Browsing by Author "Sarita Pandey"

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A new arsenate reductase involved in arsenic detoxification in Anabaena sp. PCC7120
(2013) Sarita Pandey; A.K. Shrivastava; V.K. Singh; R. Rai; P.K. Singh; S. Rai; L.C. Rai
In silico analysis followed by experimental validation leads us to propose that the predicted protein All0195 of Anabaena sp. PCC7120 showing enhanced expression under sodium arsenate (Na2HAsO4) stress belongs to the thioredoxin superfamily with structural similarity to bacterial arsenate reductase. The All0195 protein demonstrated C-X-TC-X-K, NTSG-X2-YR, and D-X2-L-X-KRP as functional motifs that show similarity to seven known bacterial arsenate reductase family protein homologs with Cys, Arg, and Pro as conserved residues. In view of physicochemical properties, such as aliphatic index, ratio of Glu + Lys to Gln + His, and secondary structure, it was evident that All0195 was also a thermostable protein. The predicted three-dimensional structure on molecular docking with arsenate oxyanion (HAsO4-2) revealed its interaction with conserved Cys residue as also known for other bacterial arsenate reductase. In silico derived properties were experimentally attested by cloning and heterologous expression of all0195. Furthermore, this protein functionally complemented the arsenate reductase-deficient sodium arsenate-hypersensitive phenotype of Escherichia coli strainWC3110 (ΔarsC) and depicted arsenate reductase activity on purification. In view of the above properties, All0195 appears to be a new arsenate reductase involved in arsenic detoxification in Anabaena sp. PCC7120. © 2012 Springer-Verlag Berlin Heidelberg.
A novel alkyl hydroperoxidase (AhpD) of Anabaena PCC7120 confers abiotic stress tolerance in Escherichia coli
(Springer Verlag, 2015) Alok Kumar Shrivastava; Shilpi Singh; Prashant Kumar Singh; Sarita Pandey; L.C. Rai
In silico analysis together with cloning, molecular characterization and heterologous expression reports that the hypothetical protein All5371 of Anabaena sp. PCC7120 is a novel hydroperoxide scavenging protein similar to AhpD of bacteria. The presence of E(X)11CX HC(X)3H motif in All5371 confers peroxidase activity and closeness to bacterial AhpD which is also reflected by its highest 3D structure homology with Rhodospirillum rubrum AhpD. Heterologous expression of all5371 complimented for ahpC and conferred resistance in MJF178 strain (ahpCF::Km) of Escherichia coli. All5371 reduced the organic peroxide more efficiently than inorganic peroxide and the recombinant E. coli strain following exposure to H2O2, CdCl2, CuCl2, heat, UV-B and carbofuron registered increased growth over wild-type and mutant E. coli transformed with empty vector. Appreciable expression of all5371 in Anabaena sp. PCC7120 as measured by qRT-PCR under selected stresses and their tolerance against H2O2, tBOOH, CuOOH and menadione attested its role in stress tolerance. In view of the above, All5371 of Anabaena PCC7120 emerged as a new hydroperoxide detoxifying protein. © 2014, Springer-Verlag Berlin Heidelberg.
Alr0882 encoding a hypothetical protein of Anabaena PCC7120 protects Escherichia coli from nutrient starvation and abiotic stresses
(2012) Alok Kumar Shrivastava; Sarita Pandey; Prashant Kumar Singh; Snigdha Rai; Lal Chand Rai
This study is the first to demonstrate cloning of alr0882, a hypothetical protein gene of Anabaena PCC7120, its heterologous expression in Escherichia coli strain LN29MG1655 (uspA::Kan) and functional complementation of abiotic stress tolerance of E. coli UspA. The recombinant vector pGEX-5X-2-alr0882 was used to transform uspA E. coli strain. The IPTG induced expression of a 56.6kDa GST fusion protein was visualized on SDS-PAGE and attested by immunoblotting. E. coli uspA strain harboring pGEX-5X-2-alr0882 when grown under carbon, nitrogen, phosphorus and sulphur limitation and abiotic stresses e.g. nalidixic acid, cycloserine, CdCl2, H2O2, UV-B, phenazine methosulphate (PMS), dinitrophenol (DNP), NaCl, heat, carbofuron and CuCl2 demonstrated about 22.6-51.6% increase in growth over the cells transformed with empty vector. Expression of alr0882 gene in mutant E. coli as measured by semi-quantitative RT-PCR at different time points under selected treatments reaffirmed its role in tolerance against stresses employed in this study. Thus the results of this study vividly demonstrated that the novel protein alr0882, although appreciably different from the known UspA of E. coli, offers tolerance to abiotic stresses hence holds potential for the development of transgenic cyanobacteria. © 2012 Elsevier B.V.
