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Shailja Misra-bhattacharya - One of the best experts on this subject based on the ideXlab platform.
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RNA interference mediated knockdown of Brugia malayi UDP-Galactopyranose mutase severely affects Parasite Viability, embryogenesis and in vivo development of infective larvae.
Parasites & vectors, 2017Co-Authors: Sweta Misra, Jyoti Gupta, Shailja Misra-bhattacharyaAbstract:Galactofuranose is an essential cell surface component present in bacteria, fungi and several nematodes such as Caenorhabditis spp., Brugia spp., Onchocerca spp. and Strongyloides spp. This sugar maintains the integrity of Parasite surface and is essential for virulence. UDP-Galactopyranose mutase (bmugm) plays a key role in Galf biosynthesis by catalyzing conversion of UDP-Galactopyranose into UDP-galactofuranose and knockout studies of the gene in Leishmania major, Mycobacterium and Aspergillus fumigatus displayed attenuated virulence while RNA interference study in C. elegans exhibited detrimental effects. Presence of UGM in several prokaryotic and eukaryotic microbial pathogens and its absence in higher eukaryotes renders it an attractive drug target. In the present study, RNA interference studies have been carried out to validate bmugm as an antifilarial drug target. RNA interference studies using two different sequences of siRNAs targeting bmugm were carried out. The in vitro gene silencing of adult B. malayi Parasites was undertaken to observe the effects on Parasites. Infective larvae were also exposed to siRNAs and their in vivo development in jirds was observed. The in vitro gene silencing induced by siRNA1 and 2 individually as well as together knocked down the bmugm gene expression causing impaired Viability of the exposed worms along with extremely reduced motility, abridged microfilarial release and adversely effected embryogenesis. The combinatorial in vitro gene silencing revealed marginally better results than both the siRNAs individually. Thus, infective larvae were treated with siRNA combination which showed downregulation of bmugm mRNA expression resulting into sluggish larval movements and/or death. The siRNA-treated actively motile larvae when inoculated intraperitoneally into jirds demonstrated highly reduced transformation of these larvae into adult worms with detrimental effects on embryogenesis. The effects of gene silencing were long-lasting as the adult worms developed from siRNA-treated larvae showed noticeable knockdown in the target gene expression. The validation studies undertaken here conclude that bmugm is essential for the proper development and survival of the Parasite and support its candidature as an antifilarial drug target.
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In vitro gene silencing of independent phosphoglycerate mutase (iPGM) in the filarial Parasite Brugia malayi.
Infectious diseases of poverty, 2013Co-Authors: Prashant Kumar Singh, Susheela Kushwaha, Shahab Mohd, M. Pathak, Shailja Misra-bhattacharyaAbstract:Background The phosphoglycerate mutase (PGM) enzyme catalyzes the interconversion of 2- and 3-phosphoglycerate in the glycolytic /gluconeogenic pathways that are present in the majority of cellular organisms. They can be classified as cofactor-dependent PGM (dPGM) or cofactor-independent PGM (iPGM). Vertebrates, yeasts, and many bacteria have only dPGM, while higher plants, nematodes, archaea, and many other bacteria have only iPGM. A small number of bacteria, including Escherichia coli and certain archaea and protozoa, contain both forms. The silencing of ipgm in Caenorhabditis elegans (C. elegans) has demonstrated the importance of this enzyme in Parasite Viability and, therefore, its potential as an anthelmintic drug target. In this study, the role of the Brugia malayi (B. malayi) ipgm in Parasite Viability, microfilaria release, embryogenesis, and in vivo development of infective larvae post-gene silencing was explored by applying ribonucleic acid (RNA) interference studies.
