Human serum paraoxonase 1 (PON1) is known as an antioxidant and is also involved in the detoxific... more Human serum paraoxonase 1 (PON1) is known as an antioxidant and is also involved in the detoxification of many compounds. In this study, a novel purification strategy was employed to purify the PON1 by using cholesterol-conjugated magnetic nanoparticles. Magnetic nanoparticles were synthesized and conjugated with cholesterol through diazotized p-aminohippuric acid. In Fourier transform infrared spectrum of cholesterol-p-aminohippuric acid-Fe 3 O 4 nanoparticles, the appearance of peaks at 3,358.3, 1,645 cm −1 , and at 2,334.9 cm −1 confirmed the conjugation. The molecular weight of purified PON1 was nearly 45 kDa on sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE), and isoelectric point was 5.3. The specific activity was 438 U mg −1 protein, and the purification fold was 515 with 73% yield. The K m values were 1.3 and 0.74 mM with paraoxon and phenyl acetate, respectively. Western blot of 2D-PAGE confirmed the homogeneity and stability of the enzyme. Mg +2 , Mn +2 , glycerol, (NH 4) 2 SO 4 , PEG 6000, Triton X-100, and phenylmethylsulfonyl fluoride did not show any effect on activity. Pb +2 , Co +2 , Zn 2+ , ethanol, β-mercaptoethanol, and acetone reduced the activity while Ni 2+ , Cd 2+ , Cu 2+ , iodoacetic acid, SDS, dimethylformamide, DMSO inhibited the activity. In vitro enzyme activity was slightly reduced by acetyl salicylic and acetaminophen and reduced 50% with amino glycosides and ampicillin antibiotics at concentrations of 0.6 and 30 mg ml −1 , respectively. This is the first report for the synthesis of cholesterol-conjugated magnetic nanoparticles for simple purification of PON1 enzyme.
Deployment of next generation sequencing tools has made it possible to sequence the whole genome ... more Deployment of next generation sequencing tools has made it possible to sequence the whole genome within limited time period. At the moment, more than 100 plant genomes have been sequenced, of these 63% are crop species. Next challenge is to define function to these sequences (genes) and their regulation and interaction with the other genes. Though the efforts towards the identification of genes function were started in early 1990s, however, after the sequencing of Arabidopsis thaliana (a model plant species), a mega project towards the exploration of genes function was initiated. Both the reverse (gene to phenotype) and forward genetics (phenotype to gene) approaches were used. For example, microarray, virus-induced gene silencing (VIGS), gene knockout, RNA interference (RNAi), insertional mutagenesis, TILLING, EcoTILLING, next-generation sequencing (NGS) technologies etc. have been extensively used for discovering genes and their functions. All these technologies with their own inherent merits and demerits were supplemented by the emerging bioinformatics tools in translating the information generating on model species to less-studied species—effective tool in predicting function of unknown sequences within the shortest possible time through homology search. All these efforts set a stage for unravelling the functions of unknown genes involved in conferring various mechanisms including growth, grain development, response to various stresses, etc. Thus the functional genomic studies would lead to sustain crop productivity on this planet by providing a comprehensive knowledge about the function of genes—a huge genetic resource that can be utilized for improving the crop varieties using nonconventional (genetic transformation) and conventional means assisted with DNA markers. 2
In 1912, the first evidence of leaf curl disease (CLCuD) was observed in Nigeria, and since then ... more In 1912, the first evidence of leaf curl disease (CLCuD) was observed in Nigeria, and since then it has spread to Pakistan, India, and China. The disease is of viral origin— transmitted by a whitefly (Bemisia tabaci), which is difficult to control because of the prevalence of multiple virulent viral strains or related species. The problem is further complicated as the CLCuD-causing virus complex has a higher recombination rate. Also, the ability of infecting other important crops, for example, tomato, okra, etc., and practicing mixed-type farming systems act synergistically toward the evolution of new viral strains and vectors. Efforts to combat this disease using host-plant resistance remained successful using two gene-based resistance that was broken by the evolution of new resistance-breaking strain called as Burewala virus strain. Development of transgenic cotton using both the pathogen- andnonpathogenic-derived approaches is in progress. In future, screening for new forms of host resistance and use of DNA markers for the rapid incorporation of resistance into adapted cultivars overlaid withtransgenics would be instrumental in adding multiple layers of defense to combat the disease—thus fiber production will be sustained.