Arsenic-induced changes in morphological, physiological, and biochemical attributes and artemisinin biosynthesis in Artemisia annua, an antimalarial plant
(2011) Rashmi Rai; Sarita Pandey; Shashi Pandey Rai
Present study is the first to explore physiological, biochemical and molecular changes in the medicinal plant Artemisia annua under arsenic (As) stress. A. annua grown hydroponically in a nutrient solution was spiked with increasing doses of As (0, 1,500, 3,000 and 4,500 μg l -1) for 7 days. Plants accumulated As in a dose dependent manner with bioconcentration factor 13.4 and translocation factor 0.97. While a similar trend of As accumulation was observed under soil culture experiments, the transfer factor went up to 2.1, depicting high efficiency of As translocation from roots to shoots by A. annua. Plants raised in 0-3,000 μg l -1 As containing nutrient solution registered increase in root length, biomass, and carotenoid contents without any visual toxicity symptoms. A dose dependent increase in the activities of enzymes such as superoxide dismutase, ascorbate peroxidase, glutathione reductase and guaiacol peroxidase followed by a gradual decline at higher concentrations suggested their role in alleviating oxidative stress. Significant increase in the levels of thiols, GSH, and pcs gene transcript up to 3,000 μg l -1 As attested their roles in As detoxification. Enhanced artemisinin production (an antimalarial compound) under As stress and upregulation of the transcripts (measured by RT-PCR) of the genes HMGR, FDS, ADS, and CYP71AV1 involved in artemisinin biosynthesis reaffirmed induction of artemisinin biosynthesis in A. annua under As stress. The results of the present study vividly suggested that A. annua has considerable As tolerance, and thus can be successfully cultivated in As contaminated fields. © 2011 Springer Science+Business Media, LLC.
Biochemical and molecular basis of arsenic toxicity and tolerance in microbes and plants
(Elsevier, 2023) Sarita Pandey; Rashmi Rai; Lal Chand Rai
Arsenic is a ubiquitous toxic metalloid abundant in Earth's crust. It is of major concern with respect to its increased accumulation in soils, in the food chain, or in drinking water. This chapter will focus on recent progress on the mechanisms of its uptake, toxicity, and detoxification in microbes and in planta. Due to widespread occurrence in nature, both microbes and plants have evolved a wide range of tolerance and detoxification mechanisms such as reduced uptake, immobilization, chelation, reduction/oxidation, methylation, and efflux. Among microbes, the ars operon is a well-characterized genetic system for arsenic detoxification. The mechanisms proposed for metal detoxification and hyperaccumulation within the plant involve chelation of the metal cation by ligands and binding with thiol groups or sequestration of metals away from sites or metabolism in the cytoplasm, notably into the vacuole or cell wall. Finally, this chapter will also shed light on hyperaccumulators and mechanisms of hyperaccumulation. © 2023 Elsevier Inc. All rights reserved.
