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USE OF CERIUM OXIDE NANOPARTICLES FOR PROTECTION AGAINSTRADIATION-INDUCED CELL DEATH
Title
USE OF CERIUM OXIDE NANOPARTICLES FOR PROTECTION AGAINSTRADIATION-INDUCED CELL DEATH.
Creator
Colon, Jimmie, Kolattukudy, Pappachan, University of Central Florida
Abstract / Description
The ability of engineered cerium oxide nanoparticles to confer radioprotection was examined. Rat astrocytes were treated with cerium oxide nanoparticles to a final concentration of 10 nanomolar, irradiated with a single 10 Gy dose of ionizing radiation and cell death was evaluated by propidium iodine uptake at 24 and 48 hours after radiation insult. Treatment of rat astrocytes with nanoceria resulted in an approximate 3-fold decrease in radiation induced death. These results suggest that the...
Show moreThe ability of engineered cerium oxide nanoparticles to confer radioprotection was examined. Rat astrocytes were treated with cerium oxide nanoparticles to a final concentration of 10 nanomolar, irradiated with a single 10 Gy dose of ionizing radiation and cell death was evaluated by propidium iodine uptake at 24 and 48 hours after radiation insult. Treatment of rat astrocytes with nanoceria resulted in an approximate 3-fold decrease in radiation induced death. These results suggest that the nanoceria are conferring protection from radiation induced cell death. Further experiments with human cells were conducted. Human normal and tumor cells (MCF-7 and CRL8798) were treated with the same dosage of cerium oxide nanoparticles, irradiated and evaluated for cell survival. Treatment of normal cells (MCF-7) conferred nearly 99% protection from radiation-induced cell death while the same concentration of nanoceria showed almost no protection in tumor cells (CRL8798). TUNEL analysis results of similarly treated cells demonstrated that nanoceria reduced radiation-induced cell death by 3-fold in normal breast cells but not in MCF-7 tumor cell lines when cultured under the same conditions. We concluded that cerium oxide nanoparticles confer radioprotection in a normal human breast line (CRL 8798) but not in a human breast tumor line (MCF-7). It is hoped that the outcome of this study will guide future endeavors toward a better elucidation of the molecular pathways involved in the protection of cells with nanoceria against radiation-induced cell death, as well as the minimization of the bystander effect in radiation therapy.
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Date Issued
2006
Identifier
CFE0001048, ucf:46823
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0001048
ZINC-FINGER PROTEIN MCPIP IN CELL DEATH AND DIFFERENTIATION
Title
ZINC-FINGER PROTEIN MCPIP IN CELL DEATH AND DIFFERENTIATION.
Creator
Younce, Craig, Kolattukudy, Pappachan, University of Central Florida
Abstract / Description
Monocyte chemotactic protein-1 (MCP-1) plays a critical role in the development of cardiovascular diseases. How MCP-1 contributes to the development of heart disease is not understood. We present evidence that MCP-1 causes death in cardiac myoblasts, H9c2 by inducing oxidative stress, ER stress and autophagy via a novel Zn-finger protein, MCP-1 induced protein (MCPIP). MCPIP expression caused cell death and knockdown of MCPIP, attenuated MCP-1 induced cell death. Expression of MCPIP resulted...
