Browsing by Author "Rajnikant Mishra"

Now showing 1 - 20 of 46

A Study of Arginase Expression in Chronic Non-healing Wounds
(SAGE Publications Inc., 2023) Ruhi Dixit; Abhik Debnath; Suman Mishra; Rajnikant Mishra; Satyanam K. Bhartiya; Arvind Pratap; Vijay K. Shukla
Arginase expression has been recently shown to increase in numerous disease states like neurodegeneration, inflammation, and malignancies. Although it has been found to be functionally important in various disease pathologies, little is known about its role in wound healing. Here, we look at the expression of arginase and its isoforms in chronic non-healing wounds and also study the expression of nitric oxide synthase (NOS) and oxidative stress enzymes in them. Wound tissues and blood samples were collected at the time of index presentation and follow-up from 61 chronic non-healing wound cases. The expression patterns of arginase isoenzymes, NOS, superoxide dismutases (SOD), lactic acid dehydrogenase (LDH), and catalase were examined by using enzyme-linked immunosorbent assay, immunohistochemistry, and western blot analysis at the transcript and protein level. We reported a significant decrease of serum arginase levels in chronic nonhealing wounds in the progress of wound healing. Interestingly, tissue arginase levels were found to be increased with improved wound condition at follow-up. Tissue NOS, LDH, and catalase activity were also found to be increased with the progress of healing, whereas SOD levels were downregulated. Our findings reported increased expression at the transcript level of arginase-I and arginase-II in chronic non-healing wounds for the first time. In conclusion, we observed decreased serum arginase levels in completely healed patients as compared to non-healed cases. Our study findings support the hypothesis that inhibition of the activity of arginase delays wound healing. Arginase and iNOS may also find their place in the future as possible biomarkers for wound healing. © The Author(s) 2021.
Age-dependent alterations in expression and co-localization of Pax6 and Ras-GAP in brain of aging mice
(Elsevier B.V., 2018) Khushboo Srivastava; Ratnakar Tripathi; Rajnikant Mishra
As the brain ages, the survival and plasticity of neurons and glia are compromised. The data-mining and in silico studies suggest interactions of Pax6 with Ras and binding sites in Ras-GAP promoter. The Pax6 also shows age-dependent alterations. Therefore, it is presumed that Pax6 may be associated with the Ras-GAP, a synaptic protein, either directly or indirectly in brain. The expression, co-localization and interaction of Pax6 and Ras-GAP in different regions of brain of mice during aging were investigated through immunofluorescence assay, co-immunoprecipitation and western blotting, respectively. The co-localization of Pax6 and Ras-GAP were observed in dentate gyrus (DG) and sub-granular zone (SGZ) of hippocampus, in glomerular (GlLa) and mitral cells (MiCe) of olfactory lobe, granular cells (GrCe), Purkinje cell (PuCe) and molecular cell layer (MoLa) of cerebellum, internal plexiform layer (InPl), molecular layer (MoLa) of cerebral cortex and in intercalated cells of amygdala (ITC), caudate nucleus regions in brain of aging mice. The expression of Pax6 and Ras-GAP was altered in hippocampus, amygdala, caudate nucleus, olfactory lobe, cerebral cortex and cerebellum from young to old mice. The Pax6 interacts with Ras-GAP in brain of mice. Results indicate impact of Pax6 on Ras-GAP-mediated activities of synapses, learning and memory, emotions and fear as well as motor functions. Alterations in expression and co-localization of Pax6 and Ras-GAP during aging may be responsible for age-associated compromised survival and plasticity of neurons and glia. © 2018 Elsevier B.V.
Aging-associated modulation in the expression of pax6 in mouse brain
(2012) Ratnakar Tripathi; Rajnikant Mishra
Symptoms like mental retardation, depression, and anxiety have been observed during aging. Almost similar phenotypes have been evident in patients having haploinsufficiency or mutations in Pax6, a transcriptional regulator. Since Pax6 regulates axon guidance, differentiation of neurons from glia, and neuronal migration, it has been considered as a marker of newly generated neurons. The immunohistochemical analysis of Pax6 positive cells and expression pattern of Pax6 in olfactory lobe, hippocampus, and cerebellum of aging mouse brain have been investigated. The number of Pax6 positive cells and level of Pax6 were reduced progressively in olfactory lobe, cerebellum, and hippocampus from postnatal day-zero (P0) to old age mice. Pax6 positive cells were significantly lower in dentate gyrus, CA1, CA2, and CA3 regions of hippocampus, in mitral cell (MiCe), and internal plexiform (InPl) layers of olfactory lobe, and in granular cell (GrLa), and Purkinje's cell (PuCe) layers of cerebellum from P0 to old age. Thus, modulation in the expression of Pax6 and reduction in Pax6 positive cells show direct association of Pax6 with aging-related neuronal dystrophy. © Springer Science+Business Media, LLC 2011.
