Rohit Saluja

Publications of Rohit Saluja

• Molecular and biochemical characterization of nitric oxide synthase isoforms and their intracellular distribution in human peripheral blood mononuclear cells.

  Authors: Rohit Saluja, Anupam Jyoti, Madhumita Chatterjee, Saman Habib, Anupam Verma, Kalyan Mitra, Manoj Kumar Barthwal, Virendra K Bajpai, Madhu Dikshit

  Biochimica et biophysica acta. 06/2011; 1813(10):1700-7.

  Nitric oxide synthase (NOS) expression and catalytic status in human peripheral blood mononuclear cells (PBMCs) is debatable, while its sub-cellular distribution remains unascertained. The presentNitric oxide synthase (NOS) expression and catalytic status in human peripheral blood mononuclear cells (PBMCs) is debatable, while its sub-cellular distribution remains unascertained. The present study characterizes NOS transcripts by real time PCR, NOS protein by immunoprecipitation (IP)/Western blot (WB), nitric oxide (NO) generation by DAF-2DA and NOS sub-cellular distribution by immunogold electron microscopy in resting PBMCs, monocytes and lymphocytes obtained from healthy donors. We observed constitutive expression of full length NOS isoforms (nNOS, iNOS and eNOS) in PBMCs: with the highest expression of iNOS in comparison to nNOS and eNOS. Isolated monocytes expressed more eNOS transcript and protein as compared to nNOS and iNOS. Lymphocytes however had more iNOS transcripts and protein than nNOS and eNOS. NOS was catalytically active in PBMCs, monocytes as well as in lymphocytes as evident by NO generation in the presence of substrate and cofactors, which was significantly reduced in the presence of NOS inhibitor. Immunogold electron microscopy and morphometric analysis revealed the distinct pattern of NOS distribution in monocytes and lymphocytes and also exhibited differences in the nuclear-cytoplasmic ratio. nNOS localization was much more in the cytosol than in the nucleus among both monocytes and lymphocytes. Interestingly, iNOS distribution was comparable in both cytosol and nucleus among monocytes, but in lymphocytes iNOS was predominantly localized to the cytosol. The present study exhibits constitutive presence of all the NOS isoforms in PBMCs and reports the distinct pattern of NOS distribution among monocytes and lymphocytes.

• Nitric oxide donors release extracellular traps from human neutrophils by augmenting free radical generation.

  Authors: Satyananda Patel, Sachin Kumar, Anupam Jyoti, Bangalore Suresh Srinag, Ravi Shankar Keshari, Rohit Saluja, Anupam Verma, Kalyan Mitra, Manoj Kumar Barthwal, Hanumanthappa Krishnamurthy, Virendra K Bajpai, Madhu Dikshit

  Nitric oxide : biology and chemistry / official journal of the Nitric Oxide Society. 04/2010; 22(3):226-34.

  High availability of NO, oxidative stress and neutrophil extracellular trap (NETs) contents are often noticed at the site of inflammation/infection. Studies from this lab and others have reported NOHigh availability of NO, oxidative stress and neutrophil extracellular trap (NETs) contents are often noticed at the site of inflammation/infection. Studies from this lab and others have reported NO mediated free radical generation from neutrophils; role of NO in NETs formation however remains undefined so far. The present study was therefore undertaken to explore the effect of NO donors on NET release from human neutrophils (PMNs), using confocal/scanning microscopy, measuring the extracellular DNA content and NET-bound elastase activity. Addition of NO donors (SNAP and SNP) to adhered PMNs led to a time and concentration dependent NETs release, which was blocked by N-acetyl cysteine, suggesting involvement of free radicals in NETs formation. Free radical formation by NO donors was assessed by using DCF-DA, DMPO-nitrone antibody and by p47 phox migration to the neutrophils membrane. NO mediated formation of free radicals and NETs was significantly reduced by the pretreatment of neutrophils with diphenyleneiodonium (DPI), a NADPH-oxidase inhibitor and 4-aminobenzoic acid hydrazide (ABAH), a myeloperoxidase inhibitor, suggesting role of enzymatic free radical generation by NO donors. We thus demonstrate that NO by augmenting free radical formation in human neutrophils mediates NETs release.

• Ultrastructural immunogold localization of nitric oxide synthase isoforms in rat and human eosinophils.

