John Whitin
Senior Research Scientist, Pediatrics
Current Role at Stanford
Senior Research Scientist
Education & Certifications
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Ph.D., Boston University, Biochemistry
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B.A., Tufts University, Chemistry
https://orcid.org/0000-0002-6229-4628All Publications
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Global metabolomics revealed deviations from the metabolic aging clock in colorectal cancer patients.
Theranostics
2024; 14 (4): 1602-1614
Abstract
Background: Markers of aging hold promise in the context of colorectal cancer (CRC) care. Utilizing high-resolution metabolomic profiling, we can unveil distinctive age-related patterns that have the potential to predict early CRC development. Our study aims to unearth a panel of aging markers and delve into the metabolomic alterations associated with aging and CRC. Methods: We assembled a serum cohort comprising 5,649 individuals, consisting of 3,002 healthy volunteers, 715 patients diagnosed with colorectal advanced precancerous lesions (APL), and 1,932 CRC patients, to perform a comprehensive metabolomic analysis. Results: We successfully identified unique age-associated patterns across 42 metabolic pathways. Moreover, we established a metabolic aging clock, comprising 9 key metabolites, using an elastic net regularized regression model that accurately estimates chronological age. Notably, we observed significant chronological disparities among the healthy population, APL patients, and CRC patients. By combining the analysis of circulative carcinoembryonic antigen levels with the categorization of individuals into the "hypo" metabolic aging subgroup, our blood test demonstrates the ability to detect APL and CRC with positive predictive values of 68.4% (64.3%, 72.2%) and 21.4% (17.8%, 25.9%), respectively. Conclusions: This innovative approach utilizing our metabolic aging clock holds significant promise for accurately assessing biological age and enhancing our capacity to detect APL and CRC.
View details for DOI 10.7150/thno.87303
View details for PubMedID 38389840
View details for PubMedCentralID PMC10879879
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Correction: Exploring the feasibility of using long-term stored newborn dried blood spots to identify metabolic features for congenital heart disease screening.
Biomarker research
2023; 11 (1): 101
View details for DOI 10.1186/s40364-023-00546-w
View details for PubMedID 37993911
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Exploring the feasibility of using long-term stored newborn dried blood spots to identify metabolic features for congenital heart disease screening.
Biomarker research
2023; 11 (1): 97
Abstract
Congenital heart disease (CHD) represents a significant contributor to both morbidity and mortality in neonates and children. There's currently no analogous dried blood spot (DBS) screening for CHD immediately after birth. This study was set to assess the feasibility of using DBS to identify reliable metabolite biomarkers with clinical relevance, with the aim to screen and classify CHD utilizing the DBS. We assembled a cohort of DBS datasets from the California Department of Public Health (CDPH) Biobank, encompassing both normal controls and three pre-defined CHD categories. A DBS-based quantitative metabolomics method was developed using liquid chromatography with tandem mass spectrometry (LC-MS/MS). We conducted a correlation analysis comparing the absolute quantitated metabolite concentration in DBS against the CDPH NBS records to verify the reliability of metabolic profiling. For hydrophilic and hydrophobic metabolites, we executed significant pathway and metabolite analyses respectively. Logistic and LightGBM models were established to aid in CHD discrimination and classification. Consistent and reliable quantification of metabolites were demonstrated in DBS samples stored for up to 15 years. We discerned dysregulated metabolic pathways in CHD patients, including deviations in lipid and energy metabolism, as well as oxidative stress pathways. Furthermore, we identified three metabolites and twelve metabolites as potential biomarkers for CHD assessment and subtypes classifying. This study is the first to confirm the feasibility of validating metabolite profiling results using long-term stored DBS samples. Our findings highlight the potential clinical applications of our DBS-based methods for CHD screening and subtype classification.
View details for DOI 10.1186/s40364-023-00536-y
View details for PubMedID 37957758
View details for PubMedCentralID PMC10644604
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Development of a Urine Metabolomics Biomarker-Based Prediction Model for Preeclampsia during Early Pregnancy.
