Vrajesh Tripathi - Academia.edu (original) (raw)

Papers by Vrajesh Tripathi

The aim of the study was to demonstrate the presence of nitric oxide synthase (NOS) isoforms and ... more The aim of the study was to demonstrate the presence of nitric oxide synthase (NOS) isoforms and role of nitric oxide (NO) in ovary of H. fossilis. The immunohistochemistry (IHC) showed eNOS and iNOS in granulosa and theca cells and nNOS in ooplasm of early previtellogenic follicles. Western blot confirmed the presence of three types of NOS in ovary. The ovarian concentrations of nitric oxide were significantly higher in pre-vitellogenic than vitellogenic phase. The in vitro study showed the inhibitory action of NO in oocytes maturation. The results suggest a role of NO in follicular development in H. fossilis.

Influence of nitric oxide on in vitro growth, survival, steroidogenesis, and apoptosis of follicl... more Influence of nitric oxide on in vitro growth, survival, steroidogenesis, and apoptosis of follicle stimulating hormone stimulated buffalo (Bubalus bubalis) preantral follicles

Our previous studies show that a decrease in endogenous nitric oxide (NO) is involved in the blun... more Our previous studies show that a decrease in endogenous nitric oxide (NO) is involved in the blunted outward K ϩ currents in carotid body (CB) glomus cells from chronic heart failure (CHF) rabbits. In the present study, we measured the effects of the neuronal nitric oxide synthase (nNOS) transgene on the K ϩ currents in CB glomus cells from pacing-induced CHF rabbits. Using single-cell real-time RT-PCR and immunofluorescent techniques, we found that nNOS mRNA and protein are expressed in the rabbit CB glomus cells and CHF decreased the expression of nNOS mRNA and protein in CB glomus cells. After 3 days of an adenoviral nNOS (Ad.nNOS) gene transfection, the expression of nNOS protein was increased to the level found in sham CB glomus cells. In whole cell patch-clamp experiments, Ad.nNOS markedly reversed the attenuated K ϩ currents in CB glomus cells from CHF rabbits. The specific nNOS inhibitor (S-methyl-L-thiocitrulline [SMTC]) and large-conductance Ca 2ϩ-activated K ϩ (BK) channel blocker (iberiotoxin) fully abolished the effect of Ad.nNOS on the K ϩ currents in the CB glomus cells from CHF rabbits. However, neither CHF nor Ad.nNOS altered the protein expression of BK channel ␣-subunit. These results suggest that a decrease of NO induced by an attenuated nNOS activity lowers the activation of the BK channels but not the protein expression of the BK channel ␣-subunit in the CB glomus cells during CHF.

International Journal of Immunopathology and Pharmacology, 2003

Bioactive novel polypeptide of Anurans skin has a wide range of antimicrobial properties against ... more Bioactive novel polypeptide of Anurans skin has a wide range of antimicrobial properties against the infection and tumour cell. Macrophages are known to produce the Nitric oxide (NO) by a variety of cells upon activation. NO produced by the activated macrophages an important mediator for antimicrobial and tumoricidal activity. In-vitro macrophage exposed with medium alone, containing LPS, containing polypepeptides and LPS + polypeptides for 24 h showed enhanced production of NO with respect to control and LPS treated and significant increase in NO production in LPS + polypeptide. Western blot and PCR analysis also showed that increased production of protein expression and mRNA expression of inducible nitric oxide synthase (iNOS). These findings suggest that novel polypeptides are potent activating agent for enhanced production of NO through activation of iNOS gene.

