Unveiling the interactions among BMPR-2, ALK-1 and 5-HTT genes in the pathophysiology of HAPE.

CONTEXT: Few potential candidate genes coding for type I and II receptors of transforming growth factor beta signaling pathway and the serotonin transporter have been associated with pulmonary hypertension (PH). The latter being a phenotype for high altitude pulmonary edema (HAPE), these genes are hypothesized to be crucial markers to investigate under the hypobaric hypoxic environment of high altitude. AIMS: We hence aimed to investigate bone-morphogenetic protein-2 (BMP2), bone morphogenetic protein receptor type-II (BMPR-2), activin receptor-like kinase-1 (ALK-1), serotonin transporter (5-HTT) and serotonin (5-HT) for their contribution, individually/epistatically, to clinical endpoints by altering downstream signaling molecules. METHODS AND MATERIALS: In a case-control design, interactions between/among polymorphisms of BMPR-2, ALK-1 and 5-HTT were screened in 200 HAPE-patients (HAPE-p) and 200 HAPE-free sojourners (HAPE-f). Plasma biomarker BMP-2 and 5-HT were estimated. The relative gene expression was also witnessed in 20 humans/10 rats followed by correlation analyses. RESULTS: The genotype/allele models revealed the prevalence of BMPR-2 rs6717924A-rs4303700A-rs1048829A; ALK-1 rs11169953T-rs3759178C-rs706816C and 5-HTT rs6354C in HAPE (P≤0.05). Multifactor dimensionality reduction for interactions among genes revealed a 4-locus model of BMPR-2 rs6717924G/A; ALK-1 rs11169953C/T-rs706816T/C and 5-HTT rs6354A/C as the best disease predicting (P≤0.001); whereas HapEvolution analysis confirmed the alleles rs6717924A, rs4303700A and rs6354C as the best interacting (P≤0.01). Plasma levels of BMP-2 and 5-HT were elevated in HAPE (P≤0.0001). The expression of BMP-2, ALK-1, 5-HT, 5-HTT was elevated and of BMPR-2 decreased in humans and rats (P≤0.05). The risk alleles BMPR-2 rs6717924A-rs4303700G-rs1048829A; ALK-1 rs11169953T-rs706816C and 5-HTT rs6354C correlated inversely with arterial oxygen saturation (SaO2) and positively with mean arterial pressure (MAP), BMP-2 and 5-HT in HAPE. Likewise, haplotypes BMPR-2 GGGCGAAAA, AAATAGGGA and ALK-1 CCTCAAAG, CCTTAAAG correlated with clinical markers and biomarkers (P≤0.01). BMP-2 and 5-HT correlated positively with MAP and negatively with SaO2 (P≤0.01). CONCLUSIONS: The genetic-interactions among BMPR-2, ALK-1, and 5-HTT polymorphisms, elevated BMP-2 and 5-HT levels and differential gene expression substantiated the strong genetic contribution in HAPE pathophysiology.

ROCK2 and MYLK variants under hypobaric hypoxic environment of high altitude associate with high altitude pulmonary edema and adaptation.

OBJECTIVE: To date, a major class of kinases, serine-threonine kinase, has been scantly investigated in stress-induced rare, fatal (if not treated early), and morbid disorder, high altitude pulmonary edema (HAPE). This study examined three major serine-threonine kinases, ROCK2, MYLK, and JNK1, along with six other genes, tyrosine hydroxylase, G-protein subunits GNA11 and GNB3, and alpha1 adrenergic receptor isoforms 1A, 1B, and 1D as candidate gene markers of HAPE and adaptation. METHODS: For this, 57 variants across these nine genes were genotyped in HAPE patients (n=225), HAPE controls (n=210), and highlanders (n=259) by Sequenom MS (TOF)-based MassARRAY® platform using iPLEX™ Gold technology. In addition, to study the gene expression, quantitative real-time polymerase chain reaction was performed in human peripheral blood mononuclear cells of the three study groups. RESULTS: A significant association was observed for C allele (ROCK2 single-nucleotide polymorphism, rs10929728) with HAPE (P=0.03) and C, T, and A alleles (MYLK single-nucleotide polymorphisms, rs11717814, rs40305, and rs820336) with both HAPE and adaptation (P=0.001, P=0.006, and P=0.02, respectively). ROCK2 88 kb GGGTTGGT haplotype was associated with lower risk of HAPE (P=0.0009). MYLK 7 kb haplotype CTA, composed of variant alleles, was associated with higher risk of HAPE (P=0.0006) and lower association with adaptation (P=1E-06), whereas haplotype GCG, composed of wild-type alleles, was associated with lower risk of HAPE (P=0.001) and higher association with adaptation (P=1E-06). Haplotype-haplotype and gene-gene interactions demonstrated a correlation in working of ROCK2 and MYLK. CONCLUSION: The data suggest the association of ROCK2 with HAPE and MYLK with HAPE and adaptation in Indian population. The outcome has provided new insights into the physiology of HAPE and adaptation.