Biochemical and Molecular Basis of Arsenic Toxicity and Tolerance in Microbes and Plants
(Elsevier Inc., 2015) Sarita Pandey; Rashmi Rai; Lal Chand Rai
Arsenic is a ubiquitous toxic metalloid abundant in Earth's crust. It is of major concern with respect to its increased accumulation in soils, in the food chain, or in drinking water. This chapter will focus on recent progress on the mechanisms of its uptake, toxicity, and detoxification in microbes and in planta. Due to widespread occurrence in nature, both microbes and plants have evolved a wide range of tolerance and detoxification mechanisms such as reduced uptake, immobilization, chelation, reduction/oxidation, methylation, and efflux. Among microbes, the ars operon is a well-characterized genetic system for arsenic detoxification. The mechanisms proposed for metal detoxification and hyperaccumulation within the plant involve chelation of the metal cation by ligands and binding with thiol groups or sequestration of metals away from sites or metabolism in the cytoplasm, notably into the vacuole or cell wall. Finally, this chapter will also shed light on hyperaccumulators and mechanisms of hyperaccumulation. © 2015 Elsevier Inc. All rights reserved.
Cadmium toxicity in diazotrophic Anabaena spp. adjudged by hasty up-accumulation of transporter and signaling and severe down-accumulation of nitrogen metabolism proteins
(Elsevier, 2015) Prashant Kumar Singh; Alok Kumar Shrivastava; Antra Chatterjee; Sarita Pandey; Snigdha Rai; Shilpi Singh; L.C. Rai
Present study demonstrates interspecies variation in proteome and survival strategy of three Anabaena species i.e., Anabaena L31, Anabaena sp. PCC 7120 and Anabaena doliolum subjected to respective LC50 doses of Cd at 0, 1, 3, 5 and 7day intervals. The proteome coverage with 452 differentially accumulated proteins unveiled species and time specific expression and interaction network of proteins involved in important cellular functions. Statistical analysis of protein abundance across Cd-treated proteomes clustered their co-expression pattern into four groups viz., (i) early (days 1 and 3) accumulated proteins, (ii) proteins up-accumulated for longer duration, (iii) late (days 5 and 7) accumulated proteins, and (iv) mostly down-accumulated proteins. Appreciable growth of Cd treated A L31 over other two species may be ascribed to proteins contained in the first and second groups (belonging to energy and carbohydrate metabolism (TK, G6-PI, PGD, FBA, PPA, ATP synthase)), sulfur metabolism (GR, GST, PGDH, PAPS reductase, GDC-P, and SAM synthetase), fatty acid metabolism (AspD, PspA, SQD-1), phosphorous metabolism (PhoD, PstB and SQD1), molecular chaperones (Gro-EL, FKBP-type peptidylprolyl isomerase), and antioxidative defense enzymes (SOD-A, catalase). Anabaena sp. PCC 7120 harboring proteins largely from the third group qualified as a late accumulator and A. doliolum housing majority of proteins from the fourth group emerged as the most sensitive species. Thus early up-accumulation of transporter and signaling category proteins and drastic reduction of nitrogen assimilation proteins could be taken as a vital indicator of cadmium toxicity in Anabaena spp. This article is part of a Special Issue entitled: Proteomics in India. © 2015 .
Comparative proteomics of wild type, An+ahpC and An∆ahpC strains of Anabaena sp. PCC7120 demonstrates AhpC mediated augmentation of photosynthesis, N2-fixation and modulation of regulatory network of antioxidative proteins
(Elsevier, 2016) Alok K. Shrivastava; Sarita Pandey; Shivam Yadav; Yogesh Mishra; Prashant K. Singh; Ruchi Rai; Shilpi Singh; Snigdha Rai; L.C. Rai
Alkylhydroperoxide reductase (AhpC), a 1-Cys peroxiredoxin is well known for maintaining the cellular homeostasis. Present study employs proteome approach to analyze and compare alterations in proteome of Anabaena PCC7120 in overexpressing (An+ahpC), deletion (An∆ahpC) and its wild type. 2-DE based analysis revealed that the major portion of identified protein belongs to energy metabolism, protein folding, modification and stress related proteins and carbohydrate metabolism. The two major traits discernible from An+ahpC were (i) augmentation of photosynthesis and nitrogen fixation (ii) modulation of regulatory network of antioxidative proteins. Increased accumulation of proteins of light reaction, dark reaction, pentose phosphate pathway and electron transfer agent FDX for nitrogenase in An. +. ahpC and their simultaneous downregulation in AnδahpC demonstrates its role in augmenting photosynthesis and nitrogen fixation. Proteomic data was nicely corroborated with physiological, biochemical parameters displaying upregulation of nitrogenase (1.6 fold) PSI (1.08) and PSII (2.137) in An + ahpC. Furthermore, in silico analysis not only attested association of AhpC with peroxiredoxins but also with other players of antioxidative defense system viz. thioredoxin and thioredoxin reductase. Above mentioned findings are in agreement with 33-40% and 40-60% better growth performance of An+ahpC over wild type and An∆ahpC respectively under abiotic stresses, suggesting its role in maintenance of metabolic machinery under stress. Significance: Present work explores key role of AhpC in mitigating stress in Anabaena PCC7120 through combined proteomic, biochemical and in silico investigations. This study is the first attempt to analyze and compare alterations in proteome of Anabaena PCC7120 following addition (overexpressing strain An+ahpC) and deletion (mutantAn∆ahpC) of AhpC against its wild type. The effort resulted in two major traits in An+ahpC as (i) augmentation of photosynthesis and nitrogen fixation (ii) modulation of regulatory network of antioxidative proteins. © 2016 Elsevier B.V.