Show moreMonocyte chemotactic protein-1 (MCP-1) plays a critical role in the development of cardiovascular diseases. How MCP-1 contributes to the development of heart disease is not understood. We present evidence that MCP-1 causes death in cardiac myoblasts, H9c2 by inducing oxidative stress, ER stress and autophagy via a novel Zn-finger protein, MCP-1 induced protein (MCPIP). MCPIP expression caused cell death and knockdown of MCPIP, attenuated MCP-1 induced cell death. Expression of MCPIP resulted in induction of iNOS and production of reactive oxygen (ROS). It caused induction of NADPH oxidase subunit phox47 and its translocation to the cytoplasmic membrane. Oxidative stress led to the induction of ER stress markers HSP40, PDI, GRP78 and IRE1α. ER stress lead to autophagy as indicated by beclin-1 induction, cleavage of LC3 to LCII and autophagolysosome formation. Here, MCPIP-induced processes lead to apoptosis as indicated by caspase 3 activation and TUNEL assay. This cell death involved caspase 2 and caspase 12 as specific inhibitors of these caspases prevented MCPIP-induced cell death. Inhibitors of oxidative stress inhibited ER stress, and cell death. Specific inhibitors of ER stress inhibited autophagy and cell death. Inhibition of autophagy inhibited cell death. Microarray analysis showed that MCPIP expression caused induction of a variety of genes known to be involved in cell death. MCPIP caused activation of JNK and p38 and induction of p53 and PUMA. These results collectively suggest that MCPIP induces ROS/RNS production that causes ER stress which leads to autophagy and apoptosis through caspase 2/12 and IRE1α –JNK/p38-p53-PUMA pathway. These results provide the first molecular insights into the mechanism by which elevated MCP-1 levels associated with chronic inflammation may contribute to the development of heart failure. A role for inflammation and MCP-1 in obesity and diabetes has been implicated. Adipogenesis is a key process involved in obesity and associated diseases such as type 2 diabetes. This process involves temporally regulated genes controlled by a set of transcription factors, C/EBPβ, C/EBPδ, C/EBPα, and PPARγ. Currently PPARγ is considered the master regulator of adipogenesis as no known factor can induce adipogenesis without PPARγ. We present evidence that a novel Zn-finger protein, MCPIP, can induce adipogenesis without PPARγ. Classical adipogenesis-inducing medium induces MCP-1 production and MCPIP expression in 3T3-L1 cells before the induction of the C/EBP family of transcription factors and PPARγ. Knockdown of MCPIP prevents their expression and adipogenesis. Treatment of 3T3-L1 cells with MCP-1 or forced expression of MCPIP induces expression of C/EBPβ, C/EBPδ, C/EBPα, PPARγ and adipogenesis without any other inducer. Forced expression of MCPIP induces adipogenesis in PPARγ-/- fibroblasts. Thus, MCPIP is a newly identified master controller that can induce adipogenesis without PPARγ. Heart failure is a major cause of death in diabetic patients. Hyperglycemia is a major factor associated with diabetes that causes cardiomyocyte apoptosis that leads to diabetic cardiomyopathy. Cardiomyoycte apoptosis is a key event involved in the pathophysiological progression of diabetic cardiomyopathy. We have recently found that in ischemic hearts, MCP-1 can induce the zinc-finger protein, MCP-1 induced protein (MCPIP) that causes cardiomyocyte apoptosis. Although there is evidence that inflammation may play a role in diabetic cardiomyopathy, the underlying mechanisms are poorly understood. In this study, we show that treatment of H9c2 cardiomyoblasts and Neonatal Rat Ventricular Myocytes (NRVM) with 28mmol/L glucose concentration results in the induction of both transcript and protein levels of MCP-1 and MCPIP. Inhibition of MCP-1 interaction with CCR2 via specific antibody or with the G-coupled receptor inhibitors propagermanium and pertussis toxin attenuated glucose-induced cell death. Knockdown of MCPIP with specific siRNA yielded similar results. Treatment of cells with 28mmol/L glucose resulted in increased ROS production and phox47 activation. Knockdown of MCPIP attenuated these effects. The increased ROS production observed in H9c2 cardiomyoblasts and NRVM's resulted in increased ER stress proteins GRP78 and PDI. Knockdown of MCPIP attenuated expression of both GRP78 and PDI. Inhibition of ER stress with TUDC and 4'PBA prevented high glucose-induced cell death death. Treatment of cells with 28mmol/l glucose resulted in autophagy as determined by an increase in expression of beclin-1 and through increased cleavage of LC3I to LC3II. Knockdown of MCPIP attenuated expression of beclin-1 and prevented cleavage of LC3. Addition of the autophagy inhibitors 3'methyladenine and LY294002 attenuated high glucose-induced H9c2 cardiomyoblast death. We conclude that high glucose-induced H9c2 cardiomyoblast death is mediated via MCP-1 induction of MCPIP that results in ROS that leads to ER stress that causes autophagy and eventual apoptosis.
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Date Issued
2009
Identifier
CFE0002888, ucf:48027
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0002888
IDENTIFICATION OF GENES ENCODING ACYL-COA REDUCTASES AND ALDEHYDE REDUCTASES IN MYCOBACTERIAL GENOME BY CHARACTERIZATION OF THE REDUCTASES EXPRESSED IN E.COLI.