Independent associations of low 25 hydroxy vitamin D and high parathyroid hormonal levels with nonalcoholic fatty liver disease in Asian Indians residing in north India.

OBJECTIVE: We analysed the associations of 25 hydroxy vitamin D [25(OH) D] and parathyroid hormone (PTH) levels with clinical, anthropometric, biochemical and body composition parameters in Asian Indians with nonalcoholic fatty liver disease (NAFLD). METHODS: In this case-control study, 162 cases and 173 age and sex matched controls were recruited. Clinical, anthropometric, biochemical parameters and liver ultrasound were done. Percentage body fat (%BF), lean body mass and bone mineral density (BMD) were assessed by dual energy X-ray absorptiometry (DXA). Fasting insulin levels, value of homeostasis model assessment of insulin resistance (HOMA-IR), serum 25(OH) D, calcium and PTH levels were measured. RESULTS: Subjects with NAFLD had lower serum 25(OH) D (19.4 ± 8.5 vs. 27.8 ± 9.4 ng/ml, p = 0.0001) and higher serum PTH (54.9 ± 19.5 vs.41.5 ± 18.3 pg/ml, p = 0.0001) levels as compared to controls. We observed significantly high values of systolic blood pressure (p = 0.002), waist circumference (p = 0.05), serum triglycerides (p = 0.002), total cholesterol (p = 0.002), alanine transaminase (p = 0.05), fasting insulin (p = 0.02) and HOMA-IR (p = 0.03) in the lowest 25(OH) D quartile. Multivariable-logistic regression showed that low serum 25(OH) D [OR (95%CI): 4.46 (2.58-7.72), p = 0.0001] and high PTH [OR (95%CI): 2.21 (1.50-3.30), p = 0.0001] level were independently associated with NAFLD. CONCLUSION: Low serum 25(OH) D and high PTH levels were independently associated with the presence of NAFLD in Asian Indians residing in north India.

Association of the Myostatin gene with obesity, abdominal obesity and low lean body mass and in non-diabetic Asian Indians in north India.

BACKGROUND: To determine the association of the A55T and K153R polymorphisms of the Myostatin gene with obesity, abdominal obesity and lean body mass (LBM) in Asian Indians in north India. MATERIALS AND METHODS: A total of 335 subjects (238 men and 97 women) were assessed for anthropometry, % body fat (BF), LBM and biochemical parameters. Associations of Myostatin gene polymorphisms were evaluated with anthropometric, body composition and biochemical parameters. In A55T polymorphism, BMI (p=0.04), suprailiac skinfold (p=0.05), total skinfold (p=0.008), %BF (p=0.002) and total fat mass (p=0.003) were highest and % LBM (p=0.03) and total LBM (Kg) were lowest (p=0.04) in subjects with Thr/Thr genotype as compared to other genotypes. Association analysis of K153R polymorphism showed that subjects with R/R genotype had significantly higher BMI (p=0.05), waist circumference (p=0.04), %BF (p=0.04) and total fat mass (p=0.03), and lower %LBM (p=0.02) and total LBM [(Kg), (p=0.04)] as compared to other genotypes. Using a multivariate logistic regression model after adjusting for age and sex, subjects with Thr/Thr genotype of A55T showed high risk for high %BF (OR, 3.92, 95% Cl: 2.61-12.41), truncal subcutaneous adiposity (OR, 2.9, 95% Cl: 1.57-6.60)] and low LBM (OR, 0.64, 95% CI: 0.33-0.89) whereas R/R genotype of K153R showed high risk of obesity (BMI; OR, 3.2, 95% CI: 1.2-12.9; %BF, OR, 3.6, 95% CI: 1.04-12.4), abdominal obesity (OR, 2.12, 95% CI: 2.71-14.23) and low LBM (OR, 0.61, 95% CI: 0.29-0.79). CONCLUSIONS/SIGNIFICANCE: We report that variants of Myostatin gene predispose to obesity, abdominal obesity and low lean body mass in Asian Indians in north India.

