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Featured researches published by John C. Whitin.


Archives of Biochemistry and Biophysics | 1987

Purification and characterization of human plasma glutathione peroxidase: A selenoglycoprotein distinct from the known cellular enzyme☆

K Takahashi; Nelly E. Avissar; John C. Whitin; Harvey J. Cohen

Glutathione peroxidase (GSHPx), (glutathione:H2O2 oxidoreductase, EC 1.11.1.9) was purified to homogeneity from human plasma. This resulted in a 6800-fold purification of the enzyme with a 2.8% yield. The purification process involved ammonium sulfate fractionation, DEAE-cellulose batch and column chromatographies, hydroxyapatite, and Sephadex G-200 and DEAE-Sephadex A-25 chromatographies. The major peak on DEAE-Sephadex A-25 column chromatography was found to be homogeneous on polyacrylamide gel electrophoresis in the presence or absence of sodium dodecyl sulfate (SDS). Relative mobility in nondenaturing polyacrylamide gel electrophoresis at pH 8.2 was 0.5 for the purified enzyme as detected by both protein staining and enzyme activity compared with 0.38 for erythrocyte GSHPx. The molecular weight of the plasma enzyme as determined by gel filtration was found to be approximately 100,000. SDS-gel electrophoresis of the plasma enzyme gave a subunit molecular weight of approximately 23,000. This suggests that the plasma enzyme exists as a tetramer in its native state, similar to that seen for the erythrocyte enzyme, but with slightly different mobility on SDS-gel electrophoresis. Plasma GSHPx, like the erythrocyte enzyme, was found to contain approximately four atoms of selenium per mole of protein. Utilizing iodinated concanavalin A, it was found that plasma GSHPx, but not the erythrocyte GSPx, is a glycoprotein. Purified plasma enzyme catalyzes both the reduction of tertiary butyl hydroperoxide and hydrogen peroxide. The apparent Km of plasma GSHPx for GSH is 5.3 mM and for tertiary butyl hydroperoxide it is 0.57 mM. Copper, mercury, and zinc strongly inhibit the enzyme activity of plasma GSHPx. Rabbit antibodies directed against the human erythrocyte GSHPx do not precipitate the enzyme activity of the purified plasma enzyme. Radioimmunoassay utilizing erythrocyte GSHPx and anti-erythrocyte GSHPx antibodies showed that less than 0.13% of the antigenically detectable protein is found in the purified GSHPx from plasma.


Journal of Clinical Oncology | 1993

Comparative pharmacokinetic studies of three asparaginase preparations.

Barbara L. Asselin; John C. Whitin; David J. Coppola; I P Rupp; Stephen E. Sallan; Harvey J. Cohen

PURPOSE As part of pharmacologic studies of asparaginase (ASNase), we determined the half-life of ASNase activity and protein, and the effect of dose, repeated doses, different drug preparations, and hypersensitivity reactions on the half-life (t1/2) of serum ASNase activity. PATIENTS AND METHODS We measured ASNase activity (spectrophotometric assay) in serum samples obtained from patients with acute lymphoblastic leukemia (ALL) at various times during their therapy with intramuscular ASNase. ASNase protein was measured by enzyme-linked immunoadsorbent assay (ELISA). RESULTS Studies following the initial dose of Escherichia coli-derived ASNase demonstrated no difference in apparent t1/2 following 25,000 IU/m2 versus 2,500 IU/m2 (1.24 v 1.35 days, P = .2). The apparent t1/2s following maintenance doses of E coli ASNase (middle dose t1/2, 1.28 days, or last dose t1/2, 1.14 days) showed no difference when compared with the initial dose of ASNase (P = .3 to .9). There was no significant difference between the apparent t1/2s of ASNase activity and ASNase protein (n = 8, P = .2 to .6). The serum t1/2 was 0.65 and 5.73 days for patients receiving Erwinia or polyethylene glycol (PEG)-modified E coli ASNase, respectively, as the induction dose. ASNase activity was undetectable in sera of four patients studied in the week following an anaphylactic reaction to E coli ASNase and the t1/2 was significantly shorter in five patients with a history of allergic reaction to E coli ASNase who were studied following a dose of PEG ASNase, (t1/2, 1.80 days). CONCLUSION We conclude that (1) the apparent t1/2 of ASNase is dependent on enzyme preparation used, but is not affected by dose or by repeated use; (2) the apparent t1/2 of E coli ASNase as a protein is the same as the apparent t1/2 of enzymatic activity; and (3) patients who have had a hypersensitivity reaction to E coli ASNase have a decreased apparent t1/2 with both E coli and PEG ASNase.