Asian-Australasian Journal of Animal Sciences, 2011

Objective of this study was to examine the effect of sodium nitroprusside (SNP), a nitric oxide (... more Objective of this study was to examine the effect of sodium nitroprusside (SNP), a nitric oxide (NO) donor on steroid synthesis, growth and apoptosis of buffalo granulosa cells (GCs) in vitro. Follicular fluid of antral follicles (3-5 mm diameter) was aspirated and GCs were cultured in 0 (control), 10-3 , 10-5 , 10-7 , 10-9 M of SNP for 48 h. To evaluate whether this effect was reversible, GCs were cultured in presence of 10-5 M SNP+1.0 mM N ω-nitro-L-arginine methyl ester (L-NAME) a NO synthase (NOS) inhibitor or hemoglobin (Hb, 1.0 μg) as NO scavenger. Nitrate/nitrite concentration was evaluated by Griess method, progesterone and estradiol concentrations by RIA and apoptosis by TUNEL assay. SNP (10-3 , 10-5 , 10-7 M) significantly (p<0.05) inhibited estradiol and progesterone synthesis, growth, disorganized GCs aggregates and induced apoptosis in a dose dependent manner. However, 10-9 M SNP induced the progesterone synthesis and stimulated GCs to develop into a uniform monolayer. Combination of SNP 10-5 M+L-NAME strengthened the inhibitory effect while, SNP+Hb together reversed these inhibitory effects. In conclusion, SNP at greater concentrations (10-3 , 10-5 and 10-7 M) has a cytotoxic effect and it may lead to cell death whereas, at a lower concentration (10-9 M) induced progesterone synthesis and growth of GCs. These findings have important implications that NOS derived NO are involved at physiological level during growth and development of buffalo GCs which regulates the steroidogenesis, growth and apoptosis.

Reproduction in Domestic Animals, 2012

In the present study, the expression profile of luteinizing hormone receptor (LHR) was investigat... more In the present study, the expression profile of luteinizing hormone receptor (LHR) was investigated in the ovary, magnum and uterus and in hierarchcal follicles (F-1, F-2, F-3 and F-4) of hens subjected to moulting to establish their involvement in moulting and presence in non-gonadal tissues. Fifty-two layers (72 weeks) were subjected to moult for a period of 14 days. Four birds were sacrificed each time on 0,

Journal of Veterinary Science, 2011

Effect of sodium nitroprusside (SNP), a nitric oxide (NO) donor, on in vitro survival, growth, st... more Effect of sodium nitroprusside (SNP), a nitric oxide (NO) donor, on in vitro survival, growth, steroidogenesis, and apoptosis of buffalo preantral follicles (PFs) was investigated. PFs (200∼250 μm) were isolated by micro-dissection and cultured in 0 (control), 10 3 , 10 5 , 10 7 , and 10 9 M SNP. To examine the reversible effect of SNP, PFs were cultured with 10 5 M SNP ＋ 1 mM N ω-nitro-L-arginine methyl ester (L-NAME) or 1.0 μg hemoglobin (Hb). The results showed that greater concentrations of SNP (10 3 , 10 5 , 10 7 M) inhibited (p ＜ 0.05) FSH-induced survival, growth, antrum formation, estradiol production, and oocyte apoptosis in a dose-dependent manner. However, a lower dose of SNP (10 9 M) significantly stimulated (p ＜ 0.05) the survival, growth, antrum formation, follicular oocyte maturation, and stimulated progesterone secretion compared to the control. A combination of SNP ＋ L-NAME promoted the inhibitor effect of SNP while a SNP ＋ Hb combination reversed this effect. Nitrate and nitrite concentrations in the culture medium increased (p ＜ 0.05) in a dosedependent manner according to SNP concentration in the culture medium. At higher concentrations, SNP had a cytotoxic effect leading to follicular oocyte apoptosis whereas lower concentrations have stimulatory effects. In conclusion, NO exerts a dual effect on its development of buffalo PFs depending on the concentration in the culture medium.

Journal of Endocrinology, 2008

The purpose of the study was to demonstrate the presence of nitric oxide (NO) synthase (NOS) isof... more The purpose of the study was to demonstrate the presence of nitric oxide (NO) synthase (NOS) isoforms (neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS)) and the role of NO in the ovary of Heteropneustes fossilis. In one half of the ovary collected during different reproductive stages, NOS isoforms were localized immunohistochemically in paraffin sections whereas the other half was processed for NOS and NO quantification using western blot followed by densitometry and nitrate/nitrite assay respectively. The role of NO on oocyte maturation was studied by examining the effect of NO donor (sodium nitroprusside; SNP) and NOS inhibitor (Nω-nitro-l-arginine methyl ester) on 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-P)-induced germinal vesicle breakdown (GVBD) in the cultured oocyte collected during prespawning phase. NOS immunostaining was predominantly localized in previtellogenic follicles, with nNOS detected in the nucleus and cytoplasm of oocytes whereas iNOS and ...