Multi-locus interactions of vascular homeostasis genes in essential hypertension: a gender-based study.

BACKGROUND: Studies on genes of endothelial and vascular homeostasis are inadequate in females. METHODS: We investigated the role of 7 variants of ACE, AGT and NOS3 and their correlation with NO(x) levels and ACE activity in hypertension susceptibility in 910 case-controls of both genders. RESULTS: Prevalence of alleles D of ACE I/D; -6A of AGT -6G/A; -786C, 894T and 4a of NOS3 -786T/C, 894G/T and 4b/4a polymorphisms was observed in patients (P< or =0.05). The 3 genotypes-combinations containing 6+5 wild-type alleles of AGT and NOS3 were significantly less prevalent in patients (P< or =0.0003). The haplotypes 235T/174T/-6A of AGT (P=4E-3) and -786T/894G/4a and -786C/894G/4a of NOS3 (P=2E-3, P=0.011, respectively) were significantly more prevalent in patients. The AGT and NOS3 findings were similar in males. Genotypes-combinations with 6+5 wild-type alleles of AGT correlated with higher NO(x) levels (P=0.03). The NOS3 genotypes-combinations having 6 and 6+5 wild-type alleles correlated with decreased ACE activity (P=0.025, P=0.0015, respectively) and increased NO(x) levels (P=0.001, P=0.0001, respectively) in patients. In gene-gene interactions, ACE D allele associated with < or =4 wild-type alleles containing genotypes-combinations of AGT and NOS3 in patients (P< or =0.04). CONCLUSION: Within gene and between genes interactions of variants influence ACE activity and NO(x) levels and associate with EH.

Endoplasmic reticulum stress response in murine kidney exposed to acute hypobaric hypoxia.

Any perturbation in the normal functioning of endoplasmic reticulum (ER), such as due to hypoxia, triggers the unfolded protein response (UPR). We studied the temporal variation in gene expression in murine kidney exposed to acute hypobaric hypoxia. Molecular chaperones like Grp78, Grp94, Canx and Calr in the ER were transcriptionally downregulated. Further, the splicing of Xbp1 mRNA decreased, whereas transcription of the unspliced mRNA increased. This step produces Xbp1 protein, which is negatively regulated by the unspliced protein. Hence, the decreased splicing of Xbp1 along with decreased transcription of ER chaperones in kidney is a definite indication of reduced stress.

Correlation of oxidative status with BMI and lung function in COPD.

OBJECTIVES: The imbalance in oxidative status together with nutrition depletion and low body weight play a vital role in the pathogenesis and severity of chronic obstructive pulmonary disease (COPD). The study was undertaken to ascertain if a relationship existed between oxidative status and BMI in COPD. In addition, association of oxidative status and BMI with lung function of the disease was also examined. MATERIALS AND METHODS: In 202 COPD patients and 136 healthy controls plasma lipid peroxidation (LPO), reduced glutathione (GSH), glutathione peroxidase (GPx), catalase (CAT) activities, BMI and FEV(1)% predicted were looked for interactions. RESULTS: The patients had increased LPO (p=0.006) and decreased antioxidants (GSH, p=0.005; GPx, p=0.035 and CAT, p=0.008, respectively). Of note are the correlations of oxidative stress markers with BMI and FEV(1)% predicted in the patients. LPO inversely and GSH, GPx, and CAT positively correlated with both BMI (p=0.007, p<0.001, p=0.045 and p=0.009, respectively), and FEV(1)% of predicted (LPO, p=0.001; GSH, p<0.001; GPx, p=0.043 and CAT, p<0.001) in the patients. Further, a positive correlation existed between BMI and FEV(1)% predicted (p=0.016) in COPD. CONCLUSION: The intimate relationship of oxidative status with BMI and lung function, and the direct correlation between BMI and FEV(1) may potentiate severity of the disease.

