A highly efficient cowpea stable transformation system was developed with a 44 % increase in effi... more A highly efficient cowpea stable transformation system was developed with a 44 % increase in efficiency compared to the existing transformation methods by employing an improved kanamycin selection regime, and fertile transgenic plants expressing Btcry1Ac were generated. Agrobacterium-cocultivated 4-day-old cotyledonary node explants were selected on medium containing 150 mg/l kanamycin for 20 days. Surviving explants were shifted to kanamycin-free media supplemented with a reduced dosage (2.5 μM) of 6-benzyl aminopurine resulting in profuse proliferation of kanamycin-resistant shoots with a 63.6 % increase in shoot length within 15 days of culture. The elongated shoots readily developed roots on kanamycin-free rooting media and fertile plants were obtained following transfer to soil. β-glucuronidase histochemical assay, polymerase chain reaction, and reverse-transcriptase PCR analyses of T0 and T1 generations confirmed the transgenic nature of the selected plants. Of the four select...
In silico studies with uracil phosphoribosyltransferase from Arabidopsis thaliana (AtUPRT) reveal... more In silico studies with uracil phosphoribosyltransferase from Arabidopsis thaliana (AtUPRT) revealed its lower binding energies for uracil and 5-fluorouracil (5-FU) as compared to those of bacterial UPRT indicating the prospective of AtUPRT in gene therapy implications. Hence, AtUPRT was cloned and stably expressed in cervical cancer cells (HeLa) to investigate the effect of prodrug 5-FU on these transfected cancer cells. The treatment of AtUPRT-expressing HeLa (HeLa-UPP) cells with 5-FU for 72h resulted in significant decrease in cell viability. Moreover, 5-FU was observed to induce apoptosis and perturb mitochondrial membrane potential in HeLa-UPP cells. While cell cycle analysis revealed significant S-phase arrest as a result of 5-FU treatment in HeLa-UPP cells, quantitative gene expression analysis demonstrated simultaneous upregulation of important cell cycle related genes, cyclin D1 and p21. The survival fractions of non-transfected, vector-transfected and AtUPRT-transfected He...
Riverbank erosion is a global problem with significant socio-economic impacts. Microbially induce... more Riverbank erosion is a global problem with significant socio-economic impacts. Microbially induced calcite precipitation (MICP) has recently emerged as a promising technology for improving the mechanical properties of soils. The present study investigates the potential of selectively enriched native calcifying bacterial community and its supplementation into the riverbank soil of the Brahmaputra river for reducing the erodibility of the soil. The ureolytic and calcium carbonate cementation abilities of the enriched cultures were investigated with reference to the standard calcifying culture of Sporosarcina pasteurii (ATCC 11859). 16S rRNA analysis revealed Firmicutes to be the most predominant calcifying class with Sporosarcina pasteurii and Pseudogracilibacillus auburnensis as the prevalent strains. The morphological and mineralogical characterization of carbonate crystals confirmed the calcite precipitation potential of these communities. The erodibility of soil treated with nativ...
Permanent wilting point (PWP) is generally used to ascertain plant resistance against abiotic dro... more Permanent wilting point (PWP) is generally used to ascertain plant resistance against abiotic drought stress and designated as the soil water content (θ) corresponding to soil suction (ψ) at 1500 kPa obtained from the soil water retention curve. Determination of PWP based on only pre-assumed ψ may not represent true wilting condition for soils with contrasting water retention abilities. In addition to ψ, there is a need to explore significance of additional plant parameters (i.e., stomatal conductance and photosynthetic status) in determining PWP. This study introduces a new framework for determining PWP by integrating plant leaf response and ψ during drought. Axonopus compressus were grown in two distinct textured soils (clayey loam and silty sand), after which drought was initiated till wilting. Thereafter, ψ and θ within the root zone were measured along with corresponding leaf stomatal conductance and photosynthetic status. It was found that coarse textured silty sand causes wil...