Analysis of SET and MYND Domain-Containing Protein 3 (SMYD3) Expression in Gallbladder Cancer: a Pilot Study
(Springer, 2021) Pushkar Chandra; Ruhi Dixit; Arvind Pratap; Suman Mishra; Rajnikant Mishra; Vijay Kumar Shukla
The Suvar, Enhancer of zeste, and Trithorax (SET) and myeloid-Nervy-DEAF-1 (MYND) domain-containing protein 3 (SMYD3) is a histone lysine methyltransferase and has been recently unveiled to play significant roles in the progression of human cancer via regulating various key cancer-associated genes and pathways. The role of SMYD3 in gallbladder cancer (GBC) still needs to be studied. In the present study, we examined the SMYD3 gene expression at mRNA and protein level to look its impact on risk for developing gallbladder carcinogenesis. SMYD3 expression was evaluated by immunohistochemistry and reverse transcriptase PCR (RT-PCR) from 30 cases each of GBC and cholelithiasis patients. The expression was compared with different clinicopathological parameters. The SMYD3 expression was found to be significantly upregulated in GBC than cholelithiasis group (p < 0.05). The SMYD3 with increased expression level was observed in 73.3% of the GBC cases (p < 0.05). Moreover, mRNA SMYD3 expression was observed in 73.3% of GBC and 10% of control (p < 0.05). Our results indicated that the overexpression of SMYD3 plays an important role in the GBC progression, and SMYD3 may represent useful biomarker for gallbladder cancer patients. © 2020, Indian Association of Surgical Oncology.
Anti-leishmanial activity of Ni(II), Pd(II) and Pt(II) β-oxodithioester complexes
(Royal Society of Chemistry, 2015) Manoj Kumar Yadav; Gunjan Rajput; Khushboo Srivastava; Rakesh K. Singh; Rajnikant Mishra; Michael G. B. Drew; Nanhai Singh
New functionalized planar β-oxodithioester cis-chelate complexes, [M(L)2] (L = L1, methyl-3-hydroxy-3-(p-bromophenyl)-2-propenedithioate, M = Ni 1, Pd 5, Pt 9; L2, methyl-3-hydroxy-3-(p-fluorophenyl)-2-propenedithioate, Ni 2, Pd 6, Pt 10; L3, methyl-3-hydroxy-3-(naphthyl)-2-propenedithioate, Ni 3, Pd 7, Pt 11; methyl-3-hydroxy-3-(p-methoxyphenyl)-2-propenedithioate, Ni 4, Pd 8, Pt 12), have been synthesized and characterized by elemental analysis, IR, UV-Vis, 1H and 13C NMR spectroscopy; the structures of 2-4, 8 and 11 have been elucidated by X-ray crystallography. In all crystal structures, the metal has four-coordinate slightly distorted square planar geometry with a cis-configuration of the ligands. These complexes have been assessed for their use as anti-leishmanial agents; 7 and 9 showed impressive anti-promastigote and anti-amastigote efficacy with IC50 values of 0.59 ± 0.10 μg mL-1, 0.56 ± 0.10 μg mL-1 and IC50 0.85 ± 0.27, 1.99 ± 0.08 μg mL-1, respectively. Cytotoxicity assays on both compounds displayed toxicity on the promastigotes but less toxicity against RAW 264.7 cell lines at different concentrations. The Pd and Pt complexes exhibit luminescent characteristics in solution, originating from the intraligand charge transfer state. © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2015.