T lymphocyte subset profile and serum alpha-1-antitrypsin in pathogenesis of chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is an inflammatory disorder characterized by the presence of non-fully reversible airflow limitation. The study was undertaken to investigate the involvement of alpha-1-antitrypsin (alpha(1)AT) and T lymphocyte subsets in the pathogenesis of COPD. Blood samples of 50 subjects, including 25 healthy volunteers and 25 patients with COPD, were analysed. Serum trypsin inhibitory capacity (STIC) was determined by enzymatic assay. CD4(+) and CD8(+) T lymphocytes were enumerated in heparinized blood using a fluorescence activated cell sorter counter. The STIC in COPD patients was found to be decreased significantly than in controls (P < 0.01). In COPD patients with lower expression levels of alpha(1)AT, a highly significant decrease in the number of CD4(+) T lymphocytes (P < 0.0009) and CD4/CD8 ratio was observed compared with control subjects (P < 0.008). The mean +/- standard error of CD8(+) lymphocytes was found to be little different (only marginally decreased) in COPD patients compared to healthy controls; however, an alteration in the individual count of CD8(+) lymphocytes cells was observed in COPD patients. Using linear regression analysis, a negative correlation was observed between STIC and CD4(+) lymphocytes and CD8(+) lymphocytes (r = -0.40, P < 0.04; r = -0.42, P < 0.03, respectively) in COPD patients. An alteration in alpha(1)AT and T lymphocyte subsets in COPD patients suggested that interplay of these factors may be responsible for the progression of COPD.

Genetic polymorphisms of GSTP1 and mEPHX correlate with oxidative stress markers and lung function in COPD.

The genetic susceptibility to COPD might depend on variations in detoxification enzymes that activate and detoxify cigarette smoke products, which otherwise generate oxidative stress causing pathogenesis. In a case-control study of 202 COPD patients and 136 normals, we examined the association of polymorphisms I105V, A114V of GSTP1 and Y113H, H139R of mEPHX individually or in combination with disease and their contribution to oxidative stress markers such as MDA, GSH, GPx and airflow obstruction. Patients were over-represented by the alleles 105V, 114V of GSTP1 and 113H, 139H of mEPHX (chi(2)=10.63, p=0.001, chi(2)=13.92, p<0.001, chi(2)=13.02, p<0.001 and chi(2)=4.48, p=0.034, respectively) and the haplotypes of same alleles i.e. 105V-114V and 113H-139H (chi(2)=14.58, p<0.001 and chi(2)=23.14, p<0.001). Moreover, there was marked over-representation of combination of genotypes, I105I+A114A of GSTP1 (53% vs. 36%) in controls; whereas, the combinations with 105V/114V alleles (64% vs. 47%) of GSTP1 (OR=1.99; 95% CI=1.28-3.09; p=0.002) and the homozygotes H113H+H139H (27% vs.10%) of mEPHX (OR=3.26; 95% CI=1.73-6.15; p=0.0001) in patients. Patients had significantly elevated MDA level (p<0.001) and decreased GSH level (p<0.001) and GPx activity (p=0.035), respectively. Of note, the genotypes, I105V/V105V, A114V/V114V of GSTP1 and Y113H/H113H of mEPHX associated with increased MDA level (p=0.04, p=0.03 and p=0.003), decreased GSH level (p=0.019, p=0.007 and p=0.0006) and lower FEV1 (p=0.23, p=0.037 and p=0.029), respectively, in patients; so was the correlation of these biomarkers and lung function with the combinations of the genotypes. In conclusion, 105V/114V alleles of GSTP1 and 113H/139H alleles of mEPHX and the combination of genotypes with same alleles associated with imbalanced oxidative stress and lung function in patients, signifying the importance in the disease.