Mungbean is an important pulse crop extensively cultivated in Southeast Asia for supply of easily... more Mungbean is an important pulse crop extensively cultivated in Southeast Asia for supply of easily digestible protein. Salinity severely limits the growth and productivity of mungbean, and weeding poses nutritional and disease constraints to mungbean cultivation. To pyramid both salt tolerance and protection against herbicide in mungbean, the AtNHX1 encoding tonoplast Na(+)/H(+) antiporter from Arabidopsis, and bar gene associated with herbicide resistance were co-expressed through Agrobacterium-mediated transformation. Stress inducible expression of AtNHX1 significantly improved tolerance under salt stress to ionic, osmotic, and oxidative stresses in transgenic mungbean plants compared to the wild type (WT) plants, whereas constitutive expression of bar provided resistance to herbicide. Compared to WT, transgenic mungbean plants grew better with higher plant height, foliage, dry mass and seed yield under high salt stress (200 mM NaCl) in the greenhouse. The improved performance of t...
To investigate the potential of recombinant phytaspase loaded manganese (Mn) doped zinc sulphide ... more To investigate the potential of recombinant phytaspase loaded manganese (Mn) doped zinc sulphide (ZnS) quantum dots embedded chitosan nanoparticles for augmenting cisplatin induced chemotherapy of HeLa cells. The recombinant phytaspase was cloned into bacterial expression vector PGEX-4T-2. The expressed and purified recombinant plant phytaspase protein from Escherichia coli BL21 was immobilized onto the cationic nanocomposite. Confocal microscopy elucidated the delivery of these luminescent nanocomposites inside cervical cancer HeLa cells. A 50% reduction in the viability of HeLa cells was achieved only in the case of phytaspase-nanocomposites-cisplatin combination at a dose of phytaspase (42 nM), nanocomposites (56.3 μg/ml) and cisplatin (0.44 μg/ml). Luminescent cationic nanocomposites were developed for intracellular delivery of recombinant phytaspase, which due to its caspase-like activity assisted in substantiating the chemotherapeutic activity of apoptosis inducing drug-cispla...
Sesbania grandiflora (L.) Pers. is one of the fast growing tree legumes having the efficiency to ... more Sesbania grandiflora (L.) Pers. is one of the fast growing tree legumes having the efficiency to produce around 50tha(-1) above ground dry matters in a year. In this study, biomass of 2years old S. grandiflora was selected for the chemical composition, pretreatments and enzymatic hydrolysis studies. The stem biomass with a wood density of 3.89±0.01gmcm(-3) contains about 38% cellulose, 12% hemicellulose and 28% lignin. Enzymatic hydrolysis of pretreated biomass revealed that phosphoric acid (H3PO4) pretreated samples even at lower cellulase loadings [1 Filter Paper Units (FPU)], could efficiently convert about 86% glucose, while, even at higher cellulase loadings (60FPU) alkali pretreated biomass could convert only about 58% glucose. The effectiveness of phosphoric acid pretreatment was also supported by X-ray diffraction (XRD), field emission scanning electron microscopy (FE-SEM) and Fourier transform infrared spectroscopy (FTIR) analysis.
Phytaspase, a plant serine protease, has been demonstrated to play an important role in the progr... more Phytaspase, a plant serine protease, has been demonstrated to play an important role in the programmed cell death of various plants. Phytaspase is synthesized as an inactive proenzyme containing an N-terminal signal peptide followed by a pro-domain and a mature protease catalytic domain. Pre-prophytaspase autocatalytically processes itself into a pro-domain and an active mature phytaspase enzyme. We have recently demonstrated the successful expression of mature phytaspase from tobacco in a bacterial system. Herein, we focus on the expression of pre-prophytaspase as a GST-tag fusion and on its purification by affinity chromatography.
International journal of biological macromolecules, 2017
Following the cloning and expression of tobacco (Nicotiana tabacum) phytaspase gene in Escherichi... more Following the cloning and expression of tobacco (Nicotiana tabacum) phytaspase gene in Escherichia coli BL21, the recombinant protease was purified by affinity chromatography for further characterization. Circular dichroism (CD) spectroscopy and in silico analysis revealed structural similarities of recombinant phytaspase with other plant serine-proteases. Molecular docking studies showed favourable binding of synthetic peptide substrate for caspase 8 (Ac-VETD-AMC) to the reactive pocket of recombinant phytaspase indicating its potential in assessing functional activity of recombinant phytaspase. In silico findings were supported by caspase 8-like activity of purified phytaspase demonstrated in vitro. The Michaelis constant (KM) and specificity constant (kcat/KM) of phytaspase for hydrolyzing Ac-VETD-AMC were found to be 1.587μM and 4.67×10(3)M(-1)min(-1), respectively. Transient expression of phytaspase in lung epithelial adenocarcinoma cells (A549) resulted in reduced IC50 value o...