Human serum paraoxonase 1 (PON1) is known as an antioxidant and is also involved in the detoxific... more Human serum paraoxonase 1 (PON1) is known as an antioxidant and is also involved in the detoxification of many compounds. In this study, a novel purification strategy was employed to purify the PON1 by using cholesterol-conjugated magnetic nanoparticles. Magnetic nanoparticles were synthesized and conjugated with cholesterol through diazotized p-aminohippuric acid. In Fourier transform infrared spectrum of cholesterol-p-aminohippuric acid-Fe 3 O 4 nanoparticles, the appearance of peaks at 3,358.3, 1,645 cm −1 , and at 2,334.9 cm −1 confirmed the conjugation. The molecular weight of purified PON1 was nearly 45 kDa on sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE), and isoelectric point was 5.3. The specific activity was 438 U mg −1 protein, and the purification fold was 515 with 73% yield. The K m values were 1.3 and 0.74 mM with paraoxon and phenyl acetate, respectively. Western blot of 2D-PAGE confirmed the homogeneity and stability of the enzyme. Mg +2 , Mn +2 , glycerol, (NH 4) 2 SO 4 , PEG 6000, Triton X-100, and phenylmethylsulfonyl fluoride did not show any effect on activity. Pb +2 , Co +2 , Zn 2+ , ethanol, β-mercaptoethanol, and acetone reduced the activity while Ni 2+ , Cd 2+ , Cu 2+ , iodoacetic acid, SDS, dimethylformamide, DMSO inhibited the activity. In vitro enzyme activity was slightly reduced by acetyl salicylic and acetaminophen and reduced 50% with amino glycosides and ampicillin antibiotics at concentrations of 0.6 and 30 mg ml −1 , respectively. This is the first report for the synthesis of cholesterol-conjugated magnetic nanoparticles for simple purification of PON1 enzyme.
Deployment of next generation sequencing tools has made it possible to sequence the whole genome ... more Deployment of next generation sequencing tools has made it possible to sequence the whole genome within limited time period. At the moment, more than 100 plant genomes have been sequenced, of these 63% are crop species. Next challenge is to define function to these sequences (genes) and their regulation and interaction with the other genes. Though the efforts towards the identification of genes function were started in early 1990s, however, after the sequencing of Arabidopsis thaliana (a model plant species), a mega project towards the exploration of genes function was initiated. Both the reverse (gene to phenotype) and forward genetics (phenotype to gene) approaches were used. For example, microarray, virus-induced gene silencing (VIGS), gene knockout, RNA interference (RNAi), insertional mutagenesis, TILLING, EcoTILLING, next-generation sequencing (NGS) technologies etc. have been extensively used for discovering genes and their functions. All these technologies with their own inherent merits and demerits were supplemented by the emerging bioinformatics tools in translating the information generating on model species to less-studied species—effective tool in predicting function of unknown sequences within the shortest possible time through homology search. All these efforts set a stage for unravelling the functions of unknown genes involved in conferring various mechanisms including growth, grain development, response to various stresses, etc. Thus the functional genomic studies would lead to sustain crop productivity on this planet by providing a comprehensive knowledge about the function of genes—a huge genetic resource that can be utilized for improving the crop varieties using nonconventional (genetic transformation) and conventional means assisted with DNA markers. 2
In 1912, the first evidence of leaf curl disease (CLCuD) was observed in Nigeria, and since then ... more In 1912, the first evidence of leaf curl disease (CLCuD) was observed in Nigeria, and since then it has spread to Pakistan, India, and China. The disease is of viral origin— transmitted by a whitefly (Bemisia tabaci), which is difficult to control because of the prevalence of multiple virulent viral strains or related species. The problem is further complicated as the CLCuD-causing virus complex has a higher recombination rate. Also, the ability of infecting other important crops, for example, tomato, okra, etc., and practicing mixed-type farming systems act synergistically toward the evolution of new viral strains and vectors. Efforts to combat this disease using host-plant resistance remained successful using two gene-based resistance that was broken by the evolution of new resistance-breaking strain called as Burewala virus strain. Development of transgenic cotton using both the pathogen- andnonpathogenic-derived approaches is in progress. In future, screening for new forms of host resistance and use of DNA markers for the rapid incorporation of resistance into adapted cultivars overlaid withtransgenics would be instrumental in adding multiple layers of defense to combat the disease—thus fiber production will be sustained.
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transgenic cotton using both the pathogen- andnonpathogenic-derived approaches is in progress. In future, screening for new forms of host resistance and use of DNA
markers for the rapid incorporation of resistance into adapted cultivars overlaid withtransgenics would be instrumental in adding multiple layers of defense to combat the disease—thus fiber production will be sustained.
transgenic cotton using both the pathogen- andnonpathogenic-derived approaches is in progress. In future, screening for new forms of host resistance and use of DNA
markers for the rapid incorporation of resistance into adapted cultivars overlaid withtransgenics would be instrumental in adding multiple layers of defense to combat the disease—thus fiber production will be sustained.