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डॉ मधु दीक्षित, एफएनए, एफएएससी, एफएनएएससी, जेसी बोस नेशनल फेलो |
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Dr. Madhu Dikshit, FNA, FASc, FNASc, JC Bose National Fellow |
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निदेशक/Director
सीएसआईआर-केन्द्रीय औषधि अनुसंधान संस्थान
CSIR-Central Drug Research Institute
सेक्टर 10, जानकीपुरम विस्तार, सीतापुर रोड
Sector 10, Jankipuram Extn., Sitapur Road
लखनऊ - २२६०३१ उ0प्र0, भारत
Lucknow - 226 031, UP, India
दूरभाष / Tel: 091-522-2771940; फैक्स / Fax: 091-522-2771941
ईमेल / Email: [email protected], [email protected]
Lucknow 226 031 |
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| Educational qualifications |
MSc (Biochemistry), Ph.D. |
| Date of Birth |
21.11.1957 |
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| RESEARCH AREAS |
Investigation of the molecular mechanisms involved in the regulation of NADPH-oxidase (NOX-2) and nitric oxide synthase (NOS) activity in the neutrophils.
Neutrophils (PMNs), the most abundant, are very short lived terminally differentiated circulating leukocytes. They comprise more than 60% of the total circulating leukocyte population, and are primarily recognized for the effective elimination of invading pathogens. Migration of PMNs from blood to specific tissues following pathological insult is a key feature of the host inflammatory response, which is often localized and protective. Conversely, success of thrombolysis to treat acute coronary obstructions unmasked their active role in the oxidative injury following reperfusion. PMNs in circulation are constantly exposed to diverse pathogenic and inflammatory stimuli, and constantly respond to their microenvironment. PMNs are involved in the clearance of viral and intracellular microbes and their interaction with various components of the immune system such as B and T cells, dendritic cells and NK cells have been recognized. Neutrophils, the suicide killers, possess efficient oxidative and non-oxidative machineries to execute the engulfed pathogens. Activated PMNs generate highest amount of reactive oxygen species in human body through NADPH-oxidase (NOX-2) and myeloperoxidase (MPO), while resting and activated cells generate appreciable amounts of nitric oxide (NO). |
Research in my lab is focused to understand the - |
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Effect of NO on human and rodent neutrophils associated ROS/RNS generation and neutrophils extracellular trap formation
.Neutrophils (PMNs) when added to platelet suspension significantly reduced platelet aggregation, characterization of the labile inhibitory factor, led to the identification of NO. Even though the biological functions of NO were the subject of much research, its role in PMNs functions was very less defined at that time. I found it incredibly interesting why nature has devised such short
lived cells to generate NO as well as reactive oxygen species (ROS). Investigations in our lab were thus focused on the interaction of NO with superoxide radicals and examined the possibility of NO regulating the generation of ROS in PMNs. We have demonstrated that NO modulates PMNs free radical generation in a biphasic manner, inhibiting NOX-2 activity and scavenging radicals at higher concentrations while, at lower concentrations NO augmented free radical generation. NO activated
NOX-2 activity by facilitating the migration of p47phox to the neutrophil membranes, and interaction of NO with MPO generate nitrogen dioxide (.NO2) and nitryl chloride (NO2Cl). NO also mediated release of neutrophil extracellular traps (NETs) which was regulated by ERK and MAP kinase signaling. These findings have direct implications in medical science and suggest the role of NO in innate immunity as an important modulator of neutrophil functions.
Blood, 1994; Brit J Pharmacol 1996; Blood 1999; Thromb Res 2000; J Leuko Biol, 2004; Free Rad Res, 2009; Cytometry Part A, 2010; NO Biol. Chem, 2010; PLOS One, 2012; Cytometry A, 2012; J Cell Biochem. 2013
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Biochemical and molecular characterization of NOS isoforms, their subcellular distribution and regulation in human and rodent leukocytes.
 Large stores of ascorbate are present in PMNs, we demonstrated that ascorbate prevented oxidation of tetrahydrobiopterin, a cofactor of NOS, to support the enzymatic synthesis of NO. Studies on PMNs from various species, including human, have thus provided a convincing explanation as to why PMNs store exceptionally large amounts of ascorbate. It was evidenced that ascorbate deficiency led to a decline in both expression (both nNOS and iNOS expression) and catalysis of NOS in neutrophils among scorbutic guinea pigs. This was owing to instability of tetrahydrobiopterin a vital co-factor for NOS catalysis, and a rise in corticosterone levels as evidenced in scurvy, thereby interfering with iNOS expression among the scorbutics. These studies support the contention of Sir Pauling that vitamin C enhances immunity against infections.
We demonstrated that rodents and human PMNs constitutively express iNOS & nNOS, and do not possess eNOS. Similar to neurons, neutrophils also express full length PDZ domain containing nNOS. In resting human neutrophils, iNOS protein was distributed in the cytosol, granules containing elastase & gelatinase, and also in other subcellular organelles. Studies on bone marrow derived PMNs in rodents exhibited differential expression of nNOS and iNOS during maturation. Interestingly iNOS expression was consistently augmented during the neutrophil maturation in the bone marrow.
NO Biol. Chem, 2001; NO Biol. Chem, 2002; BBRC, 2003; J Leuko Biol, 2004; J Leuko Biol, 2006, Free Rad Biol Med, 2008; Cytometry Part A, 2010;Cell & Tissue Res, 2010; BBA Mol Cell Res, 2011
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Identification of NOS interacting proteins in human and rodent neutrophils. The novel interaction of RAC2 with iNOS proteins in human PMNs is a potential mechanism to carry iNOS to the phagosomes for the generation of both ROS/RNS to kill the phagocytosed bacteria and has been investigated in detail.