Title
IDENTIFICATION OF GENES ENCODING ACYL-COA REDUCTASES AND ALDEHYDE REDUCTASES IN MYCOBACTERIAL GENOME BY CHARACTERIZATION OF THE REDUCTASES EXPRESSED IN E.COLI.
Creator
Singh, Harminder, Kolattukudy, Pappachan, University of Central Florida
Abstract / Description
Mycobacterium tuberculosis has been long known to produce wax esters. However, the enzymes involved in their biosynthesis have not been identified. Here we report the identification of Rv3391 and Rv1543 as genes that encode fatty acyl-CoA reductases and Rv1544 as one that encodes an aldehyde reductase. When expressed in E.coli, the products of Rv3391 and Rv1543 catalyzed reduction of fatty acyl-CoA to fatty alcohol with the corresponding aldehyde as an intermediate with an optimal pH of 7.0....
Show moreMycobacterium tuberculosis has been long known to produce wax esters. However, the enzymes involved in their biosynthesis have not been identified. Here we report the identification of Rv3391 and Rv1543 as genes that encode fatty acyl-CoA reductases and Rv1544 as one that encodes an aldehyde reductase. When expressed in E.coli, the products of Rv3391 and Rv1543 catalyzed reduction of fatty acyl-CoA to fatty alcohol with the corresponding aldehyde as an intermediate with an optimal pH of 7.0. Both enzymes showed a strong preference for NADPH over NADH as a reductant. Apparent Km for NADPH was 38 M for Rv3391 product and 202 M for Rv1543 product. Both enzymes reduced saturated fatty acyl-CoA such as palmitoyl-CoA and stearyl-CoA but showed a preference for oleoyl-CoA. Apparent Km for oleoyl-CoA was 13 M for Rv3391 product and 7 M for Rv1543 product. The Rv1544 product catalyzed fatty aldehyde reduction to fatty alcohol but not acyl-CoA reduction. The optimal pH for aldehyde reduction was 8.0. This aldehyde reductase showed a strong preference for NADPH with an apparent Km of 83 M. All three reductases were inhibited by SH directed reagents.
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Date Issued
2007
Identifier
CFE0001903, ucf:47495
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0001903
Expression of Trichoderma reesei (&)#223;-Mannanase in Tobacco Chloroplasts and its Utilization in Lignocellulosic Woody Biomass Hydrolysis
Title
Expression of Trichoderma reesei (&)#223;-Mannanase in Tobacco Chloroplasts and its Utilization in Lignocellulosic Woody Biomass Hydrolysis.
Creator
Agrawal, Pankaj, Daniell, Henry, Kolattukudy, Pappachan, Self, William, University of Central Florida
Abstract / Description
Lignocellulosic ethanol offers a promising alternative to conventional fossil fuels. One among the major limitations in the lignocellulosic biomass hydrolysis is unavailability of efficient and environmentally biomass degrading technologies. Plant-based production of these enzymes on large scale offers a cost effective solution. Cellulases, hemicellulases including mannanases and other accessory enzymes are required for conversion of lignocellulosic biomass into fermentable sugars. ?...
