Expression of NADPH Oxidase (NOX) 5 in Rabbit Corneal Stromal Cells 英文参考文献.docVIP

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Expression of NADPH Oxidase (NOX) 5 in Rabbit Corneal Stromal Cells 英文参考文献.doc

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Expression of NADPH Oxidase (NOX) 5 in Rabbit Corneal Stromal Cells 英文参考文献

ExpressionofNADPHOxidase(NOX)5inRabbitCorneal StromalCells FarhanRizvi1*,TomHeimann1,WilliamJ.O’Brien1,2* 1Departments of Ophthalmology, Medical College of Wisconsin, Milwaukee, Wisconsin, United States of America, 2Department of Microbiology/Molecular Genetics, MedicalCollegeofWisconsin,Milwaukee,Wisconsin,UnitedStatesofAmerica Abstract Purpose: To determine whether NOX 5 is expressed in rabbit corneal stromal cells (RCSC). NADPH oxidases (NOXes) are enzymes that preferentially use NADPH as a substrate and generate superoxide. Several isoforms of NOXes function as multi-proteincomplexeswhileNOX5andDUOXsdonotrequiretheaccessoryproteinsfortheiractivityandpossesscalcium bindingEFhands. Methods:HumanNOX5primerswereusedtoamplifytherabbitNOX5byRT-PCR.Amplifiedproductwassequencedto confirmitsidentity.TheproteinencodedbytheNOX5wasidentifiedbywesternblotanalysis.NOX5siRNAwasusedto reduce transcript, protein, and calcium stimulated activity. In silico analyses were performed to establish the putative structure,functions,andevolutionofrabbitNOX5. Results: NOX activity was measured in RCSC with NADPH rather than NADH as a substrate. RT-PCR with NOX5 primers amplified288bpproductusingRCSCcDNA,which,whensequenced,confirmeditsidentitytohumanNOX5mRNA.This sequencewasusedtopredicttherabbit(Oryctolaguscuniculus)NOX5gene.NOX5siRNAreducedamountsofNOX5mRNA inRCSCandreducedionomycinstimulatedsuperoxideproduction.Aproteinofabout65to70kDaencodedbytheNOX5 was detected by western blot analysis. In silico analysis predicted a putative rabbit NOX5 protein containing 801 amino acids.Motifsearchespredictedthepresenceofatleast3putativeEF-handsinN-terminusandaNOXdomaininCterminal region. Conclusions: The data document that the NOX5 gene was expressed in cells of lagomorphs unlike rodents, making the rabbitaninterestingmodeltostudyNOX5functions.TheactivityoftherabbitNOX5wascalciumstimulated,atraitofNOX5 ingeneral.NOX5mayalsoprovetobeausefulgeneticmarkerforstudyingthetaxonomicpositionoflagomorphs