Browsing by Author "Garima Srivastava"

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B-amylase from starchless seeds of Trigonella foenum-graecum and its localization in germinating seeds
(Public Library of Science, 2014) Garima Srivastava; Arvind M. Kayastha
Fenugreek (Trigonella foenum-graecum) seeds do not contain starch as carbohydrate reserve. Synthesis of starch is initiated after germination. A β-amylase from ungerminated fenugreek seeds was purified to apparent electrophoretic homogeneity. The enzyme was purified 210 fold with specific activity of 732.59 units/mg. Mr of the denatured enzyme as determined from SDS-PAGE was 58 kD while that of native enzyme calculated from size exclusion chromatography was 56 kD. Furthermore, its identity was confirmed to be β-amylase from MALDI-TOF analysis. The optimum pH and temperature was found to be 5.0 and 50°C, respectively. Starch was hydrolyzed at highest rate and enzyme showed a Km of 1.58 mg/mL with it. Antibodies against purified Fenugreek β-amylase were generated in rabbits. These antibodies were used for localization of enzyme in the cotyledon during different stages of germination using fluorescence and confocal microscopy. Fenugreek β-amylase was found to be the major starch degrading enzyme depending on the high amount of enzyme present as compared to α-amylase and also its localization at the periphery of amyloplasts. A new finding in terms of its association with protophloem was observed. Thus, this enzyme appears to be important for germination of seeds. © 2014 Srivastava, Kayastha.
Cicer a-galactosidase immobilization onto functionalized graphene nanosheets using response surface method and its applications
(Elsevier Ltd, 2014) Neelesh Singh; Garima Srivastava; Mahe Talat; Himanshu Raghubanshi; Onkar Nath Srivastava; Arvind M. Kayastha
Cicer a-galactosidase was immobilized onto functionalized graphene with immobilization efficiency of 84% using response surface methodology (Box-Behnken design). The immobilized enzyme had higher thermal stability than the soluble one, attractive for industrial applications. Immobilization of the enzyme lowered the Km to 1/3rd compared to the soluble enzyme. Raffinose family oligosaccharides (RFOs) are mainly responsible for flatulence by taking soybean derived food products. The immobilized enzyme can be used effectively for the hydrolysis of RFOs. After ten successive runs, the immobilized enzyme still retained approximately 60% activity, with soybean RFOs. The easy availability of enzyme source, ease of its immobilization on matrices, non-toxicity, increased stability of immobilized enzyme and effective hydrolysis of RFOs increase the Cicer a-galactosidase application in food processing industries. © 2013 Elsevier Ltd.
Functionalized graphene sheets as immobilization matrix for fenugreek β-Amylase: Enzyme kinetics and stability studies
(Public Library of Science, 2014) Garima Srivastava; Kritika Singh; Mahe Talat; Onkar Nath Srivastava; Arvind M. Kayastha
β-Amylase finds application in food and pharmaceutical industries. Functionalized graphene sheets were customised as a matrix for covalent immobilization of Fenugreek β-amylase using glutaraldehyde as a cross-linker. The factors affecting the process were optimized using Response Surface Methodology based Box-Behnken design of experiment which resulted in 84% immobilization efficiency. Scanning and Transmission Electron Microscopy (SEM, TEM) and Fourier Tansform Infrared (FTIR) spectroscopy were employed for the purpose of characterization of attachment of enzyme on the graphene. The enzyme kinetic studies were carried out for obtaining best catalytic performance and enhanced reusability. Optimum temperature remained unchanged, whereas optimum pH showed shift towards acidic range for immobilized enzyme. Increase in thermal stability of immobilized enzyme and non-toxic nature of functionalized graphene can be exploited for production of maltose in food and pharmaceutical industries. © 2014 Srivastava et al.