Journal of Immunology | 2003

Intracellular mediators of granulysin-induced cell death.

Satoshi Okada; Qing Li; John C. Whitin; Carol Clayberger; Alan M. Krensky

Granulysin, a molecule present in the granules of CTL and NK cells, is cytolytic against microbes and tumors. Granulysin induces apoptosis of mammalian cells by damaging mitochondria and causing the release of cytochrome c and apoptosis-inducing factor, resulting in DNA fragmentation. Here we show that Ca2+ and K+ channels as well as reactive oxygen species are involved in granulysin-mediated Jurkat cell death. The Ca2+ channel blockers, nickel and econazole, and the K+ channel blockers, tetraethylammonium chloride, apamin, and charybdotoxin, inhibit the granulysin-induced increase in intracellular Ca2+ ([Ca2+]i), the decrease in intracellular K+, and apoptosis. Thapsigargin, which releases Ca2+ from the endoplasmic reticulum, prevents a subsequent granulysin-induced increase in [Ca2+]i in Jurkat cells, indicating that the initial increase in [Ca2+]i is from intracellular stores. The rise in [Ca2+]i precedes a decrease in intracellular K+, and elevated extracellular K+ prevents granulysin-mediated cell death. In granulysin-treated cells, electron transport is uncoupled, and reactive oxygen species are generated. Finally, an increase in intracellular glutathione protects target cells from granulysin-induced lysis, indicating the importance of the redox state in granulysin-mediated cell death.


The New England Journal of Medicine | 1981

A Variant of Chronic Granulomatous Disease: Deficient Oxidative Metabolism Due to a Low-Affinity NADPH Oxidase

P. Daniel Lew; Frederick S. Southwick; Thomas P. Stossel; John C. Whitin; Elizabeth R. Simons; Harvey J. Cohen

OUR defense against bacterial infections depends in part on the action of phagocytic leukocytes, which encounter and kill potentially pathogenic microorganisms. A severe reduction in the number or ...


American Journal of Physiology-renal Physiology | 2010

Extracellular glutathione peroxidase (Gpx3) binds specifically to basement membranes of mouse renal cortex tubule cells

Gary E. Olson; John C. Whitin; Kristina E. Hill; Virginia P. Winfrey; Amy K. Motley; Lori M. Austin; Jacqualyn Deal; Harvey J. Cohen; Raymond F. Burk

Glutathione peroxidase-3 (Gpx3), also known as plasma or extracellular glutathione peroxidase, is a selenoprotein secreted primarily by kidney proximal convoluted tubule cells. In this study Gpx3(-/-) mice have been produced and immunocytochemical techniques have been developed to investigate Gpx3 metabolism. Gpx3(-/-) mice maintained the same whole-body content and urinary excretion of selenium as did Gpx3(+/+) mice. They tolerated selenium deficiency without observable ill effects. The simultaneous knockout of Gpx3 and selenoprotein P revealed that these two selenoproteins account for >97% of plasma selenium. Immunocytochemistry experiments demonstrated that Gpx3 binds selectively, both in vivo and in vitro, to basement membranes of renal cortical proximal and distal convoluted tubules. Based on calculations using selenium content, the kidney pool of Gpx3 is over twice as large as the plasma pool. These data indicate that Gpx3 does not serve in the regulation of selenium metabolism. The specific binding of a large pool of Gpx3 to basement membranes in the kidney cortex strongly suggests a need for glutathione peroxidase activity in the cortical peritubular space.