  Authors: Rohit Saluja, Rashmi Saini, Kalyan Mitra, Virendra K Bajpai, Madhu Dikshit

  Cell and tissue research. 03/2010; 340(2):381-8.

  The involvement of nitric oxide (NO) as both pro and anti-inflammatory agent in allergic, airway inflammatory, and asthmatic diseases and the active participation of eosinophils in such ailments haveThe involvement of nitric oxide (NO) as both pro and anti-inflammatory agent in allergic, airway inflammatory, and asthmatic diseases and the active participation of eosinophils in such ailments have been previously suggested. NO modulates eosinophil number, migration and their survival. The microenvironment of NO synthase (NOS) in subcellular organelles determines its rate and efficiency of catalysis, which in turn influences NO generation at distinct intracellular locales. The present study was undertaken to assess the intracellular distribution of NOS isoforms by transmission electron microscopy followed by morphometric analysis in human and rat eosinophils. Rat eosinophils were explored in parallel, and since they are widely used as model systems to mimic human diseases, a comparative study on NOS localization patterns might provide useful information in deciphering NO role in diverse aspects of eosinophil-related inflammatory ailments. The results demonstrated predominance of neuronal NOS (nNOS) in the eosinophilic granules and even distribution of inducible NOS (iNOS) and nNOS in the cytoplasm and nucleus of human eosinophils. In rat eosinophils, however, iNOS was mainly localized in the eosinophilic granules and nucleus, while nNOS was distributed evenly in cytoplasm and nucleus. Distribution of endothelial NOS (eNOS) in eosinophils was scanty. Differences in NOS isoforms and their localization in human and rat cells might have implications in differential mode of catalysis and functional contribution to eosinophil physiology and pathology, warranting detailed investigations. The present study highlights species-specific differences in the relative abundance and distribution pattern of NOS isoforms in rat and human eosinophils, which should be considered cautiously in interpreting the rat data to humans.

• Augmented nitric oxide generation in neutrophils: Oxidative and pro-inflammatory implications in hypertension.

  Authors: Madhumita Chatterjee, Rohit Saluja, Shikha Tewari, Manoj Kumar Barthwal, Sudhir Kumar Goel, Madhu Dikshit

  Free radical research. 12/2009; 43(12):1195-204.

  Abstract The present study explores expression of NOS and pro-inflammatory cytokines, NOS catalysis and NO mediated modulation of oxidative response and apoptosis in neutrophils from spontaneouslyAbstract The present study explores expression of NOS and pro-inflammatory cytokines, NOS catalysis and NO mediated modulation of oxidative response and apoptosis in neutrophils from spontaneously hypertensive rats (SHR). Neutrophils from SHR showed approximately 3-fold increments in iNOS expression, 1.5-fold increments in nOS expression and calcium independent NOS catalysis, whereas GTPCH expression was unaltered. Although phagocytic potential was comparable, neutrophils from SHR demonstrated augmented oxidative burst, which was reduced by NOS inhibition or in the presence of NO scavenger. SHR neutrophils also exhibited enhanced MPO catalysis and [Ca(2+)](i) levels. Levels of TNF-alpha and IFN-gamma were comparable, but IL-1beta and CRP levels in SHR plasma were (p<0.05) elevated. This study evidenced significantly enhanced expression of IL-1beta in SHR neutrophils whereas those of TNF-alpha and IFN-gamma were unaltered. Moreover neutrophils from SHR exhibited (p<0.01) delayed apoptotic response and sustained NO generation, as evident from elevated nitrite levels in neutrophil culture supernatant above the control levels. Results obtained indicate an augmented NO generation from neutrophils during hypertension which might fortify their attribute to the oxidative and inflammatory stress in SHR, emphasizing the importance of neutrophils in hypertension.

• Ascorbate sustains neutrophil NOS expression, catalysis, and oxidative burst.