Effect of magnetically induced water structure on the oestrous cycles of albino female mice mus musculus
(Marcel Dekker Inc., 1996) Sarita Pandey; T.K. Garg; K.P. Singh; Subas Rai
The effect of magnetically pregenerated water structures was studied on oestrous cycles (OC) and body weight changes in cycling female albino mice. The result showed that exposure time-dependent, magnetically generated water structures produced by different polarities of a magnet produce differential effects on the OC and body weight of cycling mice. We believe that magnetically induced water structures may have caused these effects by influencing states of the central nervous system, activities of the gonadal hormones, and thus altering the physiological processes of cycling mice.
Effect of magnetically induced water structure on the oestrous cycles of albino female mice Mus Musculus
(Marcel Dekker Inc., 1996) Sarita Pandey; T.K. Garg; K.P. Singh; Subas Rai
The effect of magnetically pregenerated water structures was studied on oestrous cycles (OC) and body weight changes in cycling female albino mice. The result showed that exposure time-dependent, magnetically generated water structures produced by different polarities of a magnet produce differential effects on the OC and body weight of cycling mice. We believe that magnetically induced water structures may have caused these effects by influencing states of the central nervous system, activities of the gonadal hormones, and thus altering the physiological processes of cycling mice.
Molecular characterization of Alr1105 a novel arsenate reductase of the diazotrophic cyanobacterium Anabaena sp. PCC7120 and decoding its role in abiotic stress management in Escherichia coli
(Kluwer Academic Publishers, 2013) Sarita Pandey; Alok K. Shrivastava; Rashmi Rai; Lal Chand Rai
This paper constitutes the first report on the Alr1105 of Anabaena sp. PCC7120 which functions as arsenate reductase and phosphatase and offers tolerance against oxidative and other abiotic stresses in the alr1105 transformed Escherichia coli. The bonafide of 40.8 kDa recombinant GST+Alr1105 fusion protein was confirmed by immunoblotting. The purified Alr1105 protein (mw 14.8 kDa) possessed strong arsenate reductase (Km 16.0 ± 1.2 mM and Vmax 5.6 ± 0.31 μmol min-1 mg protein-1) and phosphatase activity (Km 27.38 ± 3.1 mM and Vmax 0.077 ± 0.005 μmol min-1 mg protein-1) at an optimum temperature 37 °C and 6.5 pH. Native Alr1105 was found as a monomeric protein in contrast to its homologous Synechocystis ArsC protein. Expression of Alr1105 enhanced the arsenic tolerance in the arsenate reductase mutant E. coli WC3110 ({increment}arsC) and rendered better growth than the wild type W3110 up to 40 mM As (V). Notwithstanding above, the recombinant E. coli strain when exposed to CdCl2, ZnSO4, NiCl2, CoCl2, CuCl2, heat, UV-B and carbofuron showed increase in growth over the wild type and mutant E. coli transformed with the empty vector. Furthermore, an enhanced growth of the recombinant E. coli in the presence of oxidative stress producing chemicals (MV, PMS and H2O2), suggested its protective role against these stresses. Appreciable expression of alr1105 gene as measured by qRT-PCR at different time points under selected stresses reconfirmed its role in stress tolerance. Thus the Alr1105 of Anabaena sp. PCC7120 functions as an arsenate reductase and possess novel properties different from the arsenate reductases known so far. © 2013 Springer Science+Business Media Dordrecht.