Autoregulation of Pax6 in neuronal cells is mediated by Pax6(5a), Pax6(ΔPD), SPARC, and p53
(Springer Science and Business Media B.V., 2022) Sachin Shukla; Rajnikant Mishra
Background: Pax6, a multifunctional protein and a transcriptional regulator is critical for optimal functioning of neuronal cells. It is known that alternatively spliced Pax6 isoforms and co-expressed interacting proteins mediate cell/tissue specific autoregulation of Pax6, however, underlying mechanism(s) are poorly understood. Methods and Results: We used Neuro-2a cells to explore the mechanism of autoregulation of Pax6 in neuronal cells whereas NIH/3T3 cells were used as control. We first studied the transcript expression of the three Pax6 isoforms: Pax6, Pax6(5a), and Pax6(ΔPD); and the two co-expressed Pax6-interacting partners: SPARC and p53 in normal and overexpressed conditions, through the semi-quantitative RT-PCR. Further, we used the luciferase reporter assay to study the binding and transactivation of the three Pax6 isoforms: Pax6, Pax6(5a), and Pax6(ΔPD) to their respective promoters: P0, P1, and Pα; followed by that of the two co-expressed Pax6-interacting partners: SPARC and p53 to the Pax6-P1 promoter. Expression and distribution of Pax6, Pax6(5a) and Pax6(ΔPD), their binding to Pax6-promoters (P0, P1, and Pα) and transactivation were modulated in transfected Neuro-2a cells. Conclusion: Our results suggest that autoregulation of Pax6 in neuronal cells is driven by a promoter dependent mechanism which is mediated by spliced variants [Pax6(5a) and Pax6(ΔPD)] and interacting proteins (SPARC and p53) of Pax6. © 2022, The Author(s), under exclusive licence to Springer Nature B.V.
Biochemical and molecular characterization of mitochondrial membrane-bound arginase in Heteropneustes fossilis
(Springer Netherlands, 2016) Suman Mishra; Rajnikant Mishra
The two predominant forms of arginase, cytosolic Arginase-I and mitochondrial Arginase-II, catalyze hydrolysis of arginine into ornithine and urea. Based on presence of arginase activity in extracts using potassium chloride (KCl), mitochondrial membrane-bound arginase has also been suggested. However, the activity of arginase in fractions obtained after KCl-treatment may be either due to leakage of mitochondrial arginase or release of adhered cytosolic arginase to cell organelles having altered net charge. Therefore, it has been intended to analyse impact of KCl on ultra-structural properties of mitochondria, and biochemical analysis of mitochondrial membrane-bound proteins and arginase of Heteropneustes fossilis. Liver of H. fossilis was used for isolating mitochondria for analysis of ultrastructural properties, preparing cytosolic, mitochondrial, and mitochondrial-membrane bound extracts after treatment of KCl. Extracts were analysed for arginase activity assay, protein profiling through SDS-PAGE and MALDI MS/MS. The KCl-mediated modulation in polypeptides and arginase were also evaluated by PANTHER, MitoProt and IPSORT servers. The effects of KCl on ultra-structural integrity of mitochondria, activity of arginase, modulation on mitochondrial proteins and enzymes including arginase were observed. The 48 kDa polypeptide of mitochondrial fraction, that showed KCl-dependent alteration matched with Myb binding protein and 30 kDa bands resembles to arginase after MALDI MS/MS analysis. Results indicate KCl-dependent ultrastructural changes in mitochondria and release of mitochondrial arginase. The proposed membrane bound mitochondrial arginase could be mitochondrial arginase-II or altered form of cytosolic arginase-I contributing to KCl-induced arginase activity in H. fossilis. © 2016, Springer Science+Business Media Dordrecht.
Co-expression and Interaction of Pax6 with Genes and Proteins of Immunological Surveillance in the Brain of Mice
(Springer, 2022) Shashank Kumar Maurya; Rajnikant Mishra
The Pax6 binds to promoter sequence elements of genes involved in immunological surveillance and interacts with Iba1, p53, Ras-GAP, and Sparc in the brain of mice. The Pax6 also affects the expression pattern of genes involved in neurogenesis and neurodegeneration. However, the expression and association of Pax6 in the brain under immunologically challenged conditions are still elusive. Therefore, it has been intended to analyze the association of Pax6 in the immunity of the brain using the immune-challenged Dalton’s lymphoma (DL) mice model. The expressions of Pax6, Iba1, and Tmem119 decreased, but expressions of Ifn-γ, Tnf-α, Bdnf, and Tgf-β increased in the brain of immune-challenged mice as compared to the control. The level of co-expression of Pax6 decreased in dual positive cells with Iba1, Tmem119, Sparc, p53, Bdnf, and Tgf-β in the brain of immune-challenged mice. Binding of Pax6 to multiple sites of the promoter sequences of Bdnf and Tgf-β indicates their Pax6-associated differential expression and association with immune responsive gene. The levels of binding of Pax6 to Tmem119, Iba1, Ifn-γ, and Tnf-α got altered during the immune-challenged state as compared to control. Results provide the first evidence of the association of Pax6 in brain-specific immunity. © 2022, The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.