Identification of active site residues of Fenugreek β-amylase: Chemical modification and in silico approach
(Elsevier Masson SAS, 2014) Garima Srivastava; Vinay K. Singh; Arvind M. Kayastha
The amino acid sequence of Fenugreek β-amylase is not available in protein data bank. Therefore, an attempt has been made to identify the catalytic amino acid residues of enzyme by employing studies of pH dependence of enzyme catalysis, chemical modification and bioinformatics. Treatment of purified Fenugreek β-amylase with EDAC in presence of glycine methyl ester and sulfhydryl group specific reagents (IAA, NEM and p-CMB), followed a pseudo first-order kinetics and resulted in effective inactivation of enzyme. The reaction with EDAC in presence of NTEE (3-nitro-l-tyrosine ethylester) resulted into modification of two carboxyl groups per molecule of enzyme and presence of one accessible sulfhydryl group at the active site, per molecule of enzyme was ascertained by titration with DTNB. The above results were supported by the prevention of inactivation of enzyme in presence of substrate. Based on MALDI-TOF analysis of purified Fenugreek β-amylase and MASCOT search, β-amylase of Medicago sativa was found to be the best match. To further confirm the amino acid involved in catalysis, homology modelling of β-amylase of M.sativa was performed. The sequence alignment, superimposition of template and target models, along with study of interactions involved in docking of sucrose and maltose at the active site, led to identification of Glu187, Glu381 and Cys344 as active site residues. © 2014 Elsevier Masson SAS.
Immobilisation of Fenugreek β-amylase on chitosan/PVP blend and chitosan coated PVC beads: A comparative study
(Elsevier Ltd, 2015) Garima Srivastava; Sonam Roy; Arvind M. Kayastha
A Box-Behnken design of Response Surface Methodology (RSM) was utilised for optimisation of parameters affecting immobilisation of Fenugreek β-amylase on chitosan coated PVC (polyvinyl chloride) beads and beads made from chitosan/PVP (polyvinylpyrrolidone) blend, which resulted in 85.2% and 81% immobilisation efficiency, respectively. Immobilisation resulted in shift of pH optima while the optimum temperature remained unaffected. Enhancement in thermal stability of the enzyme was observed on conjugation with both the matrices. The immobilised enzyme appeared suitable for industrial applications due to the non-toxic nature of chosen matrices, ease of immobilisation procedure, enhanced stability and reusability with retention of 72% and 60% residual activity after 10 uses for the enzyme immobilised on chitosan coated PVC beads and on the beads of chitosan/PVP blend, respectively. © 2014 Elsevier Ltd. All rights reserved.
Involvement of NADPH oxidase and glutathione in zinc-induced dopaminergic neurodegeneration in rats: Similarity with paraquat neurotoxicity
(2012) Ashutosh Kumar; Brajesh Kumar Singh; Israr Ahmad; Smriti Shukla; Devendra Kumar Patel; Garima Srivastava; Vinod Kumar; Haushila Prasad Pandey; Chetna Singh
An association between excessive zinc (Zn) accumulation in brain and incidences of Parkinson's disease (PD) has been shown in several epidemiological and experimental investigations. The involvement of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and glutathione (GSH) in the pathogenesis of PD has also been proposed in a few studies. Despite the implicated role of oxidative stress in PD, the entire mechanism of Zn-induced dopaminergic neurodegeneration has not yet been clearly understood. The present study aimed to investigate the involvement of NADPH oxidase and GSH in Zn-induced dopaminergic neurodegeneration and also to assess its similarity with paraquat (PQ)-induced rat model of PD. Male Wistar rats were treated either with Zn (20 mg/kg; i.p.) or PQ (5 mg/kg; i.p.) in the presence and absence of NADPH oxidase inhibitor, apocynin (10 mg/kg; i.p.) and a GSH precursor, N-acetyl cysteine (NAC; 200 mg/kg; i.p.) either alone or in combination along with the respective controls. Apocynin and/or NAC pre-treatment significantly alleviated Zn- and PQ-induced changes in neurobehavioral deficits, number of dopaminergic neurons and contents of the striatal dopamine and its metabolites. Apocynin and/or NAC also mitigated Zn- and PQ-induced alterations in oxidative stress, NADPH oxidase activation and cytochrome c release, caspases-9 and -3 activation and CD11b expression. The results obtained thus suggest that Zn induces oxidative stress via the activation of NADPH oxidase and depletion of GSH, which in turn activate the apoptotic machinery leading to dopaminergic neurodegeneration similar to PQ. © 2011 Elsevier B.V. All rights reserved.