Metabolites
2023; 13 (6)
Abstract
Preeclampsia (PE) is a condition that poses a significant risk of maternal mortality and multiple organ failure during pregnancy. Early prediction of PE can enable timely surveillance and interventions, such as low-dose aspirin administration. In this study, conducted at Stanford Health Care, we examined a cohort of 60 pregnant women and collected 478 urine samples between gestational weeks 8 and 20 for comprehensive metabolomic profiling. By employing liquid chromatography mass spectrometry (LCMS/MS), we identified the structures of seven out of 26 metabolomics biomarkers detected. Utilizing the XGBoost algorithm, we developed a predictive model based on these seven metabolomics biomarkers to identify individuals at risk of developing PE. The performance of the model was evaluated using 10-fold cross-validation, yielding an area under the receiver operating characteristic curve of 0.856. Our findings suggest that measuring urinary metabolomics biomarkers offers a noninvasive approach to assess the risk of PE prior to its onset.
View details for DOI 10.3390/metabo13060715
View details for PubMedID 37367874
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Single center blind testing of a US multi-center validated diagnostic algorithm for Kawasaki disease in Taiwan.
Frontiers in immunology
2022; 13: 1031387
Abstract
Kawasaki disease (KD) is the leading cause of acquired heart disease in children. The major challenge in KD diagnosis is that it shares clinical signs with other childhood febrile control (FC) subjects. We sought to determine if our algorithmic approach applied to a Taiwan cohort.A single center (Chang Gung Memorial Hospital in Taiwan) cohort of patients suspected with acute KD were prospectively enrolled by local KD specialists for KD analysis. Our previously single-center developed computer-based two-step algorithm was further tested by a five-center validation in US. This first blinded multi-center trial validated our approach, with sufficient sensitivity and positive predictive value, to identify most patients with KD diagnosed at centers across the US. This study involved 418 KDs and 259 FCs from the Chang Gung Memorial Hospital in Taiwan.Our diagnostic algorithm retained sensitivity (379 of 418; 90.7%), specificity (223 of 259; 86.1%), PPV (379 of 409; 92.7%), and NPV (223 of 247; 90.3%) comparable to previous US 2016 single center and US 2020 fiver center results. Only 4.7% (15 of 418) of KD and 2.3% (6 of 259) of FC patients were identified as indeterminate. The algorithm identified 18 of 50 (36%) KD patients who presented 2 or 3 principal criteria. Of 418 KD patients, 157 were infants younger than one year and 89.2% (140 of 157) were classified correctly. Of the 44 patients with KD who had coronary artery abnormalities, our diagnostic algorithm correctly identified 43 (97.7%) including all patients with dilated coronary artery but one who found to resolve in 8 weeks.This work demonstrates the applicability of our algorithmic approach and diagnostic portability in Taiwan.
View details for DOI 10.3389/fimmu.2022.1031387
View details for PubMedID 36263040
View details for PubMedCentralID PMC9575935
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Early-pregnancy prediction of risk for pre-eclampsia using maternal blood leptin/ceramide ratio: discovery and confirmation.
BMJ open
2021; 11 (11): e050963
Abstract
OBJECTIVE: This study aimed to develop a blood test for the prediction of pre-eclampsia (PE) early in gestation. We hypothesised that the longitudinal measurements of circulating adipokines and sphingolipids in maternal serum over the course of pregnancy could identify novel prognostic biomarkers that are predictive of impending event of PE early in gestation.STUDY DESIGN: Retrospective discovery and longitudinal confirmation.SETTING: Maternity units from two US hospitals.PARTICIPANTS: Six previously published studies of placental tissue (78 PE and 95 non-PE) were compiled for genomic discovery, maternal sera from 15 women (7 non-PE and 8 PE) enrolled at ProMedDx were used for sphingolipidomic discovery, and maternal sera from 40 women (20 non-PE and 20 PE) enrolled at Stanford University were used for longitudinal observation.OUTCOME MEASURES: Biomarker candidates from discovery were longitudinally confirmed and compared in parallel to the ratio of placental growth factor (PlGF) and soluble fms-like tyrosine kinase (sFlt-1) using the same cohort. The datasets were generated by enzyme-linked immunosorbent and liquid chromatography-tandem mass spectrometric assays.RESULTS: Our discovery integrating genomic and sphingolipidomic analysis identified leptin (Lep) and ceramide (Cer) (d18:1/25:0) as novel biomarkers for early gestational assessment of PE. Our longitudinal observation revealed a marked elevation of Lep/Cer (d18:1/25:0) ratio in maternal serum at a median of 23 weeks' gestation among women with impending PE as compared with women with uncomplicated pregnancy. The Lep/Cer (d18:1/25:0) ratio significantly outperformed the established sFlt-1/PlGF ratio in predicting impending event of PE with superior sensitivity (85% vs 20%) and area under curve (0.92 vs 0.52) from 5 to 25 weeks of gestation.CONCLUSIONS: Our study demonstrated the longitudinal measurement of maternal Lep/Cer (d18:1/25:0) ratio allows the non-invasive assessment of PE to identify pregnancy at high risk in early gestation, outperforming the established sFlt-1/PlGF ratio test.