Show moreLignocellulosic ethanol offers a promising alternative to conventional fossil fuels. One among the major limitations in the lignocellulosic biomass hydrolysis is unavailability of efficient and environmentally biomass degrading technologies. Plant-based production of these enzymes on large scale offers a cost effective solution. Cellulases, hemicellulases including mannanases and other accessory enzymes are required for conversion of lignocellulosic biomass into fermentable sugars. ?-mannanase catalyzes endo-hydrolysis of the mannan backbone, a major constituent of woody biomass. In this study, man1 gene encoding ?-mannanase was isolated from Trichoderma reesei and expressed via the chloroplast genome. PCR and Southern hybridization analysis confirmed the site-specific transgene integration into the tobacco chloroplast genomes and homoplasmy. Transplastomic plants were fertile and set viable seeds. Germination of seeds showed inheritance of transgenes into the progeny without Mendelian segregation. Expression of the endo-?-mannanase gene for the first time in plants facilitated its characterization for use in enhanced lignocellulosic biomass hydrolysis. Gel diffusion assay for endo-?-mannanase showed the zone of clearance confirming functionality of chloroplast-derived mannanase. Endo-?-mannanase expression levels reached up to 25 units per gram of leaf (fresh weight). Chloroplast-derived mannanase had higher temperature stability (40 (&)deg;C to 70 (&)deg;C) and wider pH optima (pH 3.0 to 7.0) than E.coli enzyme extracts. Plant crude extracts showed 6-7 fold higher enzyme activity than E.coli extracts due to the formation of disulfide bonds in chloroplasts, thereby facilitating their direct utilization in enzyme cocktails without any purification. Chloroplast-derived mannanase when added to the enzyme cocktail containing a combination of different enzymes yielded 20% more glucose equivalents from pinewood than the cocktail without mannanase. Our results demonstrate that chloroplast-derived mannanase is an important component of enzymatic cocktail for woody biomass hydrolysis and should provide a cost-effective solution for its diverse applications in the paper, oil, pharmaceutical, coffee and detergent industries.
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Date Issued
2011
Identifier
CFE0004467, ucf:49309
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0004467
Expression of Lipase from Mycobacterium tuberculosis in Nicotiana tobacum and Lactuca sativa Chloroplasts
Title
Expression of Lipase from Mycobacterium tuberculosis in Nicotiana tobacum and Lactuca sativa Chloroplasts.
Creator
Lloyd, Bethany, Daniell, Henry, Kolattukudy, Pappachan, Self, William, University of Central Florida
Abstract / Description
Tuberculosis (TB), caused by the bacterium Mycobacterium tuberculosis (M. tuberculosis), is a global threat and the leading cause of death among individuals infected with HIV. TB treatment requires multi-drug cocktails, due to the increasing rates of drug resistance of the bacterium. With multi-drug cocktails, strains have been documented to be resistant to all major drugs in the fight against TB. Since the strains are drug resistant, it calls for an increasing need for vaccine and treatment...
Show moreTuberculosis (TB), caused by the bacterium Mycobacterium tuberculosis (M. tuberculosis), is a global threat and the leading cause of death among individuals infected with HIV. TB treatment requires multi-drug cocktails, due to the increasing rates of drug resistance of the bacterium. With multi-drug cocktails, strains have been documented to be resistant to all major drugs in the fight against TB. Since the strains are drug resistant, it calls for an increasing need for vaccine and treatment development for the purpose of preventing and managing the disease. The most widely distributed vaccine against TB is Bacillus Calmette-Gue(&)#180;rin (BCG). Apart from being ineffective in certain individuals, BCG offers only a limited timeframe of protection, is unable to serve as a booster for extending this timeframe and due to the intradermal route of administration requires costly refrigeration and syringes.LipY protein, a M. tuberculosis cell wall lipase, may play a potential role as not only a drug target but a potential vaccine antigen. LipY is known to be up-regulated during both active infection and dormancy. In a previous study, sera from TB patients had shown an IgG and IgM response against it. In this study transplastomic Lactuca sativa and Nicotiana tabacum plants were generated by transforming the chloroplasts through the particle delivery system with pLsDv-LipY and pLD-LipY vectors respectively. The vectors were flanked by the native trnI and trnA gene sequence to facilitate homologous recombination into the chloroplast genome. The vector also contained the 16S rRNA promoter, the selectable marker gene, aadA for specitinomycin resistance, the rbcL untranslated region, the LsPpsbA (PpsbA in N. tabacum) promoter, and LsTpsbA (tpsbA in N. tabacum) untranslated region. Site specific integration of the LipY gene into the chloroplast genome was confirmed by PCR. Homoplasmy of transplastomic plants was confirmed by Southern blot analysis. These plants showed normal growth and were fertile, producing seeds. Once germinated, these seeds did not show Mendelian segregation of the transgene. Immunoblot analysis was performed to analyze the expression of the LipY protein. A 40kDa protein was produced in E.coli, and a 25kDa protein was produced in chloroplasts; a cleaved product in chloroplasts is still valuable as an antigen for vaccine production. Future studies will include testing this chloroplast derived antigen in animal models for vaccine development. ?