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In vitro gene silencing of independent phosphoglycerate mutase (iPGM) in the filarial Parasite Brugia malayi
Infectious Diseases of Poverty, 2013Co-Authors: Prashant Kumar Singh, Susheela Kushwaha, Shahab Mohd, M. Pathak, Shailja Misra-bhattacharyaAbstract:Background The phosphoglycerate mutase (PGM) enzyme catalyzes the interconversion of 2- and 3-phosphoglycerate in the glycolytic /gluconeogenic pathways that are present in the majority of cellular organisms. They can be classified as cofactor-dependent PGM (dPGM) or cofactor-independent PGM (iPGM). Vertebrates, yeasts, and many bacteria have only dPGM, while higher plants, nematodes, archaea, and many other bacteria have only iPGM. A small number of bacteria, including Escherichia coli and certain archaea and protozoa, contain both forms. The silencing of ipgm in Caenorhabditis elegans ( C. elegans ) has demonstrated the importance of this enzyme in Parasite Viability and, therefore, its potential as an anthelmintic drug target. In this study, the role of the Brugia malayi ( B. malayi ) ipgm in Parasite Viability, microfilaria release, embryogenesis, and in vivo development of infective larvae post-gene silencing was explored by applying ribonucleic acid (RNA) interference studies. Results The in vitro ipgm gene silencing by small interfering RNA (siRNA) leads to severe phenotypic deformities in the intrauterine developmental stages of female worms with a drastic reduction (~90%) in the motility of adult Parasites and a significantly reduced (80%) release of microfilariae (mf) by female worms in vitro . Almost half of the in vitro- treated infective L3 displayed sluggish movement. The in vivo survival and development of siRNA-treated infective larvae (L3) was investigated in the peritoneal cavity of jirds where a ~45% reduction in adult worm establishment was observed. Conclusion The findings clearly suggest that iPGM is essential for both larval and adult stages of B. malayi Parasite and that it plays a pivotal role in female worm embryogenesis. The results thus validate the Bm-iPGM as a putative anti-filarial drug target.
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RNAi mediated silencing of ATPase RNA helicase gene in adult filarial Parasite Brugia malayi impairs in vitro microfilaria release and adult Parasite Viability.
Journal of biotechnology, 2011Co-Authors: Meghna Singh, Prashant Kumar Singh, Shailja Misra-bhattacharyaAbstract:The DExD/H box families of RNA helicases are a multifunctional group of proteins involved in unwinding of inter- and intra-molecular base-paired regions. Successful knockdown of DEAD box RNA helicase gene (BmL3-Helicase) of human lymphatic filarial Parasite Brugia malayi was done with specifically designed and chemically synthesized siRNA of
Larry Simpson - One of the best experts on this subject based on the ideXlab platform.
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trypanosome mitochondrial 3 terminal uridylyl transferase tutase the key enzyme in u insertion deletion rna editing
Cell, 2002Co-Authors: Ruslan Aphasizhev, Sandro Sbicego, Marian Peris, Seiheon Jang, Inna Aphasizheva, Agda M Simpson, Anatoly Rivlin, Larry SimpsonAbstract:Abstract A 3′ terminal RNA uridylyltransferase was purified from mitochondria of Leishmania tarentolae and the gene cloned and expressed from this species and from Trypanosoma brucei . The enzyme is specific for 3′ U-addition in the presence of Mg 2+ . TUTase is present in vivo in at least two stable configurations: one contains a ∼500 kDa TUTase oligomer and the other a ∼700 kDa TUTase complex. Anti-TUTase antiserum specifically coprecipitates a small portion of the p45 and p50 RNA ligases and approximately 40% of the guide RNAs. Inhibition of TUTase expression in procyclic T. brucei by RNAi downregulates RNA editing and appears to affect Parasite Viability.
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Trypanosome Mitochondrial 3′ Terminal Uridylyl Transferase (TUTase): The Key Enzyme in U-Insertion/Deletion RNA Editing
Cell, 2002Co-Authors: Ruslan Aphasizhev, Sandro Sbicego, Marian Peris, Seiheon Jang, Inna Aphasizheva, Agda M Simpson, Anatoly Rivlin, Larry SimpsonAbstract:Abstract A 3′ terminal RNA uridylyltransferase was purified from mitochondria of Leishmania tarentolae and the gene cloned and expressed from this species and from Trypanosoma brucei . The enzyme is specific for 3′ U-addition in the presence of Mg 2+ . TUTase is present in vivo in at least two stable configurations: one contains a ∼500 kDa TUTase oligomer and the other a ∼700 kDa TUTase complex. Anti-TUTase antiserum specifically coprecipitates a small portion of the p45 and p50 RNA ligases and approximately 40% of the guide RNAs. Inhibition of TUTase expression in procyclic T. brucei by RNAi downregulates RNA editing and appears to affect Parasite Viability.