Transcriptional downregulation of sterol metabolism genes in murine liver exposed to acute hypobaric hypoxia.

Ascent to high-altitude results in decreased inspired partial pressure of oxygen because of a decrease in barometric pressure. Altitude acclimatization requires physiological and metabolic changes to improve tolerance to altitude hypoxia. Cellular response to hypoxia results into changes in the profile of gene expression and the present study explored the same in murine model. Liver being the largest metabolic organ, the molecular details of acute hypobaric hypoxia (AHH) induced transcriptional changes in the tissue were investigated. Swiss albino mice were exposed to hypobaric hypoxia ( approximately 426mmHg) in a decompression chamber and cDNA microarray was used to study the transcriptional profile in liver. Notably, by the tenth hour several of the genes involved in sterol metabolism such as SREBF1, INSIG1, HMGCS1, FDFT1, SQLE, and HSD3B4 were downregulated more than 2-fold suggesting that AHH suppresses sterol biosynthesis in the liver. Real-time PCR helped validate the downregulation of SREBF1, HMGCS1, FDFT1, and HSD3B4 genes. However, no significant change was observed in the serum cholesterol levels throughout the AHH exposure. The findings are indicative of transcriptional downregulation of SREBP target genes as a part of acclimatization response to hypoxia. The study highlights the significance of SREBP in the regulation of sterol metabolism under the acute hypoxic response.

Predominance of interaction among wild-type alleles of CYP11B2 in Himalayan natives associates with high-altitude adaptation.

Sojourners visiting high-altitude (HA) (>2500 m) are susceptible to HA disorders; on the contrary, HA natives are well adapted to the extreme hypoxic environment. High aldosterone levels are believed to be involved in HA disorders, we, therefore, envisaged role of CYP11B2 gene variants in HA adaptation and therefore investigated the -344T/C, intron-2 conversion (Iw/Ic), K173R, and A5160C polymorphisms. In addition, polymorphisms in AGT, AT1R, ATP1A1, ADRB2, and GSTP1 genes were also investigated. The study comprised of 662 subjects, comprising of 426 Himalayan highlanders (HLs) and 236 lowlanders (LLs). The -344T/C and K173R polymorphisms were found to be in complete linkage disequilibrium. The wild-type allele -344T and combination of wild-type homozygous genotypes between -344T/C, Iw/Ic, and A5160C polymorphisms, containing all the six wild-type alleles were over-represented in the HLs (p < 0.0001, and p = 0.008, respectively). The wild-type haplotypes -344T-Iw, -344T-5160A, and -344T-Iw-5160A also showed over-representation in the HLs (p < 0.0001). Furthermore, greater the number of wild-type alleles, lower was the ARR (p < 0.05). The genotype distribution in remaining genes did not differ. To conclude, the over-representation of wild-type -344T allele, genotype combinations and haplotypes of CYP11B2, and their correlation with lower aldosterone levels associate with HA adaptation in the HLs. Such an allelic presentation in sojourners may help them cope with adverse HA environment.

Structure-activity relationship study between Ornithyl-Proline and Lysyl-Proline based tripeptidomimics as angiotensin-converting enzyme inhibitors.

A designed library of tripeptidomimics of Ornithyl-Proline (Orn-Pro) and Lysyl-Proline (Lys-Pro) conjugated with various unnatural amino acids and carboxylic acid derived heterocyclics was synthesized and screened for possible inhibitors of angiotensin-converting enzyme (ACE). Among the tripeptidomimics 10[MTP-Orn-Pro], 11[HTP-Orn-Pro], 14[TA-Orn-Pro] and 20[BPA-Orn-Pro] showed prominent inhibition with IC50 values in micromolar concentrations. Structure-activity relationship study indicated that C3 side chain of Orn as compared to C4 side chain of Lys at P1' position was better suited to inhibit ACE, with propionic acid (C3) derived heterocyclics and unnatural amino acids.

Endothelin-1 gene variants and levels associate with adaptation to hypobaric hypoxia in high-altitude natives.