Overexpression of AhpC enhances stress tolerance and N2–fixation in Anabaena by upregulating stress responsive genes
(Elsevier B.V., 2016) Alok Kumar Shrivastava; Sarita Pandey; Karl Josef Dietz; Prashant Kumar Singh; Shilpi Singh; Ruchi Rai; Lal Chand Rai
The study explores the significance of peroxides in regulating the CO2- and N2-fixation capacities in Anabaena sp. PCC7120. To this end Anabaena strains were generated carrying an extra copy of ahpC (An + ahpC) or by deleting from their endogenous functional ahpC (AnΔahpC). AhpC levels were 2.2- to 6.0-fold higher in An + ahpC than in wild type. An + ahpC revealed 1.4- to 2-fold upregulation of photosystems I and II, nitrogenase, superoxide dismutase and catalase activities while same activities were 1.3- to 2.5-fold downregulated in the insertional mutant (AnΔahpC) compared to the wild type. Peroxide, superoxide and malondialdehyde contents were low in An + ahpC and high in AnΔahpC. Growth was inhibited in AnΔahpC by approximately 40–60% compared to a 33–40% enhanced growth in An + ahpC under selected stresses. Most interestingly, heterocyst frequency was increased in An + ahpC. In order to address transcriptional and posttranscriptional effects, transcripts of genes including groEL, fld, kat, gor, gst, dps, bfr, tf, sodA, dnaK, prx, uspA, pcs and apx were quantified and found to be increased 1.33- to 7.70-fold in unstressed and 1.76- to 13.80-fold in stressed An + ahpC. In a converse manner, they were downregulated by 1.20- to 7.50-fold in unstressed and 1.23 to 10.20-fold in stressed AnΔahpC. It is concluded that the level of AhpC controls a major set of metabolic and developmental genes in normal and stress conditions and thus likely is in the core of the redox regulatory system of Anabaena. © 2016 Elsevier B.V.
Plant regeneration from alginate-encapsulated shoot tips of Spilanthes acmella (L.) Murr., a medicinally important and herbal pesticidal plant species
(2009) Shashi Kant Singh; Manoj K. Rai; Pooja Asthana; Sarita Pandey; V.S. Jaiswal; U. Jaiswal
This article demonstrates the plantlet regeneration from alginate-encapsulated shoot tips of Spilanthes acmella. Shoot tip explants excised from in vitro proliferated shoots were encapsulated in calcium alginate beads. The best gel complexation for encapsulation of shoot tips was achieved using 3% sodium alginate and 100 mM calcium chloride. Maximum percent response for the conversion of encapsulated shoot tips into plantlets was obtained on growth regulator-free full-strength liquid MS (Murashige and Skoog, Physiol Plant 15:473-497, 1962) medium. The addition of MS nutrients in alginate matrix was found to have pronounced effect on shoot and root emergence from alginate beads. Encapsulated shoot tips could be stored at low temperature (4°C) up to 60 days. Plantlets regenerated from encapsulated shoot tips were acclimatized successfully. The present synthetic seed technology could be useful in large-scale propagation as well as short-term conservation and germplasm distribution and exchange of Spilanthes acmella. © 2008 Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków.