View details for DOI 10.1136/bmjopen-2021-050963
View details for PubMedID 34824115
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Maternal metabolic profiling to assess fetal gestational age and predict preterm delivery: a two-centre retrospective cohort study in the US.
BMJ open
2020; 10 (12): e040647
Abstract
OBJECTIVES: The aim of this study was to develop a single blood test that could determine gestational age and estimate the risk of preterm birth by measuring serum metabolites. We hypothesised that serial metabolic modelling of serum analytes throughout pregnancy could be used to describe fetal gestational age and project preterm birth with a high degree of precision.STUDY DESIGN: A retrospective cohort study.SETTING: Two medical centres from the USA.PARTICIPANTS: Thirty-six patients (20 full-term, 16 preterm) enrolled at Stanford University were used to develop gestational age and preterm birth risk algorithms, 22 patients (9 full-term, 13 preterm) enrolled at the University of Alabama were used to validate the algorithms.OUTCOME MEASURES: Maternal blood was collected serially throughout pregnancy. Metabolic datasets were generated using mass spectrometry.RESULTS: A model to determine gestational age was developed (R2=0.98) and validated (R2=0.81). 66.7% of the estimates fell within ±1week of ultrasound results during model validation. Significant disruptions from full-term pregnancy metabolic patterns were observed in preterm pregnancies (R2=-0.68). A separate algorithm to predict preterm birth was developed using a set of 10 metabolic pathways that resulted in an area under the curve of 0.96 and 0.92, a sensitivity of 0.88 and 0.86, and a specificity of 0.96 and 0.92 during development and validation testing, respectively.CONCLUSIONS: In this study, metabolic profiling was used to develop and test a model for determining gestational age during full-term pregnancy progression, and to determine risk of preterm birth. With additional patient validation studies, these algorithms may be used to identify at-risk pregnancies prompting alterations in clinical care, and to gain biological insights into the pathophysiology of preterm birth. Metabolic pathway-based pregnancy modelling is a novel modality for investigation and clinical application development.
View details for DOI 10.1136/bmjopen-2020-040647
View details for PubMedID 33268420
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High-throughput quantitation of serological ceramides/dihydroceramides by LC/MS/MS: Pregnancy baseline biomarkers and potential metabolic messengers.
Journal of pharmaceutical and biomedical analysis
2020; 192: 113639
Abstract
Ceramides and dihydroceramides are sphingolipids that present in abundance at the cellular membrane of eukaryotes. Although their metabolic dysregulation has been implicated in many diseases, our knowledge about circulating ceramide changes during the pregnancy remains limited. In this study, we present the development and validation of a high-throughput liquid chromatography-tandem mass spectrometric method for simultaneous quantification of 16 ceramides and 10 dihydroceramides in human serum within 5 min. by using stable isotope-labeled ceramides as internal standards. This method employs a protein precipitation method for high throughput sample preparation, reverse phase isocratic elusion for chromatographic separation, and Multiple Reaction Monitoring for mass spectrometric detection. To qualify for clinical applications, our assay has been validated against the FDA guidelines for Lower Limit of Quantitation (1 nM), linearity (R2>0.99), precision (imprecision<15 %), accuracy (inaccuracy<15 %), extraction recovery (>90 %), stability (>85 %), and carryover (<0.01 %). With enhanced sensitivity and specificity from this method, we have, for the first time, determined the serological levels of ceramides and dihydroceramides to reveal unique temporal gestational patterns. Our approach could have value in providing insights into disorders of pregnancy.
View details for DOI 10.1016/j.jpba.2020.113639
View details for PubMedID 33017796
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Multicentre validation of a computer-based tool for differentiation of acute Kawasaki disease from clinically similar febrile illnesses.