Nitric oxide (NO) modulates diverse functions of neutrophils (PMNs), but localization of nitric oxide synthase (NOS) and identification of its interacting proteins still remain least defined. We discerned subcellular distribution of NOS and caveolin-1, a prominent NOS interacting protein in rat PMNs and provided first evidence of nNOS and iNOS in the nuclear compartment and suggested NOS interaction with caveolin-1 in rat PMNs.
Figure on the left: (A) Caveolin-1 localization in rat PMNs by immunoelectron microscopy, showing caveolin-1 immunolabeling. (B) Caveolin-1 immunolabeling in the nucleus, nuclear membrane and in the plasma membrane (C) Colocalization of iNOS (15 nm) with caveolin-1 (10 nm) in the nucleus and (D) cytoplasm. Bars represent 100 nm (A) and 100 mm (B-D). Figure on the right: (A) iNOS immunolabeling (i), nucleus (blue) (ii), iNOS (red) (iii), caveolin-1 (green) (iv), merge of ii and iii (v) negative control with preimmunized sera (B) nNOS immunolabeling (i), nucleus (blue) (ii), nNOS (red) (iii), caveolin-1 (green) (iv), merge of ii and iii (v) negative control with preimmunized sera. Bar represent 5 μm (A, B). (C) NOS enzyme activity in cytosolic fraction (CF) and nuclear fraction (NF) of PMNs. Addition of CaM augmented NOS activity.
J Leuko Biol, 2006; Blood 2012; Antiox Redox Signal, 2013 |
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Modulation in the circulating NO levels, NOS activity, and NOS expression in the diverse animal models of hypertension, dyslipidemia, atherosclerosis, thrombosis, ischemia/ hypoxia-reoxygenation, ulcers, neurodegeneration, blood brain permeability alterations as well as in the neutrophils from patients of various diseases [Parkinson’s disease, depression, schizophrenia, migraine, sepsis, SIRS, diabetes with cardiometabolic disorders, neutrophelia.
NOS found in PMNs resembles nNOS, we in a multi-centric study, investigated status of NOS in the CNS disorder patients (Parkinson’s disease, schizophrenia, depression and migraine in which role of NO was well documented). A novel finding was a significant increase in the basal NOS activity in the PMNs of Parkinson’s disease patients. Patients of schizophrenia and depression on the other hand exhibited a significant decrease in the PMNs nitrite content, while beta-adrenergic receptor binding sites was decreased only in the patients of depression. Migraine patients did not demonstrate change in the PMNs nitrite content but increase in the platelet NOS activity was reverted back to the normal level within few days after the migraine attack. Our studies have also shown that NO regulates free radical generation from PMNs during pathological conditions such as endotoxemia, and hypoxia-reoxygenation.
Free Rad Res, 2009; Cell Mol Biol 2010; Pharmacol Res 1997, J Pharmacol Exp Therap, 1993, Blood 1999, Redox report 2005; Brain Res., 2007, Neuroreport 1995; Free Rad Biol Med 1993, Acta Neurol Scand, 1999; Int J Neurosciences, 2003; Int J Neurosciences 2006; Neurochem Res, 2009, J Affective Disorder, 2002, Psychopharmacology, 2001, Headache 2001; Cephalgia, 2005, Acta Anaesthesiol Scand, 2012; J Crit Care, 2011, PLOS One 2012, Blood, 2012 |
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Molecular mechanisms involved in NO and nitrite mediated proliferation and apoptosis of myelocytic HL-60cell lines (Free Rad Biol Med, 2010; J Pharmacol Exp Therap, 2011)
Studies on promyelocytic HL-60 cells in this lab systematically explored the effect of NO donor (DETA-NO) and nitrite on the cell cycle. NO in lower concentrations had proliferative effect, while cells treated with higher concentrations exhibited cytostasis, apoptosis, mitochondrial membrane potential loss, caspase-3 activation and dUTP nick end labelling. The proliferative effect of NO was redox sensitive and augmented expression of various cell cycle regulators such as Cdk2, cyclin B, cyclin E, which was blocked by roscovitine, a Cdk2 inhibitor. S-Nitrosylation of Cdk2 and an increase in the Cdk2 associated kinase activity was observed for the first time in NO/nitrite treated cells.
Free Rad Biol Med, 2010, J Pharmacol Exp Ther, 2011
Ongoing studies evidenced reduced chemotaxis of NO/nitrite treated PMNs due to the glutathionylation of cytoskeleton associated proteins. These findings have important implications in the cardiovascular area as NO/nitrite therapy offer protection against ischemia. Since neutrophils have detrimental role in ischemic injury and the chemotaxis of neutrophils is reduced by NO, it is to be explored if the protective effect of nitrite therapy during cardiac ischemia is linked to reduced migration of PMNs to the site of injury. Current studies in my lab explore these possibilities.
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| PRESENT LAB MEMBERS |
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Megha Dubey
Senior Research Fellow
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Jitendra S Kanshana
Senior Research Fellow
Registered for PhD
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Deepika Awasthi
Senior Research Fellow
Registered for PhD
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K Babu Nageswararao
Senior Research Fellow
Registered for PhD
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Sheela Nagarkoti
Senior Research Fellow
Registered for PhD
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Samreen Sadaf
Junior Research Fellow
M.Sc Biotech
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Hobby Aggarwal
Junior Research Fellow
M.Pharm |
Priya Pathak
Project Assistant
M.Pharm
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Priyanka Dhankani
Project Assistant
M.Sc Biochem.
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