Co-Localization and Interaction of Pax5 with Iba1 in Brain of Mice
(Springer New York LLC, 2018) Shashank Kumar Maurya; Rajnikant Mishra
The Pax5, a B-cell-Specific Activator Protein (BSAP) and redox-sensitive transcription factor, is expressed in the immune-privileged brain, B-lymphocytes, lymph nodes and spleen. PAX5-mediated immune pathway has also been described in the progression of Glioblastoma multiforme. However, the status of Pax5 and its role in brain immunity are not yet elucidated. In silico analysis of Pax5 interacting proteins predicts its interaction with proteins of cell proliferation, differentiation of hematopoietic cells, neurogenesis and several cell signalling pathways. Promoter analysis shows multiple binding sites for Pax5 in promoter of ionized calcium-binding adapter molecule 1 (Iba1). Like Iba1, Pax5 is also associated with inflammatory and immune response, activation of leukocyte and remodelling of actin cytoskeleton. Therefore, localization and interaction of Pax5 with Iba1 in brain of mice were studied using Chromatin Immunoprecipitation (ChIP), Co-Immunoprecipitation (Co-IP) and Immuno-fluorescence assay. The Pax5- and Iba1-positive cells were observed in cerebral cortex, cerebellum, olfactory bulb, hippocampus, and ventricles of brain. The co-localization of Pax5 and Iba1 was evident in microglia in almost all evaluated regions of brain. In some regions, Pax5- and Iba1-positive were distinctly compartmentalized. The Pax5a/b interacts with Iba1 and binds to its regulatory sequences. Results indicate Pax5-associated activities of Iba1 in microglia in brain of mice. © 2017, Springer Science+Business Media, LLC, part of Springer Nature.
Cyanobacterial bioactive compound EMTAHDCA recovers splenomegaly, affects protein profile of E. coli and spleen of lymphoma bearing mice
(Springer Netherlands, 2019) Niveshika; Shashank Kumar Maurya; Balkrishna Tiwari; Sindhunath Chakraborty; Ekta Verma; Rajnikant Mishra; Arun Kumar Mishra
The antibacterial and anticancerous properties of EMTAHDCA have already been reported in our previous study. However, mode of action of EMTAHDCA is still elusive. The present study was aimed to investigate the molecular targets in Escherichia coli and spleen of lymphoma-bearing mice in response to cyanocompound 9-ethyliminomethyl-12 (morpholin-4-ylmethoxy)-5, 8, 13, 16-tetraaza -hexacene-2, 3- dicarboxylic acid (EMTAHDCA) isolated from fresh water cyanobacterium Nostoc sp. MGL001. Differential expressions of proteins were observed in both E. coli and spleen of lymphoma-bearing mice after EMTAHDCA treatment. In continuation of our previous study, the present study revealed that the antibacterial agent, EMTAHDCA causes the drastic reduction in synthesis of proteins related to replication, transcription, translation and transportation in E. coli. Probably the direct or indirect interaction of this compound with these important metabolic processes led to the reduction in growth and cell death. Furthermore, the anticancerous property of the compound EMTAHDCA reflected as down regulation in proteins of cell cycle, cellular metabolism, signalling, transcription and transport together with up regulation of apoptosis, DNA damage and immunoprotection related proteins in spleen of lymphoma-bearing mice. In this study the EMTAHDCA induced modulations in expression of proteins of key metabolic pathways in E. coli and spleen cells of lymphoma bearing mice helped in understanding the mechanism underlying the antibacterial and anti-cancerous property. © 2019, Springer Nature B.V.