Archives of disease in childhood
2020
Abstract
The clinical features of Kawasaki disease (KD) overlap with those of other paediatric febrile illnesses. A missed or delayed diagnosis increases the risk of coronary artery damage. Our computer algorithm for KD and febrile illness differentiation had a sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of 94.8%, 70.8%, 93.7% and 98.3%, respectively, in a single-centre validation study. We sought to determine the performance of this algorithm with febrile children from multiple institutions across the USA.We used our previously published 18-variable panel that includes illness day, the five KD clinical criteria and readily available laboratory values. We applied this two-step algorithm using a linear discriminant analysis-based clinical model followed by a random forest-based algorithm to a cohort of 1059 acute KD and 282 febrile control patients from five children's hospitals across the USA.The algorithm correctly classified 970 of 1059 patients with KD and 163 of 282 febrile controls resulting in a sensitivity of 91.6%, specificity of 57.8% and PPV and NPV of 95.4% and 93.1%, respectively. The algorithm also correctly identified 218 of the 232 KD patients (94.0%) with abnormal echocardiograms.The expectation is that the predictive accuracy of the algorithm will be reduced in a real-world setting in which patients with KD are rare and febrile controls are common. However, the results of the current analysis suggest that this algorithm warrants a prospective, multicentre study to evaluate its potential utility as a physician support tool.
View details for DOI 10.1136/archdischild-2019-317980
View details for PubMedID 32139365
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Kinetics of SARS-CoV-2 positivity of infected and recovered patients from a single center.
Scientific reports
2020; 10 (1): 18629
Abstract
Recurrence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) positive detection in infected but recovered individuals has been reported. Patients who have recovered from coronavirus disease 2019 (COVID-19) could profoundly impact the health care system. We sought to define the kinetics and relevance of PCR-positive recurrence during recovery from acute COVID-19 to better understand risks for prolonged infectivity and reinfection. A series of 414 patients with confirmed SARS-Cov-2 infection, at The Second Affiliated Hospital of Southern University of Science and Technology in Shenzhen, China from January 11 to April 23, 2020. Statistical analyses were performed of the clinical, laboratory, radiologic image, medical treatment, and clinical course of admission/quarantine/readmission data, and a recurrence predictive algorithm was developed. 16.7% recovered patients with PCR positive recurring one to three times, despite being in strict quarantine. Younger patients with mild pulmonary respiratory syndrome had higher risk of PCR positivity recurrence. The recurrence prediction model had an area under the ROC curve of 0.786. This case series provides characteristics of patients with recurrent SARS-CoV-2 positivity. Use of a prediction algorithm may identify patients at high risk of recurrent SARS-CoV-2 positivity and help to establish protocols for health policy.
View details for DOI 10.1038/s41598-020-75629-x
View details for PubMedID 33122706
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A proteomic clock for malignant gliomas: The role of the environment in tumorigenesis at the presymptomatic stage.
PloS one
2019; 14 (10): e0223558
Abstract
Malignant gliomas remain incurable with a poor prognosis despite of aggressive treatment. We have been studying the development of brain tumors in a glioma rat model, where rats develop brain tumors after prenatal exposure to ethylnitrosourea (ENU), and there is a sizable interval between when the first pathological changes are noted and tumors become detectable with MRI. Our aim to define a molecular timeline through proteomic profiling of the cerebrospinal fluid (CSF) such that brain tumor commitment can be revealed earlier than at the presymptomatic stage. A comparative proteomic approach was applied to profile CSF collected serially either before, at and after the time MRI becomes positive. Elastic net (EN) based models were developed to infer the timeline of normal or tumor development respectively, mirroring a chronology of precisely timed, "clocked", adaptations. These CSF changes were later quantified by longitudinal entropy analyses of the EN predictive metric. False discovery rates (FDR) were computed to control the expected proportion of the EN models that are due to multiple hypothesis testing. Our ENU rat brain tumor dating EN model indicated that protein content in CSF is programmed even before tumor MRI detection. The findings of the precisely timed CSF tumor microenvironment changes at presymptomatic stages, deviation from the normal development timeline, may provide the groundwork for the understanding of adaptation of the brain environment in tumorigenesis to devise effective brain tumor management strategies.
View details for DOI 10.1371/journal.pone.0223558
View details for PubMedID 31600288
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Phase I/IIa Trial of Atorvastatin in Patients with Acute Kawasaki Disease with Coronary Artery Aneurysm.