Molecular Reproduction and Development | 1998

Developmental expression of extracellular glutathione peroxidase suggests antioxidant roles in deciduum, visceral yolk sac, and skin

Paul D. Kingsley; John C. Whitin; Harvey J. Cohen; James Palis

Extracellular glutathione peroxidase (EGPx) is a secreted selenium‐dependent enzyme that reduces hydroperoxides and organic hydroperoxides. Selenium deficiency in females is associated with infertility and spontaneous abortion, suggesting a role for selenium‐requiring proteins during embryonic development. To gain insight into functions of EGPx in vivo, we determined sites of murine EGPx synthesis by in situ hybridization during embryogenesis and in adult tissues. At E7.5 of development, high EGPx expression was found in the maternally derived deciduum, with lower levels of accumulation in the embryonic visceral endoderm. At E9.5, the major sites of expression were the yolk sac endoderm and heart musculature. By E16.5, EGPx mRNA expression persisted in yolk sac endoderm but also accumulated significantly in atrially derived myocytes, ossification centers, adipose tissue, intestinal epithelium, and in a ventral‐to‐dorsal gradient in developing skin. Glutathione peroxidase activity due to EGPx protein was identified in the fluids surrounding the developing mouse embryo at midgestation. The expression of EGPx in tissues at the maternal‐fetal interface—deciduum, visceral yolk sac, and skin—suggests that EGPx may serve to protect the embryo from oxidant damage. In adult mice, we identified the S1 segment of the kidney proximal tubules as the primary site of EGPx mRNA accumulation, with lower EGPx levels in atrial cardiac muscle, intestine, skin, and adipose tissue. These findings suggest that EGPx may serve a wider antioxident role than previously recognized in the interstitium of multiple localized tissues, particularly those associated with the active transport of lipids. Mol. Reprod. Dev. 49:343–355, 1998.


Analytical Chemistry | 2008

Capture of Phosphopeptides Using α-Zirconium Phosphate Nanoplatelets

Songyun Xu; John C. Whitin; Tom To-Sang Yu; Houjiang Zhou; Dazhi Sun; Hung-Jue Sue; Hanfa Zou; Harvey J. Cohen; Richard N. Zare

Alpha-zirconium phosphate nanoplatelets (alpha-ZrPN) were studied as a binding agent for phosphopeptides. Nanoplatelets of alpha-zirconium phosphate were incubated overnight with zirconium oxychloride, followed by centrifugation, and washed twice with water followed by an aqueous solution of 80% acetonitrile to form the binding agent. Alpha-ZrPN were able specifically to capture phosphoserine-containing peptides from a tryptic digest of a complex peptide mixture in which its abundance was only 0.05%. Alpha-ZrPN also bound peptides containing phosphothreonine and phosphotyrosine. The limit of detection for phosphopeptides is approximately 2 fmol, based on using matrix-assisted laser desorption/ionization mass spectrometry. Alpha-ZrPN were applied for the analysis of tryptic digests of mouse liver and leukemia cell phosphoproteomes and succeeded in identifying 158 phosphopeptides (209 phosphorylation sites) from 101 phosphoproteins in mouse liver lysate and 78 phosphopeptides (104 phosphorylation sites) from 59 phosphoproteins in leukemia cell extract. For these two tryptic digests, the alpha-ZrPN approach is able to capture more phosphopeptides than that obtained from TiO2 particles or from Fe(3+)-IMAC beads, but each method is able to bind some phosphopeptides that the others do not.


BMC Medicine | 2011

A diagnostic algorithm combining clinical and molecular data distinguishes Kawasaki disease from other febrile illnesses

Xuefeng B. Ling; Kenneth Lau; John T. Kanegaye; Zheng Pan; Sihua Peng; Jun Ji; Gigi Liu; Yuichiro Sato; Tom To-Sang Yu; John C. Whitin; James Schilling; Jane C. Burns; Harvey J. Cohen