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Date Issued
2012
Identifier
CFE0004502, ucf:49289
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0004502
Molecular Mechanisms involved in inflammatory angiogenesis induced by monocyte chemotactic protein induced protein-1 (MCPIP1)
Title
Molecular Mechanisms involved in inflammatory angiogenesis induced by monocyte chemotactic protein induced protein-1 (MCPIP1).
Creator
Roy, Arpita, Kolattukudy, Pappachan, Ebert, Steven, Parthasarathy, Sampath, Self, William, University of Central Florida
Abstract / Description
Major diseases such as cardiovascular diseases, diabetes, obesity and tumor growth are known to involve inflammatory angiogenesis. MCP-induced protein 1 (MCPIP1) encoded by ZC3H12A gene, was reported to promote angiogenesis and is addressed in my dissertation as MCPIP. The mechanism/s involved in the angiogenic differentiation induced by MCPIP was however unknown. The aim of this study was to bridge this gap in our knowledge and delineate the molecular mechanisms and sequential processes...
Show moreMajor diseases such as cardiovascular diseases, diabetes, obesity and tumor growth are known to involve inflammatory angiogenesis. MCP-induced protein 1 (MCPIP1) encoded by ZC3H12A gene, was reported to promote angiogenesis and is addressed in my dissertation as MCPIP. The mechanism/s involved in the angiogenic differentiation induced by MCPIP was however unknown. The aim of this study was to bridge this gap in our knowledge and delineate the molecular mechanisms and sequential processes involved in angiogenesis mediated via MCPIP. To determine if angiogenesis induced by inflammatory cytokines, TNF-?, IL-1? and IL-8 is mediated via induction of MCPIP, knockdown of MCPIP by its specific siRNA, in human umbilical vein endothelial cells was performed. Oxidative stress, ER stress and autophagy are known to be involved in mediating inflammation. We hypothesized that MCPIP-induced angiogenic differentiation is mediated via induction of oxidative stress, ER stress and autophagy. Chemical inhibitors and specific gene knockdown approach were used to inhibit each process postulated. Oxidative stress was inhibited by apocynin or cerium oxide nanoparticles or knockdown of NADPH oxidase subunit, phox47. Endoplasmic reticulum (ER) stress was blocked by tauroursodeoxycholate or knockdown of ER stress signaling protein IRE-1 and autophagy was inhibited by the use of 3?methyl adenine, or LY 294002 or by specific knockdown of beclin1. Matrigel assay was used as an in vitro tool to assay angiogenic differentiation. Inhibition of each step inhibited the subsequent steps postulated. The results reveal that angiogenesis induced by inflammatory agents is mediated via sequential induction of MCPIP that causes oxidative and nitrosative stress resulting in ER stress leading to autophagy required for angiogenesis. MCPIP has deubiquitinase and anti-dicer RNase activities. If and how the dual enzymatic activities of MCPIP mediate angiogenesis was unknown. Our results showed that hypoxia-induced angiogenesis is mediated via MCPIP. MCPIP deubiquitinated ubiquitinated hypoxia-inducible factor (HIF-1?) and the stabilized HIF-1? entered the nucleus to promote the transcription of its target genes, cyclooxygenase-2 and vascular endothelial growth factor causing the activation of p38 MAP kinase involved in angiogenesis. MCPIP expression promoted angiogenesis by inhibition of thrombospondin-1 synthesis via induction of silent information regulator (SIRT)-1 and/or via suppression of VEG-inhibitor levels caused by inhibition of NF-?B activation. MCPIP inhibited the production of the anti-angiogenic microRNAs (miR)-20b and miR-34a that repress the translation of HIF-1? and SIRT-1, respectively. Cells expressing the RNase-dead mutant of MCPIP, D141N, that had lost the ability to induce angiogenesis had deubiquitinase activity but did not inhibit the production of miR-20b and miR-34a. Mimetics of miR-20b and miR-34a inhibited MCPIP-induced angiogenesis. These results show for the first time that both deubiquitinase and anti-dicer RNase activities of MCPIP are involved in inflammatory angiogenesis. Results from our study delineate key processes that could be potential targets for therapeutic intervention against inflammatory angiogenesis.
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Date Issued
2012
Identifier
CFE0004793, ucf:49760
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0004793