Editorial: Trends in neuroimmunology: cross-talk between brain-resident and peripheral immune cells in both health and disease
(Frontiers Media SA, 2024) Shashank K. Maurya; Janina E. Borgonovo; Suryanarayan Biswal; Verónica Martínez-Cerdeño; Rajnikant Mishra; Estela M. Muñoz
[No abstract available]
Effect of chronic jet lag after induction of Dalton's lymphoma in male and female mice
(2012) Priyoneel Basu; Ratnakar Tripathi; Rajnikant Mishra; Muniyandi Singaravel
Biological rhythms are ubiquitous phenomena that enable an organism to temporally adapt to the environment. Jet lag occurs during rapid transmeridial transport and disrupts the normal biological rhythm. Disruption of the biological rhythm is known to reduce survival and hasten tumorigenesis in male mice. Dalton's lymphoma (DL) is a spontaneous and highly invasive T-cell lymphoma that develops as an ascitic tumor in murines. DL was induced by serial implantation of live ascite cells in laboratory-acclimated age-matched male and female AKR mice. The mice were then subjected to simulated chronic jet lag (CJL) by rapidly and alternately advancing and delaying the ambient light dark cycle by 8 h every 2 days. Females, but not males, with DL became arrhythmic after exposure to simulated CJL. Survival was significantly curtailed in both male and female mice with DL + CJL compared to survival in mice with DL alone or CJL alone (∼16 days after DL induction in mice and facing jet lag, and ∼28 days after DL induction in mice bearing but not undergoing jet lag simulation). These results suggest that female mice may be more at risk from jet lag associated effects if they have cancer prior to facing jet lag, and DL + CJL may detrimentally impact the female circadian clock more than that of the males. © 2012 Taylor & Francis.
Effect of induced Dalton's lymphoma on circadian locomotor activity rhythm of adult male mice
(2012) Brij Bharti; Priyoneel Basu; Rajnikant Mishra; Muniyandi Singaravel
Dalton's lymphoma (DL) is a spontaneous and highly invasive T-cell lymphoma that develops as an ascitic tumor in murines. DL was induced by serial implantation of live ascite cells in laboratory-acclimated age-matched male AKR mice. The effects of DL on the locomotor activity rhythm were studied under 12:12h light:dark (LD) cycle. A significant decrease in the total and peak wheel-running activity was seen after the induction of DL, in comparison with the vehicle-injected control group. Durations of activity and rest were altered in DL mice, with a sharp daily increase in the duration of rest in the DL group. Circadian time period and acrophase of the rhythm remained similar in both control and DL mice. The mean estimate of statistic of rhythmicity (MESOR) and the amplitude of the locomotor activity rhythm were significantly lesser in post-DL-induced mice. A reduction in the span of the M1 and M2 motor cortex regions in DL mice and anatomical changes in the dentate gyrus were also seen. Thus, the effects of DL on the circadian clock seem restricted and mouse can still synchronize to an LD cycle. Impact of DL extends to motor cortices and may be the cause of the dampening of the amplitude of the rest-activity rhythm. © 2012 Copyright Taylor and Francis Group, LLC.
Effects of endosulfan on bioenergetic properties of liver-mitochondria from the freshwater catfish clarias batrachus
(1994) Rajnikant Mishra; S.P. Shukla
The effects of a sublethal concentration of an organochlorine pesticide endosulfan on fish liver mitochondrial oxidation of isocitrate and succinate in vivo and in vitro were investigated. Endosulfan depressed the phosphorylation efficiency of mitochondria as inferred from a decrease in RCR and ADP/O ratio with isocitrate; whereas endosulfan at this concentration reduced State 3 rates and RCR with succinate. The CCCP-uncoupled rates and State 4 rates of oxidation with succinate were unaffected. However, endosulfan-treated mitochondria exhibited a low rate of oligomycin-inhibited ADP dependent CCCP stimulation during the oxidation of succinate. The activity of the masked and activated ATPase was significantly increased in the presence of endosulfan. There was also a progressive stimulatory effect of endosulfan on mitochondrial ATPase activity. These results demonstrate that endosulfan exerts an inhibitory effect on electron transport and acts directly on ATP synthetase complex which leads to an impairment in mitochondrial bioenergetics of the fish. © 1994 by Academic Press, Inc.
Effects of endosulfan on bioenergetic properties of skeletal muscle mitochondria from the freshwater catfish (Clarias batrachus)
(1995) Rajnikant Mishra; S.P. Shukla
The effects of a sublethal concentration of an organochlorine pesticide endosulfan on fish skeletal muscle mitochondria oxidizing isocitrate and succinate in vivo and in vitro were investigated. The endosulfan depressed significantly State 3 rates and RCR with succinate, whereas it prevented completely the oxidation of isocitrate. The CCCP-uncoupled rates and State 4 rates of succinate oxidation remained unaffected by endosulfan. The activity of masked ATPase was significantly increased in presence of endosulfan. There was a progressive stimulatory effect of endosulfan on mitochondrial ATPase. The skeletal muscle fibres of endosulfan exposed fish undergo ultrastructural changes that are characterized by deformation of the myofibrils and disappearance of mitochondria. Summarizing, it can be stated that endosulfan exerts an inhibitory effect on electron transport and affects ATP synthetase complex leading to an impairment in mitochondrial bioenergetics, which can be correlated with marked ultrastructural alterations in the skeletal muscle fibres of the fish. © 1995.