Ruslan Aphasizhev - One of the best experts on this subject based on the ideXlab platform.
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trypanosome mitochondrial 3 terminal uridylyl transferase tutase the key enzyme in u insertion deletion rna editing
Cell, 2002Co-Authors: Ruslan Aphasizhev, Sandro Sbicego, Marian Peris, Seiheon Jang, Inna Aphasizheva, Agda M Simpson, Anatoly Rivlin, Larry SimpsonAbstract:Abstract A 3′ terminal RNA uridylyltransferase was purified from mitochondria of Leishmania tarentolae and the gene cloned and expressed from this species and from Trypanosoma brucei . The enzyme is specific for 3′ U-addition in the presence of Mg 2+ . TUTase is present in vivo in at least two stable configurations: one contains a ∼500 kDa TUTase oligomer and the other a ∼700 kDa TUTase complex. Anti-TUTase antiserum specifically coprecipitates a small portion of the p45 and p50 RNA ligases and approximately 40% of the guide RNAs. Inhibition of TUTase expression in procyclic T. brucei by RNAi downregulates RNA editing and appears to affect Parasite Viability.
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Trypanosome Mitochondrial 3′ Terminal Uridylyl Transferase (TUTase): The Key Enzyme in U-Insertion/Deletion RNA Editing
Cell, 2002Co-Authors: Ruslan Aphasizhev, Sandro Sbicego, Marian Peris, Seiheon Jang, Inna Aphasizheva, Agda M Simpson, Anatoly Rivlin, Larry SimpsonAbstract:Abstract A 3′ terminal RNA uridylyltransferase was purified from mitochondria of Leishmania tarentolae and the gene cloned and expressed from this species and from Trypanosoma brucei . The enzyme is specific for 3′ U-addition in the presence of Mg 2+ . TUTase is present in vivo in at least two stable configurations: one contains a ∼500 kDa TUTase oligomer and the other a ∼700 kDa TUTase complex. Anti-TUTase antiserum specifically coprecipitates a small portion of the p45 and p50 RNA ligases and approximately 40% of the guide RNAs. Inhibition of TUTase expression in procyclic T. brucei by RNAi downregulates RNA editing and appears to affect Parasite Viability.
Raquel Elisa Da Silva-lópez - One of the best experts on this subject based on the ideXlab platform.
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Effects of a marine serine protease inhibitor on Viability and morphology of Trypanosoma cruzi, the agent of Chagas disease
Acta tropica, 2013Co-Authors: Natália Pereira De Almeida Nogueira, José Andrés Morgado-díaz, Rubem Figueiredo Sadok Menna-barreto, Marcia Cristina Paes, Raquel Elisa Da Silva-lópezAbstract:It has been reported that serine peptidase activities of Trypanosoma cruzi play crucial roles in Parasite dissemination and host cell invasion and therefore their inhibition could affect the progress of Chagas disease. The present study investigates the interference of the Stichodactyla helianthus Kunitz-type serine protease inhibitor (ShPI-I), a 55-amino acid peptide, in T. cruzi serine peptidase activities, Parasite Viability, and Parasite morphology. The effect of this peptide was also studied in Leishmania amazonensis promastigotes and it was proved to be a powerful inhibitor of serine proteases activities and the Parasite Viability. The ultrastructural alterations caused by ShPI-I included vesiculation of the flagellar pocket membrane and the appearance of a cytoplasmic vesicle that resembles an autophagic vacuole. ShPI-I, which showed itself to be an important T. cruzi serine peptidase inhibitor, reduced the Parasite Viability, in a dose and time dependent manner. The maximum effect of peptide on T. cruzi Viability was observed when ShPI-I at 1×10(-5)M was incubated for 24 and 48h which killed completely both metacyclic trypomastigote and epimastigote forms. At 1×10(-6)M ShPI-I, in the same periods of time, reduced Parasite Viability about 91-95% respectively. Ultrastructural analysis demonstrated the formation of concentric membranar structures especially in the cytosol, involving organelles and small vesicles. Profiles of endoplasmic reticulum were also detected, surrounding cytosolic vesicles that resembled autophagic vacuoles. These results suggest that serine peptidases are important in T. cruzi physiology since the inhibition of their activity killed Parasites in vitro as well as inducing important morphological alterations. Protease inhibitors thus appear to have a potential role as anti-trypanosomatidal agents.