Minocycline, levodopa and MnTMPyP induced changes in the mitochondrial proteome profile of MPTP and maneb and paraquat mice models of Parkinson's disease
(2013) Anubhuti Dixit; Garima Srivastava; Divya Verma; Manisha Mishra; Pradhyumna Kumar Singh; Om Prakash; Mahendra Pratap Singh
Mitochondrial dysfunction is the foremost perpetrator of the nigrostriatal dopaminergic neurodegeneration leading to Parkinson's disease (PD). However, the roles played by majority of the mitochondrial proteins in PD pathogenesis have not yet been deciphered. The present study investigated the effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and combined maneb and paraquat on the mitochondrial proteome of the nigrostriatal tissues in the presence or absence of minocycline, levodopa and manganese (III) tetrakis (1-methyl-4-pyridyl) porphyrin (MnTMPyP). The differentially expressed proteins were identified and proteome profiles were correlated with the pathological and biochemical anomalies induced by MPTP and maneb and paraquat. MPTP altered the expression of twelve while combined maneb and paraquat altered the expression of fourteen proteins. Minocycline, levodopa and MnTMPyP, respectively, restored the expression of three, seven and eight proteins in MPTP and seven, eight and eight proteins in maneb- and paraquat-treated groups. Although levodopa and MnTMPyP rescued from MPTP- and maneb- and paraquat-mediated increase in the microglial activation and decrease in manganese-superoxide dismutase expression and complex I activity, dopamine content and number of dopaminergic neurons, minocycline defended mainly against maneb- and paraquat-mediated alterations. The results demonstrate that MPTP and combined maneb and paraquat induce mitochondrial dysfunction and microglial activation and alter the expression of a bunch of mitochondrial proteins leading to the nigrostriatal dopaminergic neurodegeneration and minocycline, levodopa or MnTMPyP variably offset scores of such changes. © 2013 Elsevier B.V.
Resveratrol potentiates cytochrome P450 2 d22-mediated neuroprotection in maneb- and paraquat-induced parkinsonism in the mouse
(2012) Garima Srivastava; Anubhuti Dixit; Sharawan Yadav; Devendra Kumar Patel; Om Prakash; Mahendra Pratap Singh
A strong association between polymorphisms of the cytochrome P450 (CYP/Cyp) 2D6 gene and risk to Parkinson's disease (PD) is well established. The present study investigated the neuroprotective potential of Cyp2d22, a mouse ortholog of human CYP2D6, in maneb- and paraquat-induced parkinsonism and the mechanisms involved therein along with the effects of resveratrol on various parameters associated with Cyp2d22-mediated neuroprotection. The animals were treated intraperitoneally with resveratrol (10 mg/kg, daily) and paraquat (10 mg/kg) alone or in combination with maneb (30 mg/kg), twice a week, for 9 weeks, along with their respective controls. The subsets of animals were also treated intraperitoneally with a Cyp2d22 inhibitor, ketoconazole (100 mg/kg, daily). Maneb and paraquat reduced Cyp2d22 and vesicular monoamine transporter type 2 (VMAT-2) expressions, the number of tyrosine hydroxylase-positive cells, and dopamine content and increased paraquat accumulation in the nigrostriatal tissues, oxidative stress, microglial activation, neuroinflammation, and apoptosis. Cyp2d22 inhibitor significantly exacerbated all these neurodegenerative indexes. Resveratrol cotreatment, partially but significantly, ameliorated the neurodegenerative changes by altering Cyp2d22 expression and paraquat accumulation. The results obtained in the study demonstrate that Cyp2d22 offers neuroprotection in maneb- and paraquat-induced dopaminergic neurodegeneration and resveratrol enhances its neuroprotective credentials by influencing Cyp2d22 expression and paraquat accumulation. © 2012 Elsevier Inc. All rights reserved.
Rodent models and contemporary molecular techniques: Notable feats yet incomplete explanations of Parkinson's disease pathogenesis
(Humana Press Inc., 2012) Sharawan Yadav; Anubhuti Dixit; Sonal Agrawal; Ashish Singh; Garima Srivastava; Anand Kumar Singh; Pramod Kumar Srivastava; Om Prakash; Mahendra Pratap Singh
Rodent models and molecular tools, mainly omics and RNA interference, have been rigorously used to decode the intangible etiology and pathogenesis of Parkinson's disease (PD). Although convention of contemporary molecular techniques and multiple rodent models paved imperative leads in deciphering the role of putative causative factors and sequential events leading to PD, complete and clear-cut mechanisms of pathogenesis are still hard to pin down. The current article reviews the implications and pros and cons of rodent models and molecular tools in understanding the molecular and cellular bases of PD pathogenesis based on the existing literature. Probable rationales for short of comprehensive leads and future possibilities in spite of the extensive applications of molecular tools and rodent models have also been discussed. © Springer Science+Business Media, LLC 2012.