A highly efficient cowpea stable transformation system was developed with a 44 % increase in effi... more A highly efficient cowpea stable transformation system was developed with a 44 % increase in efficiency compared to the existing transformation methods by employing an improved kanamycin selection regime, and fertile transgenic plants expressing Btcry1Ac were generated. Agrobacterium-cocultivated 4-day-old cotyledonary node explants were selected on medium containing 150 mg/l kanamycin for 20 days. Surviving explants were shifted to kanamycin-free media supplemented with a reduced dosage (2.5 μM) of 6-benzyl aminopurine resulting in profuse proliferation of kanamycin-resistant shoots with a 63.6 % increase in shoot length within 15 days of culture. The elongated shoots readily developed roots on kanamycin-free rooting media and fertile plants were obtained following transfer to soil. β-glucuronidase histochemical assay, polymerase chain reaction, and reverse-transcriptase PCR analyses of T0 and T1 generations confirmed the transgenic nature of the selected plants. Of the four select...
In silico studies with uracil phosphoribosyltransferase from Arabidopsis thaliana (AtUPRT) reveal... more In silico studies with uracil phosphoribosyltransferase from Arabidopsis thaliana (AtUPRT) revealed its lower binding energies for uracil and 5-fluorouracil (5-FU) as compared to those of bacterial UPRT indicating the prospective of AtUPRT in gene therapy implications. Hence, AtUPRT was cloned and stably expressed in cervical cancer cells (HeLa) to investigate the effect of prodrug 5-FU on these transfected cancer cells. The treatment of AtUPRT-expressing HeLa (HeLa-UPP) cells with 5-FU for 72h resulted in significant decrease in cell viability. Moreover, 5-FU was observed to induce apoptosis and perturb mitochondrial membrane potential in HeLa-UPP cells. While cell cycle analysis revealed significant S-phase arrest as a result of 5-FU treatment in HeLa-UPP cells, quantitative gene expression analysis demonstrated simultaneous upregulation of important cell cycle related genes, cyclin D1 and p21. The survival fractions of non-transfected, vector-transfected and AtUPRT-transfected He...
Riverbank erosion is a global problem with significant socio-economic impacts. Microbially induce... more Riverbank erosion is a global problem with significant socio-economic impacts. Microbially induced calcite precipitation (MICP) has recently emerged as a promising technology for improving the mechanical properties of soils. The present study investigates the potential of selectively enriched native calcifying bacterial community and its supplementation into the riverbank soil of the Brahmaputra river for reducing the erodibility of the soil. The ureolytic and calcium carbonate cementation abilities of the enriched cultures were investigated with reference to the standard calcifying culture of Sporosarcina pasteurii (ATCC 11859). 16S rRNA analysis revealed Firmicutes to be the most predominant calcifying class with Sporosarcina pasteurii and Pseudogracilibacillus auburnensis as the prevalent strains. The morphological and mineralogical characterization of carbonate crystals confirmed the calcite precipitation potential of these communities. The erodibility of soil treated with nativ...
Permanent wilting point (PWP) is generally used to ascertain plant resistance against abiotic dro... more Permanent wilting point (PWP) is generally used to ascertain plant resistance against abiotic drought stress and designated as the soil water content (θ) corresponding to soil suction (ψ) at 1500 kPa obtained from the soil water retention curve. Determination of PWP based on only pre-assumed ψ may not represent true wilting condition for soils with contrasting water retention abilities. In addition to ψ, there is a need to explore significance of additional plant parameters (i.e., stomatal conductance and photosynthetic status) in determining PWP. This study introduces a new framework for determining PWP by integrating plant leaf response and ψ during drought. Axonopus compressus were grown in two distinct textured soils (clayey loam and silty sand), after which drought was initiated till wilting. Thereafter, ψ and θ within the root zone were measured along with corresponding leaf stomatal conductance and photosynthetic status. It was found that coarse textured silty sand causes wil...
Mungbean is an important pulse crop extensively cultivated in Southeast Asia for supply of easily... more Mungbean is an important pulse crop extensively cultivated in Southeast Asia for supply of easily digestible protein. Salinity severely limits the growth and productivity of mungbean, and weeding poses nutritional and disease constraints to mungbean cultivation. To pyramid both salt tolerance and protection against herbicide in mungbean, the AtNHX1 encoding tonoplast Na(+)/H(+) antiporter from Arabidopsis, and bar gene associated with herbicide resistance were co-expressed through Agrobacterium-mediated transformation. Stress inducible expression of AtNHX1 significantly improved tolerance under salt stress to ionic, osmotic, and oxidative stresses in transgenic mungbean plants compared to the wild type (WT) plants, whereas constitutive expression of bar provided resistance to herbicide. Compared to WT, transgenic mungbean plants grew better with higher plant height, foliage, dry mass and seed yield under high salt stress (200 mM NaCl) in the greenhouse. The improved performance of t...