High-altitude natives are adapted to hypobaric hypoxia, suggestive of genetic basis of adaptation. Since endothelin-1 (ET-1) is of prime importance in high-altitude disorders in sojourners, we envisaged the role of allelic variants of ET-1 in high-altitude adaptation. Four ET-1 polymorphisms, viz., (CT)(n)-(CA)(n) repeat, -3A/-4A, G2288T, and Lys198Asn, were investigated in 426 highlanders (HLs) and 236 lowlanders (LLs). The plasma ET-1 levels, SBP and BMI were significantly lower in the HLs than those in LLs (p<0.0001). The Longer-repeats (31-45), G allele, Longer-repeats/GG, and Longer-repeats/Lys198Lys combinations were overrepresented in the HLs (p<0.0001, p=0.03, p<0.0001, and p<0.0001, respectively). The Longer-repeats, -3A/-3A, GG and Lys198Lys genotypes associated with significantly lower ET-1 levels in the HLs (p<0.0001, p=0.001, p<0.0001, and p<0.0001, respectively). Combinations of Longer-repeats with -3A/-3A, GG, and Lys198Lys genotypes, and -3A/-3A/Lys198Lys combination revealed association with lower ET-1 levels in the HLs (p<0.001). The study reports over-representation of Longer-repeats, G allele, and wild-type genotype combinations in high-altitude natives. Interaction between these alleles and association with lower ET-1 levels strengthen their association with high-altitude adaptation. Presence of such alleles in sojourners may help in acclimatization.

Simultaneous selection of the wild-type genotypes of the G894T and 4B/ 4A polymorphisms of NOS3 associate with high-altitude adaptation.

The routine performance of high-altitude (HA) natives in the hypoxic environment of HA exemplifies the process of adaptation mainly through natural selection. The recent therapeutic application of nitric oxide (NO) in HA disorders, for the improvement of oxygenation and vasodilation, ushered us to investigate the endothelial nitric oxide synthase gene (NOS3) with respect to HA adaptation. The study subjects, 131 HA monks (HAM), 136 HA controls (HAC), and 170 lowlanders (LLs) were screened for NOS3 G894T (Glu298Asp) and 4B/4A polymorphisms. The NO levels were estimated, for a correlation with the polymorphisms. The three groups were in Hardy-Weinberg equilibrium for the polymorphisms. The overall genotype distributions for the G894T and 4B/4A polymorphisms were significant (P = 0.01 and 0.02, respectively) in the three groups. Wild-type alleles G and 4B were significantly over-represented in the HA groups as compared to the LLs (P = 0.006 and P = 0.02, respectively). The NO levels were in the order of HAM>HAC>LLs (P < 0.0001). Furthermore, combinations of the GG and BB genotypes were distributed significantly more frequently in the HAM (P < 0.0001) and HAC (P = 0.0005) than in LLs. The NO levels contributed by the wild-type genotype combination GG, BB were significantly elevated when compared with the remaining eight genotype combinations together in the HAM, HAC and LLs (P = 0.003, P = 0.0006 and P < 0.0001, respectively). To conclude, the genotype combination of NOS3 wild-type homozygotes (GG, BB) was found significantly more frequently in HA groups than in LLs, by contributing to higher NO levels associated with HA adaptation.

Association of high-altitude systemic hypertension with the deletion allele-of the angiotensin-converting enzyme (ACE) gene.

People who visit high-altitude areas are exposed to a stressful environment and a good percentage of them suffer from high-altitude-induced diseases, including systemic hypertension. Identification of genetic markers for high-altitude-induced diseases would help to reduce the rate of morbidity/mortality from such diseases. The development of systemic hypertension on exposure to high altitude (3,500 m) for 30 days in otherwise normotensive natives of low-altitudes was investigated. The angiotensin-converting enzyme (ACE) insertion/deletion (I/D) genotypes and renin-angiotensin-aldosterone system were simultaneously studied. In the hypertensives during their stay at high altitude, the ACE D allele frequency was significantly higher than in the normotensives (0.67 versus 0.32 chi(2)(1) = 10.6, P < 0.05). In the normotensives during their stay at high altitude, there was no significant increase in plasma aldosterone levels despite increased plasma renin activity. Results of the present study suggest that environmental changes and pre-existing genetic factors, namely the ACE D allele, might be two of the factors predisposing natives of low altitudes to systemic hypertension, a polygenic disease, at high altitude.