Fertility and Sterility, 2004

correlations of adiponectin to the hormonal and metabolic parameters including measures of insuli... more correlations of adiponectin to the hormonal and metabolic parameters including measures of insulin resistance (IR). DESIGN: Case-control study. MATERIALS AND METHODS: One hundred and eighty selected women were classified as follows: 45 obese (BMI Ͻ30 kg/m 2) with PCOS; 45 lean (BMI Ͻ25 kg/m 2) with PCOS; 45 obese (BMI Ͼ30 kg/m 2) without PCOS and 45 lean (BMI Ͻ25 kg/m 2) without PCOS. Blood samples were collected from all women with or without PCOS between 8:00Ϫ11:00 a.m., after an overnight fast. Serum levels of LH, FSH, PROL, TSH, FT4, T, 17-OHP, ⌬4-A, DHEA, DHEAS, SHBG, insulin and plasma levels of adiponectin and glucose were determined. Measures of IR included: fasting serum insulin, GIR and HOMA. RESULTS: Adiponectin concentrations were found to be significantly decreased in women with PCOS and in obese without PCOS as compared to lean women without PCOS. Adiponectin concentrations correlated inversely with body weight, BMI, fasting plasma glucose, serum insulin, ⌬4-A, DHEA, DHEAS, HOMA, but correlated positively with serum T, SHBG, FAI and GIR. Multiple regression analysis showed that BMI, HOMA, ⌬4-A, and insulin were independent determinants of adiponectin concentrations. CONCLUSION: Hypoadiponectinaemia is evident in obese and lean women with PCOS with variable degree of IR; and it is suggested that IR per se and/or other metabolic abnormalities of PCOS are involved in the regulation of adiponectin concentration in women with PCOS.

Theriogenology, 2012

The present study was designed to investigate the expression of nitric oxide synthase (NOS) isofo... more The present study was designed to investigate the expression of nitric oxide synthase (NOS) isoforms in buffalo ovarian preantral (PFs), antral (AFs) and ovulatory (OFs) follicles (Experiment 1); effect of NO on in vitro survival and growth of PFs (Experiment 2) and NOS activity in immature oocytes by NADPH-diaphorase test (Experiment 3). In Experiment 1, NOS isoforms (neuronal, inducible and endothelial) were localized immunohistochemically; mRNA and protein expression was analyzed by semi-quantitative RT-PCR and western blot, respectively. In Experiment 2, PFs were isolated by micro-dissection method from buffalo ovaries and cultured in 0 (control), 10 Ϫ3 , 10 Ϫ5 , 10 Ϫ7 and 10 Ϫ9 M sodium nitroprusside (SNP). PFs were further cultured with 10 Ϫ5 M SNP ϩ 1.0 mM N-nitro-L-arginine methyl ester (L-NAME) or 1.0 g/ml hemoglobin (Hb) to examine the reversible effect of SNP. Immunohistochemical studies demonstrated that inducible nitric oxide synthase (iNOS) immunoreactivity was predominantly localized in granulosa and theca cells whereas, neuronal (nNOS) and endothelial (eNOS) nitric oxide synthase in the theca, granulosa and cumulus cells of PFs, AFs and OFs. The amount of mRNA as well as protein of nNOS and eNOS was found similar between different stages of follicles. In contrast, higher level of iNOS mRNA was observed in OFs and protein in the AFs. Higher doses of SNP (10 Ϫ3 , 10 Ϫ5 , 10 Ϫ7 M) inhibited (P Ͻ 0.05) while, lower dose of SNP (10 Ϫ9 M) stimulated (P Ͻ 0.05) the survival, growth, and antrum formation of PFs. The inhibitory effects of SNP were reversed by Hb, while L-NAME was not found effective. In conclusion, expression of NOS isoforms mRNA and protein in PFs, AFs, and OFs and NOS enzyme activity in immature follicular oocytes suggest a role for NO during ovarian folliculogenesis in buffalo. NO plays a dual role on growth and survival of PFs depending on its concentration in the culture medium.