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Effects of serine protease inhibitors on Viability and morphology of Leishmania (Leishmania) amazonensis promastigotes.
Parasitology research, 2007Co-Authors: Raquel Elisa Da Silva-lópez, José Andrés Morgado-díaz, M. A. Chávez, Salvatore Giovanni-de-simoneAbstract:To investigate the importance of serine proteases in Leishmania amazonensis promastigotes, we analyzed the effects of classical serine protease inhibitors and a Kunitz-type inhibitor, obtained from sea anemone Stichodactyla helianthus (ShPI-I), on the Viability and morphology of Parasites in culture. Classical inhibitors were selected on the basis of their ability to inhibit L. amazonensis serine proteases, previously described. The N-tosyl-L: -phenylalanine chloromethyl ketone (TPCK) and benzamidine (Bza) inhibitors, which are potential Leishmania proteases inhibitors, in all experimental conditions reduced the Parasite Viability, with regard to time dependence. On the other hand, N-tosyl-lysine chloromethyl ketone (TLCK) did not significantly affect the Parasite Viability, as it was poor Leishmania enzymes inhibitor. Ultrastructural analysis demonstrated that both Bza and TPCK induced changes in the flagellar pocket region with membrane alteration, including bleb formation. However, TPCK effects were more pronounced than those of Bza in Leishmania flagellar pocket in plasma membrane, and intracellular vesicular bodies was visualized. ShPI-I proved to be a powerful inhibitor of L. amazonensis serine proteases and the Parasite Viability. The ultrastructural alterations caused by ShPI-I were more dramatic than those induced by the classical inhibitors. Vesiculation of the flagellar pocket membrane, the appearance of a cytoplasmic vesicle that resembles an autophagic vacuole, and alterations of promastigotes shape resulted.
Anatoly Rivlin - One of the best experts on this subject based on the ideXlab platform.
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trypanosome mitochondrial 3 terminal uridylyl transferase tutase the key enzyme in u insertion deletion rna editing
Cell, 2002Co-Authors: Ruslan Aphasizhev, Sandro Sbicego, Marian Peris, Seiheon Jang, Inna Aphasizheva, Agda M Simpson, Anatoly Rivlin, Larry SimpsonAbstract:Abstract A 3′ terminal RNA uridylyltransferase was purified from mitochondria of Leishmania tarentolae and the gene cloned and expressed from this species and from Trypanosoma brucei . The enzyme is specific for 3′ U-addition in the presence of Mg 2+ . TUTase is present in vivo in at least two stable configurations: one contains a ∼500 kDa TUTase oligomer and the other a ∼700 kDa TUTase complex. Anti-TUTase antiserum specifically coprecipitates a small portion of the p45 and p50 RNA ligases and approximately 40% of the guide RNAs. Inhibition of TUTase expression in procyclic T. brucei by RNAi downregulates RNA editing and appears to affect Parasite Viability.
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Trypanosome Mitochondrial 3′ Terminal Uridylyl Transferase (TUTase): The Key Enzyme in U-Insertion/Deletion RNA Editing
Cell, 2002Co-Authors: Ruslan Aphasizhev, Sandro Sbicego, Marian Peris, Seiheon Jang, Inna Aphasizheva, Agda M Simpson, Anatoly Rivlin, Larry SimpsonAbstract:Abstract A 3′ terminal RNA uridylyltransferase was purified from mitochondria of Leishmania tarentolae and the gene cloned and expressed from this species and from Trypanosoma brucei . The enzyme is specific for 3′ U-addition in the presence of Mg 2+ . TUTase is present in vivo in at least two stable configurations: one contains a ∼500 kDa TUTase oligomer and the other a ∼700 kDa TUTase complex. Anti-TUTase antiserum specifically coprecipitates a small portion of the p45 and p50 RNA ligases and approximately 40% of the guide RNAs. Inhibition of TUTase expression in procyclic T. brucei by RNAi downregulates RNA editing and appears to affect Parasite Viability.