  Authors: Madhumita Chatterjee, Rohit Saluja, Vipul Kumar, Anupam Jyoti, Girish Kumar Jain, Manoj Kumar Barthwal, Madhu Dikshit

  Free radical biology & medicine. 08/2008;

  Previous studies from this lab have demonstrated that in vitro ascorbate augments neutrophil nitric oxide (NO) generation and oxidative burst. The present study was therefore undertaken in guineaPrevious studies from this lab have demonstrated that in vitro ascorbate augments neutrophil nitric oxide (NO) generation and oxidative burst. The present study was therefore undertaken in guinea pigs to further assess the implication of ascorbate deficiency in vivo on neutrophil ascorbate and tetrahydrobiopterin content, NOS expression/activity, phagocytosis, and respiratory burst. Ascorbate deficiency significantly reduced ascorbate and tetrahydrobiopterin amounts, NOS expression/activity, and NO as well as free radical generation in neutrophils from scorbutics. Ascorbate and tetrahydrobiopterin supplementation in vitro, though, significantly enhanced NOS catalysis in neutrophil lysates and NO generation in live cells, but could not restore them to control levels. Although phagocytic activity remained unaffected, scorbutic neutrophils were compromised in free radical generation. Ascorbate-induced free radical generation was NO dependent and prevented by NOS and NADPH oxidase inhibitors. Augmentation of oxidative burst with dehydroascorbate (DHA) was counteracted in the presence of glucose (DHA uptake inhibitor) and iodoacetamide (glutaredoxin inhibitor), suggesting the importance of ascorbate recycling in neutrophils. Ascorbate uptake was, however, unaffected among scorbutic neutrophils. These observations thus convincingly demonstrate a novel role for ascorbate in augmenting both NOS expression and activity in vivo, thereby reinforcing oxidative microbicidal actions of neutrophils.

• Translation of open reading frame in kinetoplast DNA minicircles of clinical isolates of L. donovani.

  Authors: Hema Kothari, Pranav Kumar, Rohit Saluja, Shyam Sundar, Neeloo Singh

  Parasitology research. 04/2007; 100(4):893-7.

  Till today, it remains an enigma whether the open reading frames said to be transcribed in minicircle sequences are indeed translated into protein products or not. We establish a protein-coding geneTill today, it remains an enigma whether the open reading frames said to be transcribed in minicircle sequences are indeed translated into protein products or not. We establish a protein-coding gene in minicircle variable region of kinetoplast DNA from clinical isolates of Leishmania donovani. The protein was expressed as an N-tagged green fluorescent protein (GFP) fusion protein in leishmanial expression system. Fluorescence microscopy of the transfectants carrying recombinant GFP construct showed the protein to be localized on the plasmalemma of the parasite. This shows that the minicircle transcript is indeed translated into a protein product in the parasite cell and further points toward probable biological function of minicircles in kinetoplastids.

• Nitric oxide synthase regulation and diversity: implications in Parkinson's disease.

  Authors: Ramkumar Kavya, Rohit Saluja, Sarika Singh, Madhu Dikshit

  Nitric oxide : biology and chemistry / official journal of the Nitric Oxide Society. 01/2007; 15(4):280-94.

  Nitric oxide (NO) is a janus faced chemical messenger, which, in the recent years, has been the focus of neurobiologists for its involvement in neurodegenerative disorders in particular, Parkinson'sNitric oxide (NO) is a janus faced chemical messenger, which, in the recent years, has been the focus of neurobiologists for its involvement in neurodegenerative disorders in particular, Parkinson's disease (PD). Nitric oxide synthase, the key enzyme involved in NO production exists in three known isoforms. The neuronal and inducible isoforms have been implicated in the pathogenesis of PD. These enzymes are subject to complex expressional and functional regulation involving mRNA diversity, phosphorylation and protein interaction. In the recent years, mRNA diversity and polymorphisms have been identified in the NOS isoforms. Some of these genetic variations have been associated with PD, indicating an etiological role for the NOS genes. This review mainly focuses on the NOS genes - their differential regulation and genetic heterogeneity, highlighting their significance in the pathobiology of PD.

• Nitric oxide synthase regulation and diversity: Implications in Parkinson’s disease

  Authors: Ramkumar Kavya, Rohit Saluja, Sarika Singh, Madhu Dikshit

  Nitric Oxide.