The aim of the study was to demonstrate the presence of nitric oxide synthase (NOS) isoforms and ... more The aim of the study was to demonstrate the presence of nitric oxide synthase (NOS) isoforms and role of nitric oxide (NO) in ovary of H. fossilis. The immunohistochemistry (IHC) showed eNOS and iNOS in granulosa and theca cells and nNOS in ooplasm of early previtellogenic follicles. Western blot confirmed the presence of three types of NOS in ovary. The ovarian concentrations of nitric oxide were significantly higher in pre-vitellogenic than vitellogenic phase. The in vitro study showed the inhibitory action of NO in oocytes maturation. The results suggest a role of NO in follicular development in H. fossilis.

Influence of nitric oxide on in vitro growth, survival, steroidogenesis, and apoptosis of follicl... more Influence of nitric oxide on in vitro growth, survival, steroidogenesis, and apoptosis of follicle stimulating hormone stimulated buffalo (Bubalus bubalis) preantral follicles

Our previous studies show that a decrease in endogenous nitric oxide (NO) is involved in the blun... more Our previous studies show that a decrease in endogenous nitric oxide (NO) is involved in the blunted outward K ϩ currents in carotid body (CB) glomus cells from chronic heart failure (CHF) rabbits. In the present study, we measured the effects of the neuronal nitric oxide synthase (nNOS) transgene on the K ϩ currents in CB glomus cells from pacing-induced CHF rabbits. Using single-cell real-time RT-PCR and immunofluorescent techniques, we found that nNOS mRNA and protein are expressed in the rabbit CB glomus cells and CHF decreased the expression of nNOS mRNA and protein in CB glomus cells. After 3 days of an adenoviral nNOS (Ad.nNOS) gene transfection, the expression of nNOS protein was increased to the level found in sham CB glomus cells. In whole cell patch-clamp experiments, Ad.nNOS markedly reversed the attenuated K ϩ currents in CB glomus cells from CHF rabbits. The specific nNOS inhibitor (S-methyl-L-thiocitrulline [SMTC]) and large-conductance Ca 2ϩ-activated K ϩ (BK) channel blocker (iberiotoxin) fully abolished the effect of Ad.nNOS on the K ϩ currents in the CB glomus cells from CHF rabbits. However, neither CHF nor Ad.nNOS altered the protein expression of BK channel ␣-subunit. These results suggest that a decrease of NO induced by an attenuated nNOS activity lowers the activation of the BK channels but not the protein expression of the BK channel ␣-subunit in the CB glomus cells during CHF.

International Journal of Immunopathology and Pharmacology, 2003

Bioactive novel polypeptide of Anurans skin has a wide range of antimicrobial properties against ... more Bioactive novel polypeptide of Anurans skin has a wide range of antimicrobial properties against the infection and tumour cell. Macrophages are known to produce the Nitric oxide (NO) by a variety of cells upon activation. NO produced by the activated macrophages an important mediator for antimicrobial and tumoricidal activity. In-vitro macrophage exposed with medium alone, containing LPS, containing polypepeptides and LPS + polypeptides for 24 h showed enhanced production of NO with respect to control and LPS treated and significant increase in NO production in LPS + polypeptide. Western blot and PCR analysis also showed that increased production of protein expression and mRNA expression of inducible nitric oxide synthase (iNOS). These findings suggest that novel polypeptides are potent activating agent for enhanced production of NO through activation of iNOS gene.