BackgroundKawasaki disease is an acute vasculitis of infants and young children that is recognized through a constellation of clinical signs that can mimic other benign conditions of childhood. The etiology remains unknown and there is no specific laboratory-based test to identify patients with Kawasaki disease. Treatment to prevent the complication of coronary artery aneurysms is most effective if administered early in the course of the illness. We sought to develop a diagnostic algorithm to help clinicians distinguish Kawasaki disease patients from febrile controls to allow timely initiation of treatment.MethodsUrine peptidome profiling and whole blood cell type-specific gene expression analyses were integrated with clinical multivariate analysis to improve differentiation of Kawasaki disease subjects from febrile controls.ResultsComparative analyses of multidimensional protein identification using 23 pooled Kawasaki disease and 23 pooled febrile control urine peptide samples revealed 139 candidate markers, of which 13 were confirmed (area under the receiver operating characteristic curve (ROC AUC 0.919)) in an independent cohort of 30 Kawasaki disease and 30 febrile control urine peptidomes. Cell type-specific analysis of microarrays (csSAM) on 26 Kawasaki disease and 13 febrile control whole blood samples revealed a 32-lymphocyte-specific-gene panel (ROC AUC 0.969). The integration of the urine/blood based biomarker panels and a multivariate analysis of 7 clinical parameters (ROC AUC 0.803) effectively stratified 441 Kawasaki disease and 342 febrile control subjects to diagnose Kawasaki disease.ConclusionsA hybrid approach using a multi-step diagnostic algorithm integrating both clinical and molecular findings was successful in differentiating children with acute Kawasaki disease from febrile controls.


Pediatric Research | 2002

Increased expression of extracellular glutathione peroxidase in mice with dextran sodium sulfate-induced experimental colitis.

Doris M. Tham; John C. Whitin; Harvey J. Cohen

Extracellular glutathione peroxidase (E-GPx) is a selenoenzyme that reduces hydrogen peroxide and organic peroxides. All plasma glutathione peroxidase (GPx) activity in humans is attributable to E-GPx. The gastrointestinal (GI) tract also synthesizes and secretes E-GPx into the extracellular milieu. Endogenously generated oxidants have been implicated in inflammatory bowel disease (IBD). We evaluated E-GPx levels in a mouse model of IBD using dextran sodium sulfate (DSS). Histologic lesions of the lower GI tract consisted of multifocal areas of mucosal erosion denuded of epithelial cells, reduction in goblet cells, dilated crypts, crypt collapse, submucosal edema, and transmural distribution of mixed inflammatory infiltrates. On d 7, plasma GPx activity in the DSS group increased by 61% compared with the control group (p < 0.05). Western blot analysis demonstrated a 64% increase in E-GPx protein in the plasma of the DSS group after 7 d of treatment (p < 0.01). As the major source of plasma GPx is the kidney, we determined whether the increase in plasma GPx activity and protein was caused by a change in E-GPx synthesis by the kidney. After 3 and 7 d of DSS treatment, E-GPx mRNA levels, relative to glyceraldehyde-3-phosphate dehydrogenase, increased in the kidney (p < 0.05) without a concomitant increase in cellular GPx mRNA on d 7. These results suggest that the inflammatory injury in the intestine elicits an increase in E-GPx in the plasma that is associated with an increase in E-GPx mRNA in the kidney. This implies that renal production of E-GPx may be sensitive to insults distal to the kidney.


Pediatric Research | 1999

Increase in extracellular glutathione peroxidase in plasma and lungs of mice exposed to hyperoxia

Kenneth K. Kim; John C. Whitin; Nataliya M Sukhova; Harvey J. Cohen

Extracellular glutathione peroxidase (E-GPx) is a selenium-dependent enzyme that can reduce hydrogen peroxide and phospholipid hydroperoxides. E-GPx is found in plasma and extracellular fluids such as bronchoalveolar lavage fluid. Because lung is one of the tissues that is capable of synthesizing and secreting E-GPx, the effect of exposure to hyperoxia on E-GPx in plasma and lung were studied in an injury model of hyperoxia exposure in adult mice. Exposure to 100% oxygen for 72 h resulted in an increase of 55% in plasma GPx activity and an increase of 50% in the amount of E-GPx protein in the plasma. Exposure to hyperoxia was also associated with an increase in the amount of E-GPx protein in lungs. The 7-fold increase in the amount of E-GPx protein in lungs was not due to plasma contamination of lungs from mice exposed to hyperoxia. E-GPx in the lung is calculated to account for 10% of lung GPx activity in control mice. However, E-GPx is calculated to account for 45% of lung GPx activity in the lungs of mice exposed to hyperoxia for 72 h. Further studies are needed to determine whether the increase in lung E-GPx is due to changes in translation or stability of E-GPx. The role of E-GPx in protecting the lung from oxidative damage warrants further study.

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K Takahashi

University of Rochester

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