Stevens RC

References (7)

Title : Biomarkers of organophosphorus (OP) exposures in humans - Marsillach_2011_Neurotoxicol_32_656
Author(s) : Marsillach J , Richter RJ , Kim JH , Stevens RC , Maccoss MJ , Tomazela D , Suzuki SM , Schopfer LM , Lockridge O , Furlong CE
Ref : Neurotoxicology , 32 :656 , 2011
Abstract : There are ongoing events where aircraft engine lubricant containing tricresyl phosphates (TCPs) contaminates aircraft cabins. Some individuals have experienced tremors or other neurological symptoms that may last for many months following exposures. Mass spectrometric (MS) protocols are being developed to determine the percentage of "biomarker proteins" that are modified by such exposures, specifically on active site serines. Both plasma butyrylcholinesterase (BChE) and red cell acylpeptide hydrolase (APH) are readily inhibited by 2-(ortho-cresyl)-4H-1,3,2-benzodioxaphosphoran-2-one (CBDP) or phenyl saligenin cyclic phosphate (PSP) and have the potential to provide information about the level of exposure of an individual. We have developed immunomagnetic bead-based single-step purification protocols for both BChE and APH and have characterized the active site serine adducts of BChE by MS.
ESTHER : Marsillach_2011_Neurotoxicol_32_656
PubMedSearch : Marsillach_2011_Neurotoxicol_32_656
PubMedID: 21767566

Title : Engineering human PON1 in an E. coli expression system - Suzuki_2010_Adv.Exp.Med.Biol_660_37
Author(s) : Suzuki SM , Stevens RC , Richter RJ , Cole TB , Park S , Otto TC , Cerasoli DM , Lenz DE , Furlong CE
Ref : Advances in Experimental Medicine & Biology , 660 :37 , 2010
Abstract : Expression and purification of recombinant human paraoxonase-1 (rHuPON1) from bacterial systems have proven elusive. Most systems for successful production of recombinant PON1 have relied on either eukaryotic expression in baculovirus or prokaryotic expression of synthetic, gene-shuffled rabbit-mouse-human PON1 hybrid molecules. We review here methods and protocols for the production of pure, native rHuPON1 using an E. coli expression system followed by conventional column chromatographic purification. The resulting rHuPON1 is stable, active, and capable of protecting PON1 knockout mice (PON1(-/-)) from exposure to high levels of the organophosphorus (OP) compound diazoxon. Bacterially-derived rHuPON1 can be produced in large quantities and lacks the glycosylation of eukaryotic systems that produces immunogenic complications when used as a therapeutic. The rHuPON1 should be useful for treating insecticide OP exposures and reducing risks of other diseases resulting from low PON1 status. The ease of mutagenesis in bacterial systems will also allow for the generation and screening of rHuPON1 variants with enhanced catalytic efficiencies against nerve agents and other OP compounds.
ESTHER : Suzuki_2010_Adv.Exp.Med.Biol_660_37
PubMedSearch : Suzuki_2010_Adv.Exp.Med.Biol_660_37
PubMedID: 20221869