  Nitric oxide (NO) is a janus faced chemical messenger, which, in the recent years, has been the focus of neurobiologists for its involvement in neurodegenerative disorders in particular, Parkinson’sNitric oxide (NO) is a janus faced chemical messenger, which, in the recent years, has been the focus of neurobiologists for its involvement in neurodegenerative disorders in particular, Parkinson’s disease (PD). Nitric oxide synthase, the key enzyme involved in NO production exists in three known isoforms. The neuronal and inducible isoforms have been implicated in the pathogenesis of PD. These enzymes are subject to complex expressional and functional regulation involving mRNA diversity, phosphorylation and protein interaction. In the recent years, mRNA diversity and polymorphisms have been identified in the NOS isoforms. Some of these genetic variations have been associated with PD, indicating an etiological role for the NOS genes. This review mainly focuses on the NOS genes—their differential regulation and genetic heterogeneity, highlighting their significance in the pathobiology of PD.

• Ascorbate sustains neutrophil NOS expression, catalysis, and oxidative burst

  Authors: Madhumita Chatterjee, Rohit Saluja, Vipul Kumar, Anupam Jyoti, Girish Kumar Jain, Manoj Kumar Barthwal, Madhu Dikshit

  Free Radical Biology and Medicine.

  Previous studies from this lab have demonstrated that in vitro ascorbate augments neutrophil nitric oxide (NO) generation and oxidative burst. The present study was therefore undertaken in guineaPrevious studies from this lab have demonstrated that in vitro ascorbate augments neutrophil nitric oxide (NO) generation and oxidative burst. The present study was therefore undertaken in guinea pigs to further assess the implication of ascorbate deficiency in vivo on neutrophil ascorbate and tetrahydrobiopterin content, NOS expression/activity, phagocytosis, and respiratory burst. Ascorbate deficiency significantly reduced ascorbate and tetrahydrobiopterin amounts, NOS expression/activity, and NO as well as free radical generation in neutrophils from scorbutics. Ascorbate and tetrahydrobiopterin supplementation in vitro, though, significantly enhanced NOS catalysis in neutrophil lysates and NO generation in live cells, but could not restore them to control levels. Although phagocytic activity remained unaffected, scorbutic neutrophils were compromised in free radical generation. Ascorbate-induced free radical generation was NO dependent and prevented by NOS and NADPH oxidase inhibitors. Augmentation of oxidative burst with dehydroascorbate (DHA) was counteracted in the presence of glucose (DHA uptake inhibitor) and iodoacetamide (glutaredoxin inhibitor), suggesting the importance of ascorbate recycling in neutrophils. Ascorbate uptake was, however, unaffected among scorbutic neutrophils. These observations thus convincingly demonstrate a novel role for ascorbate in augmenting both NOS expression and activity in vivo, thereby reinforcing oxidative microbicidal actions of neutrophils.

• Nitric oxide donors release extracellular traps from human neutrophils by augmenting free radical generation

  Authors: Satyananda Patel, Sachin Kumar, Anupam Jyoti, Bangalore Suresh Srinag, Ravi Shankar Keshari, Rohit Saluja, Anupam Verma, Kalyan Mitra, Manoj Kumar Barthwal, Hanumanthappa Krishnamurthy, Virendra K. Bajpai, Madhu Dikshit

  Nitric Oxide.

  High availability of NO, oxidative stress and neutrophil extracellular trap (NETs) contents are often noticed at the site of inflammation/infection. Studies from this lab and others have reported NOHigh availability of NO, oxidative stress and neutrophil extracellular trap (NETs) contents are often noticed at the site of inflammation/infection. Studies from this lab and others have reported NO mediated free radical generation from neutrophils; role of NO in NETs formation however remains undefined so far. The present study was therefore undertaken to explore the effect of NO donors on NET release from human neutrophils (PMNs), using confocal/scanning microscopy, measuring the extracellular DNA content and NET-bound elastase activity. Addition of NO donors (SNAP and SNP) to adhered PMNs led to a time and concentration dependent NETs release, which was blocked by N-acetyl cysteine, suggesting involvement of free radicals in NETs formation. Free radical formation by NO donors was assessed by using DCF-DA, DMPO-nitrone antibody and by p47 phox migration to the neutrophils membrane. NO mediated formation of free radicals and NETs was significantly reduced by the pretreatment of neutrophils with diphenyleneiodonium (DPI), a NADPH-oxidase inhibitor and 4-aminobenzoic acid hydrazide (ABAH), a myeloperoxidase inhibitor, suggesting role of enzymatic free radical generation by NO donors. We thus demonstrate that NO by augmenting free radical formation in human neutrophils mediates NETs release.