for genotyping. RESULT All the genotypes and allele distribution in study subjects were in the Hardy-Weinberg equilibrium. There was a significant difference in the distribution of DD, II, and ID genotypes of ACE polymorphism in patients and controls. In the subjects having an I allele, the odds ratio is 2.08 [1.6-2.58] at 95% confidence interval, thus suggesting an association of ACE I/D gene polymorphism with essential hypertension. CONCLUSION Our findings suggest that the I allele of ACE I/D polymorphism is associated with essential hypertension in our population.

Association of angiotensinogen (M235T) gene polymorphism with blood pressure lowering response to angiotensin converting enzyme inhibitor (Enalapril).

PURPOSE It has been suggested that genetic backgrounds, which have an association with essential hypertension, may also determine the responsiveness to ACE inhibitor. We determined the association of angiotensinogen (M235T) gene polymorphism with essential hypertension and the relationship between polymorphism in the angiotensinogen (M235T) gene and blood pressure response to ACE inhibitor (Enalapril) in patients with essential hypertension from northern Indian subjects. METHODS 250 patients with essential hypertension and 250 normal healthy controls from Delhi and surrounding areas were recruited for the investigation. Blood pressure was recorded before and after 6 weeks of treatment with ACE inhibitors, Enalapril. Genotyping were carried out by polymerase chain reaction and Restriction fragment length polymorphism technique. RESULTS Statistically significant association of T allele was observed with essential hypertension [x2 = 14.67, p = 0.00013, Odds ratio = 1.76 (1.3-2.32) at 95% CI], the relative risk at 95% CI being 1.28 (1.2-1.54). The decrease in systolic blood pressure and diastolic blood pressure after six weeks of treatment of the patients carrying TT genotype (SBP = 26 ± 17.4 mmHg, DBP = 14.83 ± 7.6 mmHg) were greater than the groups carrying MT (SBP = 3.0 ± 7.8 mmHg, DBP = 6.2 ± 3.0 mmHg) and MM genotypes (SBP = 1.2 ± 0.8 mmHg, DBP = 0.10 ± 12.1 mm Hg. CONCLUSIONS The angiotensinogen (M235T) gene polymorphism is significantly associated with essential hypertension. Patients carrying TT genotype had higher blood pressure lowering response when treated with ACE inhibitor, Enalapril than those carrying MM and MT genotypes suggesting that the T allele may be a possible genetic marker for essential hypertension.

Expression of Heat Shock Protein 70 Gene and Its Correlation with Inflammatory Markers in Essential Hypertension.

Objectives Hypertension is characterized by systemic high blood pressure and is the most common and important risk factor for the development of cardiovascular diseases. Studies have shown that the circulating levels of certain inflammatory markers such as tumor necrosis factor-alpha (TNF-alpha), interlukin-6 (IL-6), c-reactive protein (CRP), and tumor suppressor protein-53 (p53) are upregulated and are independently associated with essential hypertension. However, mechanism of increase in the levels of HSP70 protein is not clear. No such studies are reported in the blood circulation of patients with essential hypertension. In the present study, we investigated the expression of circulating HSP70 at mRNA and protein levels and its relationship with other inflammatory markers in patients with essential hypertension. Materials and Methods We recruited 132 patients with essential hypertension and 132 normal controls from similar socio-economic-geographical background. The expression of HSP70 at mRNA levels was determined by Real Time PCR and at protein levels by indirect Elisa and Western Blot techniques. Results We found a significantly higher expression of HSP70 gene expression (approximately 6.45 fold, P < 0.0001) in hypertensive patients as compared to healthy controls. A significant difference (P < 0.0001) in the protein expression of HSP70 was also observed in plasma of patients as compared to that of controls. Conclusion Higher expression of HSP70 is positively correlated with inflammatory markers in patients with essential hypertension and this correlation could play an important role in essential hypertension.

Expression of angiotensin-converting enzyme gene in whole blood in patients with essential hypertension

Abstract Objective: The present study aims to investigate the correlation of the angiotensin-converting enzyme (ACE) gene expression and protein expression in patients with essential hypertension in whole blood. Methods: ACE gene expression was analyzed by Real Time PCR and western blot in 52 patients with essential hypertension and 42 healthy controls. Results: We observed a significant increase in Delta threshold cycle (ΔCT) values in the circulating ACE gene and ACE protein expression in patients as compared to controls. Conclusions: The up-regulation in relative expression of circulating Angiotensin converting enzyme mRNA and protein in patients with respect to controls might be correlated with high blood pressure in patients with essential hypertension.

Single nucleotide polymorphisms of microRNA in cardiovascular diseases

Despite the advances in medicine and in science of diagnosis, cardiovascular diseases (CVD) remain the number one cause of morbidity and mortality worldwide. Apart from the modifiable risk factors, genetic factors are believed to also influence the outcome of this umbrella of diseases. Under the genetic factors, miRNA polymorphisms, namely miR-146a (rs2910164), miR-196 (rs11614913) and miR-499 (rs3746444), have become an important tool to study the mechanism that underlie the pathogenesis of this disease. In this review, we analyze the advances made through various research studies and the evidence provided by them in the area of miRNA polymorphisms by comparing the allelic frequencies and genotyping patterns. Interestingly, these studies have contradicting results even those conducted in same set of population. We also highlight the gap in literature search as majority of these studies have been conducted in Chinese population and data gaps are evident in Caucasian population, along with developing countries like, India, where no such data is available. This makes the daunting task of presenting a global picture and of the extent these polymorphisms play a role in CVD progression, even more difficult. Therefore, we suggest that more work needs to be done by taking various geographical domains in to consideration. Also, larger sample size calculated through statistical tools is the key to progress in establishing the genetic co-relation of miRNA polymorphisms and CVDs.

Atrial natriuretic peptide and aldosterone synthase gene in essential hypertension: a case-control study.

The renin-angiotensin-aldosterone system (RAAS) and their candidate genes are principally involved in regulation of blood pressure through salt-water homeostasis. Atrial natriuretic peptide (ANP) and Aldosterone synthase (CYP11B2) are the important RAAS mediators, play a major role in hypertension through regulation of cardiorenal homeostasis and water-electrolytes balance, respectively. Present study reports the expression of ANP and CYP11B2 gene at mRNA and proteins levels in patients with essential hypertension in North Indian subjects. Gene expression at mRNA and protein levels was carried out by Real time PCR and Western blot, respectively. We found a significant down regulation in the ANP gene expression at mRNA (85%) and protein (72.6%) levels and significant increase in the CYP11B2 protein expression in patients as compared to controls. A significant increase in Serum creatinine (14.6%), Sodium (1.15%) and decrease in the Blood urea (8.18%) and Potassium (2.32%) levels were also observed among the patients group having higher expression (based on median delta-CT value) in comparison to the lower expression of CYP11B2 gene. Our results suggest that the down-regulation of ANP gene expression at mRNA and protein levels and up-regulated CYP11B2 protein expression levels may be correlated with the essential hypertension and could serve as circulating prognostic biomarkers for essential hypertension.