M235T polymorphism in the AGT gene and A/G(I8-83) substitution in the REN gene correlate with end-stage renal disease.

BACKGROUND/AIMS: This study aimed at investigating if M235T polymorphism in the AGT gene and A/G(I8-83) polymorphism in the REN gene correlate with end-stage renal disease (ESRD). METHODS: We analyzed 173 ESRD patients and 329 individuals with normal kidney function for differences in the genotype distribution of AGT-M235T and REN-A/G(I8-83) polymorphisms between the two groups. The data for cases and controls were compared using the χ(2) test. RESULTS: We found significantly higher levels of serum creatinine and CRP in cases in comparison to controls (p < 0.0001). Data comparison showed a significant association of AGT M235T substitution with ESRD in the dominant model (p = 0.008) and in the comparison of the heterozygous substitution with the homozygous common genotype (p = 0.005). Similarly, REN A/G(I8-83) polymorphism showed a significant difference in the distribution of genotypes between cases and controls (p < 0.038) such that a heterozygous substitution was significantly more common in the ESRD cases in comparison to the homozygous common genotype (p = 0.023). CONCLUSION: We conclude that heterozygous substitutions at the AGT M235T and REN A/G(I8-83) loci correlate significantly with ESRD in a north Indian population.

Depleted TGF-ß1 levels in end stage renal disease patients from North India.

End stage renal disease is a clinical state that extends from chronic renal failure and is marked by an irreversible loss of renal function. TGF-ß1 mediated renal fibrosis is a common pathology implicated in this form of kidney disease. In this study circulating protein and mRNA levels of TGF-ß1 cytokine were investigated among ESRD patients and respective controls from North India. Physician diagnosed 192 ESRD patients, on hemodialysis, and 130 normal controls participated in the present study. TGF-ß1 circulating levels were measured by ELISA and its expression was quantified using competitive-PCR. Mean TGF-ß1 protein levels were 2.7-fold lower in ESRD patients as compared to normal controls (p<0.001). Additionally, TGF-ß1 mRNA transcripts of this cytokine were also significantly lower in the diseased population compared to controls (p<0.001). These results imply that TGF-ß1 has not played its anticipated pro-fibrotic role and anti-inflammatory function in the studied population.

An improved method for the isolation of hepatitis B virus DNA from human serum.

Studies show that hepatitis B virus (HBV) DNA isolation methods vary in their efficiency to extract DNA from serum samples. The purpose of the present study was to develop an improved method for isolation of HBV DNA and compare it with commonly used HBV DNA isolation protocols. In order to develop HBV DNA isolation protocol, serum samples were collected from patients and screened for the presence of hepatitis B surface antigen, hepatitis B e antigen and HBV DNA. Highly viremic samples were pooled and used to compare commonly used HBV DNA isolation methods; namely alkaline lysis, microwave treatment, organic, inorganic with modified inorganic method. DNA isolated by these methods was detected qualitatively by polymerase chain reaction and quantitatively with competitive polymerase chain reaction (cPCR). The modified inorganic method gave maximum yield of HBV DNA followed by inorganic, organic, microwave treatment and alkaline lysis method. Our data also demonstrated a critical role of proteinase K in HBV DNA isolation. DNA isolation method described here, in combination with a reproducible and sensitive quantitative technique would further help in accurate classification of HBV infected patients, designing suitable drug regimen for treatment and monitoring antiviral treatment as well as emergence of drug resistant mutants.

Spirulina protects against rosiglitazone induced osteoporosis in insulin resistance rats.

AIM: The study was undertaken to assess the protective effect of Spirulina fusiformis extract against Rosiglitazone induced osteoporosis and pharmacodynamic effects of Rosiglitazone with Spirulina in treating hyperglycemia and hyperlipidemia of insulin resistance rat. METHOD: For this aim, 30 Wistar albino rats were equally divided into five groups as control (C), diabetes mellitus (DM), diabetes mellitus+Rosiglitazone (DM+R), diabetes mellitus+Spirulina (DM+S), and diabetes mellitus+Rosiglitazone+Spirulina (DM+R+S). Serum glucose, triglyceride, HDL, LDL and insulin concentrations were estimated by routine standard methods in blood samples collected on 21th day. Integrity of the bone surface was examined by scanning electronic microscopy, and bone strength was measured by micro-hardness test on 45th day. RESULTS: A significant decrease in total bone mineral density was observed in group DM+R rats (p<0.05). The number and depth of resorptive pits on surface of the bone in Rosiglitazone treated rats improved clearly with Spirulina administration. The intactness and integrity of the bone surface as well as the bone strength improved due to the high content of calcium and phosphorous in Spirulina. Besides, chromium and gamma-linoleic acid in Spirulina helped to decrease the fasting serum glucose, HDL, LDL and triglycerides levels in insulin resistance rats. CONCLUSION: These findings suggest that combination therapy of Rosiglitazone with Spirulina reduced the risk of osteoporosis in insulin resistance rats. Additionally, Spirulina complemented the antihyperglycemic and antilipidemic activity of Rosiglitazone.