Title : Human PON1, a biomarker of risk of disease and exposure - Furlong_2010_Chem.Biol.Interact_187_355
Author(s) : Furlong CE , Suzuki SM , Stevens RC , Marsillach J , Richter RJ , Jarvik GP , Checkoway H , Samii A , Costa LG , Griffith A , Roberts JW , Yearout D , Zabetian CP
Ref : Chemico-Biological Interactions , 187 :355 , 2010
Abstract : Human paraoxonase 1 (PON1) is a high-density lipoprotein (HDL)-associated serum enzyme that exhibits a broad substrate specificity. In addition to protecting against exposure to some organophosphorus (OP) pesticides by hydrolyzing their toxic oxon metabolites, PON1 is important in protecting against vascular disease by metabolizing oxidized lipids. Recently, PON1 has also been shown to play a role in inactivating the quorum sensing factor N-(3-oxododecanoyl)-l-homoserine lactone (3OC12-HSL) of Pseudomonas aeruginosa. Native, untagged engineered recombinant human PON1 (rHuPON1) expressed in Escherichia coli and purified by conventional column chromatographic purification is stable, active, and capable of protecting PON1 knockout mice (PON1(-/-)) from exposure to high levels of the OP compound diazoxon. The bacterially derived rHuPON1 can be produced in large quantities and lacks the glycosylation of eukaryotic systems that can produce immunogenic complications when inappropriately glycosylated recombinant proteins are used as therapeutics. Previous studies have shown that the determination of PON1 status, which reveals both PON1(192) functional genotype and serum enzyme activity level, is required for a meaningful evaluation of PON1's role in risk of disease or exposure. We have developed a new two-substrate assay/analysis protocol that provides PON1 status without use of toxic OP substrates, allowing for use of this protocol in non-specialized laboratories. Factors were also determined for inter-converting rates of hydrolysis of different substrates. PON1 status also plays an important role in revealing changes in HDL-associated PON1 activities in male patients with Parkinson disease (PD). Immunolocalization studies of PONs 1, 2 and 3 in nearly all mouse tissues suggest that the functions of PONs 1 and 3 extend beyond the plasma and the HDL particle.
ESTHER : Furlong_2010_Chem.Biol.Interact_187_355
PubMedSearch : Furlong_2010_Chem.Biol.Interact_187_355
PubMedID: 20338154

Title : Identification and characterization of biomarkers of organophosphorus exposures in humans - Kim_2010_Adv.Exp.Med.Biol_660_61
Author(s) : Kim JH , Stevens RC , Maccoss MJ , Goodlett DR , Scherl A , Richter RJ , Suzuki SM , Furlong CE
Ref : Advances in Experimental Medicine & Biology , 660 :61 , 2010
Abstract : Over 1 billion pounds of organophosphorus (OP) chemicals are manufactured worldwide each year, including 70 million pounds of pesticides sprayed in the US. Current methods to monitor environmental and occupational exposures to OPs such as chlorpyrifos (CPS) have limitations, including low specificity and sensitivity, and short time windows for detection. Biomarkers for the OP tricresyl phosphate (TCP), which can contaminate bleed air from jet engines and cause an occupational exposure of commercial airline pilots, crewmembers and passengers, have not been identified. The aim of our work has been to identify, purify, and characterize new biomarkers of OP exposure. Butyrylcholinesterase (BChE) inhibition has been a standard for monitoring OP exposure. By identifying and characterizing molecular biomarkers with longer half-lives, we should be able to clinically detect TCP and OP insecticide exposure after longer durations of time than are currently possible. Acylpeptide hydrolase (APH) is a red blood cell (RBC) cytosolic serine proteinase that removes N-acetylated amino acids from peptides and cleaves oxidized proteins. Due to its properties, it is an excellent candidate for a biomarker of exposure. We have been able to purify APH and detect inhibition by both CPS and metabolites of TCP. The 120-day lifetime of the RBC offers a much longer window for detecting exposure. The OP-modified serine conjugate in the active site tryptic peptide has been characterized by mass spectrometry. This research uses functional proteomics and enzyme activities to identify and characterize useful biomarkers of neurotoxic environmental and occupational OP exposures.
ESTHER : Kim_2010_Adv.Exp.Med.Biol_660_61
PubMedSearch : Kim_2010_Adv.Exp.Med.Biol_660_61
PubMedID: 20221871