Endosulfan effects on muscle malate dehydrogenase of the freshwater catfish Clarias batrachus
(Academic Press, 2003) Rajnikant Mishra; S.P. Shukla
The effects of a sublethal concentration of an organochlorine pesticide endosulfan were investigated on crude and purified forms of cytoplasmic malate dehydrogenase (cMDH) and mitochondrial malate dehydrogenase (mMDH) in the muscle of the freshwater catfish Clarias batrachus. Endosulfan treatment reduced significantly the activity and the specific activity of cMDH and mMDH but had no effect on total cytoplasmic and mitochondrial protein contents. This indicates a decline in the efficiency of aerobic energy metabolism in endosulfan-treated fish. The polyacrylamide gel electrophoresis showed two distinct isoforms (C1 and C2) of cMDH and only one form of mMDH. The inhibition produced by endosulfan in vivo was of mixed noncompetitive/uncompetitive type for crude as well as purified cMDH and mMDH. However, in vitro inhibition was of simple noncompetitive and mixed competitive/noncompetitive type for purified cMDH and mMDH, respectively. Citrate was found to be an uncompetitive inhibitor of cMDH and mixed noncompetitive/uncompetitve inhibitor of mMDH. The inhibitory patterns were modulated by endosulfan. These results demonstrate inhibitory effects of endosulfan on skeletal muscle MDH of the freshwater catfish Clarias batrachus and inhibition effects are mediated through enzyme/substrate/ endosulfan complexing. © 2003 Elsevier Science (USA). All rights reserved.
Expression and regulation of Pax6 in brain of aging mice
(Springer Singapore, 2016) Shashank Kumar Maurya; Ratnakar Tripathi; Suman Mishra; Rajnikant Mishra
Aging, an inevitable complex phenomenon among organisms, exhibit progressive loss of functional anatomy but understanding on molecular mechanisms of aging remains elusive. This report presents analysis of complex network of genes and proteins, their functional cascades and hierarchy, and regulation of regulators like Pax6 (Paired box 6) because symptoms of age-associated changes match with patients having mutation in Pax6. The Pax6 has been observed in the olfactory bulb, amygdala, thalamus, and the cerebellum. It is pro-neurogenic in adult neural progenitors and specifies neuronal subtypes in developing brain and the adult. During aging in brain, the reduction in Pax6-positive cells indicates loss of neurons and affects on adult neuronal stem cells. The alteration in co-localization of Pax6, p53 and SPARC may lead to the loss of plasticity, and p53 mediated cell death pathway during aging. Since the TGF-β, interacts with Pax6 and also gets influenced by Catalase, the Pax6 appears influencing immunological surveillance of brain. The alteration in levels of S100β and genes of oxidative stress management by Pax6 also indicates involvement of Pax6-TGF-β-Catalase axis in aging. The microRNA based regulation is also reflected because the miR335 suppresses the Pax6 expression and acts as anti-oncogenic target in glioma. The implication of Pax6 could also be explored towards possibilities of modifying the rate of aging and reversal of aging clock because the Pax6 interacts with p53 and gets altered during aging. © Springer Science+Business Media Singapore 2017.