Role of secondary mediators in caffeine-mediated neuroprotection in maneb- and paraquat-induced Parkinson's disease phenotype in the mouse
(2012) Sharawan Yadav; Satya Prakash Gupta; Garima Srivastava; Pramod Kumar Srivastava; Mahendra Pratap Singh
Maneb and paraquat are known to induce Parkinson's disease (PD) phenotype, however, caffeine offers neuroprotection. Nitric oxide (NO) acts an important mediator in PD phenotype and tyrosine kinase (TK), nuclear factor kappa B (NF-kB), p38 mitogen activated protein kinase (p38 MAPK) are known to regulate its production. The present study aimed to elucidate the role of caffeine in the regulation of NO production and microglial activation and their subsequent contribution in dopaminergic neuroprotection. The animals were treated with caffeine and/or maneb and paraquat along with controls. In a few sets of experiments, the animals were also treated with aminoguanidine, an inhibitor of inducible NO synthase, pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-kB, genistein, an inhibitor of TK or SB202190, an inhibitor of p38 MAPK. Tyrosine hydroxylase (TH)-immunoreactivity and anti-integrin αM (OX-42) staining were performed to assess the number of dopaminergic neurons and activation of microglia, respectively. NO was measured in terms of nitrite, however, the expressions of p38 MAPK, interleukin (IL)-1β, NF-kB and TK were checked by western blot analyses. Maneb and paraquat induced the number of degenerating dopaminergic neurons, microglial cells, nitrite content, expressions of IL-1β, p38 MAPK, NF-kB and TK and caffeine co-treatment reduced the level of such alterations. Reductions were more pronounced in the animals co-treated with aminoguanidine, PDTC, genistein or SB202190. The results obtained thus demonstrate that caffeine down-regulates NO production, neuroinflammation and microglial activation, which possibly contribute to neuroprotection. © Springer Science+Business Media, LLC 2011.
Tiny non-coding RNAs in Parkinson's disease: Implications, expectations and hypes
(Elsevier Ltd, 2011) Garima Srivastava; Anubhuti Dixit; Om Prakash; Mahendra Pratap Singh
Parkinson's disease (PD) is the second most prevalent, progressive and aging related neurodegenerative disorder, characterized by the irreversible and selective degeneration of the nigrostriatal dopaminergic neurons. The early diagnosis, molecular explanation and permanent cure of this devastating and baffling disease have not yet been completely deciphered. Tiny non-coding RNAs, which consist of small or short interfering RNA (siRNA) and micro RNA (miRNA), intervene with and silence the expression of the specific genes through the evolutionary conserved process of RNA interference and act as post-transcriptional regulators. The differential expression patterns of miRNAs operate as key watchdogs and facilitate the identification of the potential therapeutic targets; however, miRNA modifiers aid in designing the strategies to encounter PD. Similarly, siRNA-mediated gene silencing paves the way to understand the function of the specific genes in PD pathogenesis by knocking down their expression. Applications of siRNAs and contributions of the potential miRNAs in investigating the etiology and molecular mechanisms of PD as well as in therapeutic interventions have been discussed in this article. The review also highlights the achievements, expectations and hypes associated with these tiny non-coding RNAs in PD. © 2011 Elsevier B.V. All rights reserved.
α-Amylase immobilization onto functionalized graphene nanosheets as scaffolds: Its characterization, kinetics and potential applications in starch based industries
(Elsevier B.V., 2015) Kritika Singh; Garima Srivastava; Mahe Talat; Onkar Nath Srivastava; Arvind Mohan Kayastha
α-Amylase is imperative for starch and its deriviatized industries. Functionalized graphene sheets were tailored and optimized as scaffold for α-amylase immobilization using Response Surface Methodology based on Box-Behnken design, with an overall immobilization efficiency of 85.16%. Analysis of variance provided adequacy to the mathematical model for further studies. Native and immobilized functionalized graphene were characterized using transmission and scanning electron microscopy, followed by Fourier transform infrared (FTIR) spectroscopy. Wheat α-amylase conjugated with functionalized graphene sheets were visually evident on transmission and scanning micrographs while the FTIR spectra showed interplay of various chemical interactions and bonding, during and after immobilization. Optimum pH and optimum temperature for immobilized enzyme though remained unchanged but showed broader range whereas Km showed a slight decrease (1.32mg/mL). It also showed enhanced thermal and storage stability and retained 73% residual activity after 10 uses. These ensemble of properties and non-toxic nature of functionalized graphene, makes it viable to be absorbed commercially in starch processing industries. © 2015 The Authors.