Asian-Australasian Journal of Animal Sciences, 2011

Objective of this study was to examine the effect of sodium nitroprusside (SNP), a nitric oxide (... more Objective of this study was to examine the effect of sodium nitroprusside (SNP), a nitric oxide (NO) donor on steroid synthesis, growth and apoptosis of buffalo granulosa cells (GCs) in vitro. Follicular fluid of antral follicles (3-5 mm diameter) was aspirated and GCs were cultured in 0 (control), 10-3 , 10-5 , 10-7 , 10-9 M of SNP for 48 h. To evaluate whether this effect was reversible, GCs were cultured in presence of 10-5 M SNP+1.0 mM N ω-nitro-L-arginine methyl ester (L-NAME) a NO synthase (NOS) inhibitor or hemoglobin (Hb, 1.0 μg) as NO scavenger. Nitrate/nitrite concentration was evaluated by Griess method, progesterone and estradiol concentrations by RIA and apoptosis by TUNEL assay. SNP (10-3 , 10-5 , 10-7 M) significantly (p<0.05) inhibited estradiol and progesterone synthesis, growth, disorganized GCs aggregates and induced apoptosis in a dose dependent manner. However, 10-9 M SNP induced the progesterone synthesis and stimulated GCs to develop into a uniform monolayer. Combination of SNP 10-5 M+L-NAME strengthened the inhibitory effect while, SNP+Hb together reversed these inhibitory effects. In conclusion, SNP at greater concentrations (10-3 , 10-5 and 10-7 M) has a cytotoxic effect and it may lead to cell death whereas, at a lower concentration (10-9 M) induced progesterone synthesis and growth of GCs. These findings have important implications that NOS derived NO are involved at physiological level during growth and development of buffalo GCs which regulates the steroidogenesis, growth and apoptosis.

Reproduction in Domestic Animals, 2012

In the present study, the expression profile of luteinizing hormone receptor (LHR) was investigat... more In the present study, the expression profile of luteinizing hormone receptor (LHR) was investigated in the ovary, magnum and uterus and in hierarchcal follicles (F-1, F-2, F-3 and F-4) of hens subjected to moulting to establish their involvement in moulting and presence in non-gonadal tissues. Fifty-two layers (72 weeks) were subjected to moult for a period of 14 days. Four birds were sacrificed each time on 0,

Journal of Veterinary Science, 2011

Effect of sodium nitroprusside (SNP), a nitric oxide (NO) donor, on in vitro survival, growth, st... more Effect of sodium nitroprusside (SNP), a nitric oxide (NO) donor, on in vitro survival, growth, steroidogenesis, and apoptosis of buffalo preantral follicles (PFs) was investigated. PFs (200∼250 μm) were isolated by micro-dissection and cultured in 0 (control), 10 3 , 10 5 , 10 7 , and 10 9 M SNP. To examine the reversible effect of SNP, PFs were cultured with 10 5 M SNP ＋ 1 mM N ω-nitro-L-arginine methyl ester (L-NAME) or 1.0 μg hemoglobin (Hb). The results showed that greater concentrations of SNP (10 3 , 10 5 , 10 7 M) inhibited (p ＜ 0.05) FSH-induced survival, growth, antrum formation, estradiol production, and oocyte apoptosis in a dose-dependent manner. However, a lower dose of SNP (10 9 M) significantly stimulated (p ＜ 0.05) the survival, growth, antrum formation, follicular oocyte maturation, and stimulated progesterone secretion compared to the control. A combination of SNP ＋ L-NAME promoted the inhibitor effect of SNP while a SNP ＋ Hb combination reversed this effect. Nitrate and nitrite concentrations in the culture medium increased (p ＜ 0.05) in a dosedependent manner according to SNP concentration in the culture medium. At higher concentrations, SNP had a cytotoxic effect leading to follicular oocyte apoptosis whereas lower concentrations have stimulatory effects. In conclusion, NO exerts a dual effect on its development of buffalo PFs depending on the concentration in the culture medium.

Journal of Endocrinology, 2008

The purpose of the study was to demonstrate the presence of nitric oxide (NO) synthase (NOS) isof... more The purpose of the study was to demonstrate the presence of nitric oxide (NO) synthase (NOS) isoforms (neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS)) and the role of NO in the ovary of Heteropneustes fossilis. In one half of the ovary collected during different reproductive stages, NOS isoforms were localized immunohistochemically in paraffin sections whereas the other half was processed for NOS and NO quantification using western blot followed by densitometry and nitrate/nitrite assay respectively. The role of NO on oocyte maturation was studied by examining the effect of NO donor (sodium nitroprusside; SNP) and NOS inhibitor (Nω-nitro-l-arginine methyl ester) on 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-P)-induced germinal vesicle breakdown (GVBD) in the cultured oocyte collected during prespawning phase. NOS immunostaining was predominantly localized in previtellogenic follicles, with nNOS detected in the nucleus and cytoplasm of oocytes whereas iNOS and ...