Functional analysis of missense mutations G36A and G51A in PAX6, and PAX6(5a) causing ocular anomalies
(2011) Sachin Shukla; Rajnikant Mishra
The PAX6 has been described a " master regulator of eye development" A specific ratio of PAX6, and its alternatively spliced isoform, PAX6(5a), has also been observed essential for optimal function. Mutations into PAX6 lead to a number of ocular, and neuronal defects of variable penetrance and expressivity but the mechanism is either poorly understood or underrepresented. This report describes analysis of functions of two missense mutations, G36A, and G51A, causing optic-nerve hypoplasia and optic-disc coloboma in humans, respectively. Mutations were created by site-directed mutagenesis. Products were detected by in-vitro translation and transient transfection to the cultured NIH-3T3 cells. Their DNA-binding, and transcriptional activation properties were analysed through electrophoretic mobility shift assay and luciferase reporter assay, respectively. Mutations induced changes in conformation and secondary structure of PAX6, and PAX6(5a) not only restrict to specific site of mutation in the paired-domain but extend to homeodomain, and transactivation domain. The PAX6-G36A showed reduced binding to PAX6-consensus binding sequence and PAX6(5a)-consensus binding sequence but its binding affinity to homeodomain binding sequence was unaffected. It showed significantly higher transactivation potential through PAX6-consensus binding sequence but reduced activity with PAX6(5a)-consensus binding sequence and homeodomain binding sequence containing luciferase reporters. The PAX6(5a)-G36A showed enhanced transactivation potential with PAX6-consensus binding sequence, PAX6(5a)-consensus binding sequence, and homeodomain binding sequence containing luciferase reporters. The binding affinity of PAX6(5a)-G36A was significantly higher to PAX6-consensus binding sequence, and PAX6(5a)-consensus binding sequence as compared to PAX6(5a) but remains unaffected to homeodomain binding sequence. The enhanced binding affinity was observed by PAX6-G51A to PAX6-consensus binding sequence, PAX6(5a)-consensus binding sequence, and homeodomain binding sequence. The transactivation potential was observed higher with PAX6-consensus binding sequence but significant reduction was evident with PAX6(5a)-consensus binding sequence, and homeodomain binding sequence containing luciferase reporters. The lower binding affinity to PAX6-consensus binding sequence and PAX6(5a)-consensus binding sequence was observed by PAX6(5a)-G51A but loss of binding affinity was detected to homeodomain binding sequence. However, PAX6(5a)-G51A showed significantly higher transactivation with PAX6-consensus binding sequence, PAX6(5a)-consensus binding sequence, and homeodomain binding sequence containing luciferase reporters. With the eye-specific α-A-crystallin promoter, PAX6-G36A and PAX6-G51A mutants were found to have higher ability to transactivate whereas PAX6(5a)-G36A and PAX6(5a)-G51A have lower transactivation potential compared to their respective wild type forms. Thus, variable DNA-binding and transactivation properties of the mutants with different PAX6-binding sequences provide an insight towards their variable penetrance and expressivity. © 2011 Elsevier Ltd.
Genetic and epigenetic alterations in DNA repair genes and treatment outcome of chemoradiotherapy in cervical cancer
(Elsevier Ireland Ltd, 2024) Atar Singh Kushwah; Shireen Masood; Rajnikant Mishra; Monisha Banerjee
Cervical cancer (CaCx) is the deadliest malignancy among women which is caused by human papillomavirus (HPV) and anthro-demographical/clinicopathological factors. HPV oncoproteins E6 and E7 target p53 and RB (retinoblastoma) protein degradation, Ataxia telangiectasia mutated (ATM), ATM-RAD3-related (ATR) inactivation and subsequent impairment of non-homologous end joining (NHEJ), homologous recombination, and base excision repair pathways. There is also an accumulation of genetic and epigenetic alterations in Tumor Growth Suppressors (TGS), oncogenes, and DNA repair genes leading to increased genome instability and CaCx development. These alterations might be responsible for differential clinical response to Cisplatin-based chemoradiotherapy (CRT) in patients. This review explores HPV-mediated DNA damage as a risk factor in CaCx development, the mechanistic role of genetic and epigenetic alterations in DNA repair genes and their association with CRT and outcome, It also explores new possibilities for the development of genetic and epigenetic-based biomarkers for diagnostic, prognostic, and molecular therapeutic interventions. © 2023 Elsevier B.V.
Immunochemical detection of serum LDH1: An indigenous method in the diagnosis of myocardial infarction
(1999) Rajnikant Mishra; S.P. Shukla
Immunochemical studies on lactate dehydrogenase (LDH) were carried out to develop an indigenous immunochemical method for the diagnosis of acute myocardial infarction (AMI). Heart-specific LDH1 was purified and polyclonal antibody against LDH1 was raised in rabbits. Immunodiffusion and immunoblotting identifications show the presence of LDH1 in AMI test samples. Immunochemical detection of LDH1 in human serum may substitute kinetic method of total LDH assay and electrophoretic analysis of LDH1 isozyme in the diagnosis of AMI.