Title : Crystal structure of an alpha\/beta serine hydrolase (YDR428C) from Saccharomyces cerevisiae at 1.85 A resolution -
Author(s) : Arndt JW , Schwarzenbacher R , Page R , Abdubek P , Ambing E , Biorac T , Canaves JM , Chiu HJ , Dai X , Deacon AM , DiDonato M , Elsliger MA , Godzik A , Grittini C , Grzechnik SK , Hale J , Hampton E , Han GW , Haugen J , Hornsby M , Klock HE , Koesema E , Kreusch A , Kuhn P , Jaroszewski L , Lesley SA , Levin I , McMullan D , McPhillips TM , Miller MD , Morse A , Moy K , Nigoghossian E , Ouyang J , Peti WS , Quijano K , Reyes R , Sims E , Spraggon G , Stevens RC , van den Bedem H , Velasquez J , Vincent J , von Delft F , Wang X , West B , White A , Wolf G , Xu Q , Zagnitko O , Hodgson KO , Wooley J , Wilson IA
Ref : Proteins , 58 :755 , 2005
PubMedID: 15624212
Gene_locus related to this paper: yeast-YDR428C

Title : A mass spectrometry plate reader: monitoring enzyme activity and inhibition with a Desorption\/Ionization on Silicon (DIOS) platform - Shen_2004_Chembiochem_5_921
Author(s) : Shen Z , Go EP , Gamez A , Apon JV , Fokin V , Greig M , Ventura M , Crowell JE , Blixt O , Paulson JC , Stevens RC , Finn MG , Siuzdak G
Ref : Chembiochem , 5 :921 , 2004
Abstract : A surface-based laser desorption/ionization mass spectrometry assay that makes use of Desorption/Ionization on Silicon Mass Spectrometry (DIOS-MS) has been developed to monitor enzyme activity and enzyme inhibition. DIOS-MS has been used to characterize inhibitors from a library and then to monitor their activity against selected enzyme targets, including proteases, glycotransferase, and acetylcholinesterase. An automated DIOS-MS system was also used as a high-throughput screen for the activity of novel enzymes and enzyme inhibitors. On two different commercially available instruments, a sampling rate of up to 38 inhibitors per minute was accomplished, with thousands of inhibitors being monitored. The ease of applying mass spectrometry toward developing enzyme assays and the speed of surface-based assays such as DIOS for monitoring inhibitor effectiveness and enzyme activity makes it attractive for a broad range of screening applications.
ESTHER : Shen_2004_Chembiochem_5_921
PubMedSearch : Shen_2004_Chembiochem_5_921
PubMedID: 15239048

Title : A structural view of evolutionary divergence - Spiller_1999_Proc.Natl.Acad.Sci.U.S.A_96_12305
Author(s) : Spiller B , Gershenson A , Arnold FH , Stevens RC
Ref : Proceedings of the National Academy of Sciences of the United States of America , 96 :12305 , 1999
Abstract : Two directed evolution experiments on p-nitrobenzyl esterase yielded one enzyme with a 100-fold increased activity in aqueous-organic solvents and another with a 17 degrees C increase in thermostability. Structures of the wild type and its organophilic and thermophilic counterparts are presented at resolutions of 1.5 A, 1.6 A, and 2.0 A, respectively. These structures identify groups of interacting mutations and demonstrate how directed evolution can traverse complex fitness landscapes. Early-generation mutations stabilize flexible loops not visible in the wild-type structure and set the stage for further beneficial mutations in later generations. The mutations exert their influence on the esterase structure over large distances, in a manner that would be difficult to predict. The loops with the largest structural changes generally are not the sites of mutations. Similarly, none of the seven amino acid substitutions in the organophile are in the active site, even though the enzyme experiences significant changes in the organization of this site. In addition to reduction of surface loop flexibility, thermostability in the evolved esterase results from altered core packing, helix stabilization, and the acquisition of surface salt bridges, in agreement with other comparative studies of mesophilic and thermophilic enzymes. Crystallographic analysis of the wild type and its evolved counterparts reveals networks of mutations that collectively reorganize the active site. Interestingly, the changes that led to diversity within the alpha/beta hydrolase enzyme family and the reorganization seen in this study result from main-chain movements.
ESTHER : Spiller_1999_Proc.Natl.Acad.Sci.U.S.A_96_12305
PubMedSearch : Spiller_1999_Proc.Natl.Acad.Sci.U.S.A_96_12305
PubMedID: 10535917
Gene_locus related to this paper: bacsu-pnbae