Angiotensin-converting enzyme gene polymorphism in hypertensive rural population of Haryana, India.

BACKGROUND: Essential hypertension is a complex genetic disorder influenced by diverse environmental factors. Of the various physiological pathways affecting the homeostasis of blood pressure, the renin-angiotensin system (RAS) is known to play a critical role. 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. OBJECTIVE: The present study is aimed to determine the association, if any, of ACE I/D polymorphism with essential hypertension in a rural population of Haryana, India. MATERIALS AND METHODS: The blood samples were collected from the patients visiting M. M. Institute of Medical Sciences, Mullana, Haryana. DNA from the patients (106) and control (110) specimens were isolated, amplified by PCR and analyzed employing agarose gel electrophoresis. RESULTS: There was no significant difference in the distribution of DD, II and I/D genotypes of ACE polymorphism in essential hypertensive patients (28.8, 25.5, and 46.2%) and their ethnically matched normal control (24.5, 30, and 45.5), respectively. The two groups also presented with very similar allelic frequencies and were also found to be in Hardy-Weinberg equilibrium. CONCLUSIONS: The present study demonstrates that ACE I/D polymorphism is not a risk factor for essential hypertension in the hitherto unstudied rural population of Haryana.

Tumor necrosis factor--alpha and transforming growth factor--beta1 polymorphisms in bronchial asthma.

BACKGROUND: Bronchial asthma is a complex genetic disorder regulated by the release of cytokines and inflammatory mediators. Tumor necrosis factor alpha (TNF-alpha) and transforming growth factor beta (TGF-beta1) cytokines play pivotal roles in the inflammatory response of the airways. Differential production of these two cytokines is associated with allelic variations in the transcriptional regulatory region of these genes. AIMS: The objective of the present study was to investigate G-308A TNF-alpha and C-509T TGF- beta1 polymorphisms for their association with Bronchial Asthma. MATERIALS AND METHODS: DNA isolated from 123 asthmatics and 100 normal healthy controls were screened for these polymorphisms using the amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) methods, developed in our laboratory. RESULTS: Significant allelic association was observed between G-308A TNF-alpha polymorphism and asthma (P = 0.031) while no association was observed with C-509T TGF- beta1 polymorphism (P = 0.207). Further sub-grouping based on either allergic response or family history failed to reveal any statistical significance among the groups or with controls. The interaction between these polymorphisms revealed statistically significant association between the high producer genotype alleles of TNF-alpha and TGF-beta (A/T) and asthma (P = 0.016). CONCLUSIONS: The present study reports, for the first time, the role of two polymorphisms, in concert, for their association with asthma in an Indian population. Our study supports the findings that the G-308A TNF-alpha promoter polymorphism is a risk factor for asthma and furthermore suggests that the patients with high producer alleles for TNF-alpha (-308) and TGF-beta (-509) have the highest risk of getting this disease in the Punjabi population.

Nitric oxide activates Rap1 and Ral in a Ras-independent manner.

Rap1 and Ral, the small GTPases belonging to the Ras superfamily, have recently attracted much attention; Ral because of Ral-specific guanine nucleotide exchange factors which are regulated by direct binding to Ras and Rap1 because of its proposed role as an antagonist of Ras signaling. We have previously demonstrated that nitric oxide (NO) activates Ras and proposed the structural basis of interaction between NO and Ras. In the present study we have shown that NO activates Rap1 and Ral in a time- and concentration-dependent manner. Using activation-specific probes for Rap1 and Ral, it was found that the NO-generating compounds SNP and SNAP could activate both Rap1 and Ral in Jurkat and PC12 cell lines. To investigate the involvement of Ras in NO mediated activation of Rap1 and Ral, we used PC12 cell lines expressing either the Ras mutant C118S (Cys118 mutated to Ser) or N17 (GDP-locked and inactive). We had previously shown that NO fails to activate Ras in these mutant cell lines. However, here it was found that Rap1 and Ral were activated by NO in these cell lines. The evidence presented in this study unambiguously demonstrates the existence of Ras-independent pathways for NO mediated activation of Rap1 and Ral.