To investigate the potential of recombinant phytaspase loaded manganese (Mn) doped zinc sulphide ... more To investigate the potential of recombinant phytaspase loaded manganese (Mn) doped zinc sulphide (ZnS) quantum dots embedded chitosan nanoparticles for augmenting cisplatin induced chemotherapy of HeLa cells. The recombinant phytaspase was cloned into bacterial expression vector PGEX-4T-2. The expressed and purified recombinant plant phytaspase protein from Escherichia coli BL21 was immobilized onto the cationic nanocomposite. Confocal microscopy elucidated the delivery of these luminescent nanocomposites inside cervical cancer HeLa cells. A 50% reduction in the viability of HeLa cells was achieved only in the case of phytaspase-nanocomposites-cisplatin combination at a dose of phytaspase (42 nM), nanocomposites (56.3 μg/ml) and cisplatin (0.44 μg/ml). Luminescent cationic nanocomposites were developed for intracellular delivery of recombinant phytaspase, which due to its caspase-like activity assisted in substantiating the chemotherapeutic activity of apoptosis inducing drug-cispla...
Sesbania grandiflora (L.) Pers. is one of the fast growing tree legumes having the efficiency to ... more Sesbania grandiflora (L.) Pers. is one of the fast growing tree legumes having the efficiency to produce around 50tha(-1) above ground dry matters in a year. In this study, biomass of 2years old S. grandiflora was selected for the chemical composition, pretreatments and enzymatic hydrolysis studies. The stem biomass with a wood density of 3.89±0.01gmcm(-3) contains about 38% cellulose, 12% hemicellulose and 28% lignin. Enzymatic hydrolysis of pretreated biomass revealed that phosphoric acid (H3PO4) pretreated samples even at lower cellulase loadings [1 Filter Paper Units (FPU)], could efficiently convert about 86% glucose, while, even at higher cellulase loadings (60FPU) alkali pretreated biomass could convert only about 58% glucose. The effectiveness of phosphoric acid pretreatment was also supported by X-ray diffraction (XRD), field emission scanning electron microscopy (FE-SEM) and Fourier transform infrared spectroscopy (FTIR) analysis.
Phytaspase, a plant serine protease, has been demonstrated to play an important role in the progr... more Phytaspase, a plant serine protease, has been demonstrated to play an important role in the programmed cell death of various plants. Phytaspase is synthesized as an inactive proenzyme containing an N-terminal signal peptide followed by a pro-domain and a mature protease catalytic domain. Pre-prophytaspase autocatalytically processes itself into a pro-domain and an active mature phytaspase enzyme. We have recently demonstrated the successful expression of mature phytaspase from tobacco in a bacterial system. Herein, we focus on the expression of pre-prophytaspase as a GST-tag fusion and on its purification by affinity chromatography.
International journal of biological macromolecules, 2017
Following the cloning and expression of tobacco (Nicotiana tabacum) phytaspase gene in Escherichi... more Following the cloning and expression of tobacco (Nicotiana tabacum) phytaspase gene in Escherichia coli BL21, the recombinant protease was purified by affinity chromatography for further characterization. Circular dichroism (CD) spectroscopy and in silico analysis revealed structural similarities of recombinant phytaspase with other plant serine-proteases. Molecular docking studies showed favourable binding of synthetic peptide substrate for caspase 8 (Ac-VETD-AMC) to the reactive pocket of recombinant phytaspase indicating its potential in assessing functional activity of recombinant phytaspase. In silico findings were supported by caspase 8-like activity of purified phytaspase demonstrated in vitro. The Michaelis constant (KM) and specificity constant (kcat/KM) of phytaspase for hydrolyzing Ac-VETD-AMC were found to be 1.587μM and 4.67×10(3)M(-1)min(-1), respectively. Transient expression of phytaspase in lung epithelial adenocarcinoma cells (A549) resulted in reduced IC50 value o...
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Papers by Lingaraj Sahoo