Fertility and Sterility, 2004

correlations of adiponectin to the hormonal and metabolic parameters including measures of insuli... more correlations of adiponectin to the hormonal and metabolic parameters including measures of insulin resistance (IR). DESIGN: Case-control study. MATERIALS AND METHODS: One hundred and eighty selected women were classified as follows: 45 obese (BMI Ͻ30 kg/m 2) with PCOS; 45 lean (BMI Ͻ25 kg/m 2) with PCOS; 45 obese (BMI Ͼ30 kg/m 2) without PCOS and 45 lean (BMI Ͻ25 kg/m 2) without PCOS. Blood samples were collected from all women with or without PCOS between 8:00Ϫ11:00 a.m., after an overnight fast. Serum levels of LH, FSH, PROL, TSH, FT4, T, 17-OHP, ⌬4-A, DHEA, DHEAS, SHBG, insulin and plasma levels of adiponectin and glucose were determined. Measures of IR included: fasting serum insulin, GIR and HOMA. RESULTS: Adiponectin concentrations were found to be significantly decreased in women with PCOS and in obese without PCOS as compared to lean women without PCOS. Adiponectin concentrations correlated inversely with body weight, BMI, fasting plasma glucose, serum insulin, ⌬4-A, DHEA, DHEAS, HOMA, but correlated positively with serum T, SHBG, FAI and GIR. Multiple regression analysis showed that BMI, HOMA, ⌬4-A, and insulin were independent determinants of adiponectin concentrations. CONCLUSION: Hypoadiponectinaemia is evident in obese and lean women with PCOS with variable degree of IR; and it is suggested that IR per se and/or other metabolic abnormalities of PCOS are involved in the regulation of adiponectin concentration in women with PCOS.

Theriogenology, 2012

The present study was designed to investigate the expression of nitric oxide synthase (NOS) isofo... more The present study was designed to investigate the expression of nitric oxide synthase (NOS) isoforms in buffalo ovarian preantral (PFs), antral (AFs) and ovulatory (OFs) follicles (Experiment 1); effect of NO on in vitro survival and growth of PFs (Experiment 2) and NOS activity in immature oocytes by NADPH-diaphorase test (Experiment 3). In Experiment 1, NOS isoforms (neuronal, inducible and endothelial) were localized immunohistochemically; mRNA and protein expression was analyzed by semi-quantitative RT-PCR and western blot, respectively. In Experiment 2, PFs were isolated by micro-dissection method from buffalo ovaries and cultured in 0 (control), 10 Ϫ3 , 10 Ϫ5 , 10 Ϫ7 and 10 Ϫ9 M sodium nitroprusside (SNP). PFs were further cultured with 10 Ϫ5 M SNP ϩ 1.0 mM N-nitro-L-arginine methyl ester (L-NAME) or 1.0 g/ml hemoglobin (Hb) to examine the reversible effect of SNP. Immunohistochemical studies demonstrated that inducible nitric oxide synthase (iNOS) immunoreactivity was predominantly localized in granulosa and theca cells whereas, neuronal (nNOS) and endothelial (eNOS) nitric oxide synthase in the theca, granulosa and cumulus cells of PFs, AFs and OFs. The amount of mRNA as well as protein of nNOS and eNOS was found similar between different stages of follicles. In contrast, higher level of iNOS mRNA was observed in OFs and protein in the AFs. Higher doses of SNP (10 Ϫ3 , 10 Ϫ5 , 10 Ϫ7 M) inhibited (P Ͻ 0.05) while, lower dose of SNP (10 Ϫ9 M) stimulated (P Ͻ 0.05) the survival, growth, and antrum formation of PFs. The inhibitory effects of SNP were reversed by Hb, while L-NAME was not found effective. In conclusion, expression of NOS isoforms mRNA and protein in PFs, AFs, and OFs and NOS enzyme activity in immature follicular oocytes suggest a role for NO during ovarian folliculogenesis in buffalo. NO plays a dual role on growth and survival of PFs depending on its concentration in the culture medium.