Protective Influence of Spayed and Androgen-Treated Females on Oestrous Cycle in Food-Restricted Mice
(1993) Sarita Pandey; C.J. Dominic
Diet restriction induced disruption of oestrous cycle in regularly cycling female mice. The irregularities in the oestrous cycle in females were directly related to the degree of diet restriction. The presence of spayed and androgen-treated females prevented these irregularities and helped to maintain regular oestrous cycles in underfed females. The ability of androgen-treated females to prevent the irregularities in the oestrous cycle in underfed females was inversely related to the degree of diet restriction. The results suggest that the production of the male-originating pheromone that helps to prevent the underfeeding-induced disruption of oestrous cycles in females is androgen-dependent. © 1993, J. A. Barth Verlag in Georg Thieme Verlag KG Stuttgart, New York. All rights reserved.
Proteomics combines morphological, physiological and biochemical attributes to unravel the survival strategy of Anabaena sp. PCC7120 under arsenic stress
(2012) Sarita Pandey; Rashmi Rai; Lal Chand Rai
Proteomics in conjunction with morphological, physiological and biochemical variables has been employed for the first time to unravel survival strategies of the diazotrophic cyanobacterium Anabaena sp. PCC7120 under Arsenic (As) stress. Significant reduction in growth, carbon fixation, nitrogenase activity and chlorophyll content after 1. day (1. d) and recovery after 15. days (15. d) of As exposure indicates the acclimation of the test organism against As stress. The formation of akinete like structures is a novel observation never reported before in Anabaena sp. PCC7120. Proteomic characterization using 2-DE showed average 537, 422 and 439 spots in control, 1 and 15. d treatment respectively. MALDI-TOF and LC-MS of As-treated Anabaena revealed a total of 45 differentially expressed proteins, of which 13 were novel (hypothetical) ones. Down-regulation of phosphoglycerate kinase (PGK), fructose bisphosphate aldolase II (FBA II), fructose 1,6 bisphosphatase (FBPase), transketolase (TK), and ATP synthase on day 1 and their significant recovery on the 15th day presumably maintained the glycolysis, pentose phosphate pathway (PPP) and turnover rate of Calvin cycle, hence survival of the test organism. Up-regulation of catalase (CAT), peroxiredoxin (Prx), thioredoxin (Trx) and oxidoreductase appears to protect the cells from oxidative stress. Appreciable induction in phytochelatin content (2.4 fold), GST activity (2.3 fold), and transcripts of phytochelatin synthase (5.0 fold), arsenate reductase (8.5 fold) and arsenite efflux genes - asr1102 (5.0 fold), alr1097 (4.7 fold) reiterates their role in As sequestration and shielding of the organism from As toxicity. While up-regulated metabolic and antioxidative defense proteins, phytochelatin and GST work synchronously, the ars genes play a central role in detoxification and survival of Anabaena under As stress. The proposed hypothetical model explains the interaction of metabolic proteins associated with the survival of Anabaena sp. PCC7120 under As stress. © 2011 Elsevier B.V.
Understanding the mechanisms of abiotic stress management in cyanobacteria with special reference to proteomics
(CRC Press, 2013) Snigdha Rai; Sarita Pandey; Alok Kumar Shrivastava; Prashant Kumar Singh; Chhavi Agrawal; Lal Chand Rai
Cyanobacteria are thought to have evolved around 3.5 billion years ago and have played a key role in earth’s transition from an anaerobic state to the aerobic one. They are the largest group of Gram-negative photosynthetic prokaryotes that has a close resemblance to higher plants in terms of lipid composition and protein assembly of the thylakoid membrane [1]. Cyanobacteria are ubiquitous in aquatic and terrestrial ecosystems including extreme habitats like hot springs, deserts, and polar regions. They occupy a central position in the nutrient cycling due to their unique capacity to fix atmospheric nitrogen into the assimilatory form (NH4+), thus contributing to the global nitrogen budget [2,3]. They are reported to play a key role in the maintenance of soil fertility [4] and have proved to be a potential biofertilizer in the form of plant-cyanobacterial symbiosis. Besides, many cyanobacterial species are reported to synthesize bioactive metabolites of potential therapeutic use [5]. It can therefore be concluded that cyanobacteria are a powerful model system for studying the molecular mechanisms of the responses and the acclimation strategies to abiotic stresses [6-8]. These mechanisms may provide models that may be applicable to plants as well. © 2013 by Taylor & Francis Group, LLC.