Novel peptidomimics as angiotensin-converting enzyme inhibitors: a combinatorial approach.

One of the efficient mode of treatments of chronic hypertension and cardiovascular disorders has been to restrain the formation of angiotensin-II by inhibiting the action of angiotensin-converting enzyme (ACE) on angiotensin-I. A number of ACE inhibitors (ACEIs) have been put to therapeutic use during the last two decades. The efforts continue towards achieving superior molecules or drugs with improved affinities, better bioavailability and thus long duration of action with minimum side effects. The present work evolves around similar objectives. In order to understand the mode of interaction of inhibitors with the active site of the enzyme and subsequently to have lead compounds as possible inhibitors the novel dipeptidomimics and tripeptidomimics have been designed and synthesized using combinatorial chemistry approach. A Focussed library of 10 di- and tri-peptides, eight dipeptidomemics and forty tripeptidomemics was generated. The pharmacophoric heterocyclic moieties and the amino acids have been selected to have affinities with the S1, S1', and S2' subsites of the active site of the enzyme. ACE inhibition studies clearly demonstrated the structural-activity relationships within these classes of peptidomimics. The dipeptidomimics interacted only with S1' and S2' subsites, whereas the tripeptidomemics had additional interaction with S1 subsite, which accounted for their significant ACE inhibition potencies. The in-vitro screening of these peptidomimics have resulted in identification of four promising tripeptidomimics 34[2-benzimidazolepropionyl-Val-Trp], 35[5hydroxytryptophanyl-Val-Trp], 40[2-benzimidazolepropionyl-Ile-Trp] and 45[2-benzimidazolepropionyl-Lys-Trp] with IC50 values in micromolar concentrations.

Angiotensin converting enzyme insertion allele in relation to high altitude adaptation.

Angiotensin converting enzyme (ACE) gene I/D polymorphism has been associated with high altitude (HA) disorders as well as physical performance. We, however, envisage that the polymorphism may be associated with adaptation to the hypobaric hypoxia of altitude, thus facilitating physical performance. For this purpose, three unrelated adult male groups, namely (1) the Ladakhis (HLs), who reside at and above a height of 3600 m, (2) lowlanders, who migrated to Ladakh (MLLs), and (3) resident lowlanders (LLs), have been investigated. The HLs had significantly (p & 0.001) greater numbers of the II homozygotes and the ID heterozygotes than the DD homozygotes, the genotype distribution being 0.46, 0.43 and 0.11 for II, ID and DD genotypes respectively. The MLLs comprised 60% II homozygotes, which was higher (p & 0.001) than the HLs (46%). In the LLs, the heterozygotes were greater (p & 0.001) in number than the II and DD homozygotes. The I allele frequency was 0.72 in the MLLs, 0.67 in the HLs and 0.55 in the LLs. Polymorphism study suggested that the II genotype could be associated with altitude adaptation, which might influence physical efficiency.

A rapid method for plasma ammonia estimation using an indigenously purified enzyme.

Determination of ammonia level in blood is important, especially in the diagnosis of hepatic disorders. An indigenously purified enzyme was used in the standardisation of the assay. The assay is a two reagent system, requires five minutes for completion and can be performed at temperature between 25-27°C. Performance of the assay was assessed by linearity, imprecision, functional sensitivity and interference studies. Lyophilised reagent I and reagent II were found stable for at least one year. The plasma level of ammonia for the controls was 13.7±7.3 µMol/L, whereas for subjects of hepatic disorders, it was 69.1±32.4 µMol/L (P<0.001). The functional sensitivity was between 2-1000 µMol/L. Within-run coefficient of variation was between 1.1-2.0% and between-run coefficient of variation was between 1.9-3.7%. The mean recovery after dilution was 99.6%. The present method can estimate ammonia up to 1000 µMol/L without dilution of sample. Assay time of five minute may be shortened to one minute. This method is suited for routine clinical use in treatment of hepatic disorders.