The Journal of pediatrics
2019
Abstract
To determine the safety, tolerability, pharmacokinetics, and immunomodulatory effects of a 6-week course of atorvastatin in patients with acute Kawasaki disease with coronary artery (CA) aneurysm (CAA).This was a Phase I/IIa 2-center dose-escalation study of atorvastatin (0.125-0.75 mg/kg/day) in 34 patients with Kawasaki disease (aged 2-17 years) with echocardiographic evidence of CAA. We measured levels of the brain metabolite 24(S)-hydroxycholesterol (24-OHC), serum lipids, acute-phase reactants, liver enzymes, and creatine phosphokinase; peripheral blood mononuclear cell populations; and CA internal diameter normalized for body surface area before atorvastatin treatment and at 2 and 6 weeks after initiation of atorvastatin treatment.A 6-week course of up to 0.75 mg/kg/day of atorvastatin was well tolerated by the 34 subjects (median age, 5.3 years; IQR, 2.6-6.4 years), with no serious adverse events attributable to the study drug. The areas under the curve for atorvastatin and its metabolite were larger in the study subjects compared with those reported in adults, suggesting a slower rate of metabolism in children. The 24-OHC levels were similar between the atorvastatin-treated subjects and matched controls.Atorvastatin was safe and well tolerated in our cohort of children with acute Kawasaki disease and CAA. A Phase III efficacy trial is warranted in this patient population, which may benefit from the known anti-inflammatory and immunomodulatory effects of this drug.
View details for DOI 10.1016/j.jpeds.2019.07.064
View details for PubMedID 31561960
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STUDIES ON BILIRUBIN (BR) METABOLISM IN THE BRAIN
WILEY. 2017: 14–15
View details for Web of Science ID 000405213500019
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Unique Molecular Patterns Uncovered in Kawasaki Disease Patients with Elevated Serum Gamma Glutamyl Transferase Levels: Implications for Intravenous Immunoglobulin Responsiveness
PLOS ONE
2016; 11 (12)
Abstract
Resistance to intravenous immunoglobulin (IVIG) occurs in 10-20% of patients with Kawasaki disease (KD). The risk of resistance is about two-fold higher in patients with elevated gamma glutamyl transferase (GGT) levels. We sought to understand the biological mechanisms underlying IVIG resistance in patients with elevated GGT levels.We explored the association between elevated GGT levels and IVIG-resistance with a cohort of 686 KD patients (Cohort I). Gene expression data from 130 children with acute KD (Cohort II) were analyzed using the R square statistic and false discovery analysis to identify genes that were differentially represented in patients with elevated GGT levels with regard to IVIG responsiveness. Two additional KD cohorts (Cohort III and IV) were used to test the hypothesis that sialylation and GGT may be involved in IVIG resistance through neutrophil apoptosis.Thirty-six genes were identified that significantly explained the variations of both GGT levels and IVIG responsiveness in KD patients. After Bonferroni correction, significant associations with IVIG resistance persisted for 12 out of 36 genes among patients with elevated GGT levels and none among patients with normal GGT levels. With the discovery of ST6GALNAC3, a sialyltransferase, as the most differentially expressed gene, we hypothesized that sialylation and GGT are involved in IVIG resistance through neutrophil apoptosis. We then confirmed that in Cohort III and IV there was significantly less reduction in neutrophil count in IVIG non-responders.Gene expression analyses combining molecular and clinical datasets support the hypotheses that: (1) neutrophil apoptosis induced by IVIG may be a mechanism of action of IVIG in KD; (2) changes in sialylation and GGT level in KD patients may contribute synergistically to IVIG resistance through blocking IVIG-induced neutrophil apoptosis. These findings have implications for understanding the mechanism of action in IVIG resistance, and possibly for development of novel therapeutics.
View details for DOI 10.1371/journal.pone.0167434
View details for Web of Science ID 000392853100008
View details for PubMedID 28002448
View details for PubMedCentralID PMC5176264
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A Novel Truncated Form of Serum Amyloid A in Kawasaki Disease
PLOS ONE
2016; 11 (6)
Abstract
Kawasaki disease (KD) is an acute vasculitis in children that can cause coronary artery abnormalities. Its diagnosis is challenging, and many cytokines, chemokines, acute phase reactants, and growth factors have failed evaluation as specific biomarkers to distinguish KD from other febrile illnesses. We performed protein profiling, comparing plasma from children with KD with febrile control (FC) subjects to determine if there were specific proteins or peptides that could distinguish the two clinical states.Plasma from three independent cohorts from the blood of 68 KD and 61 FC subjects was fractionated by anion exchange chromatography, followed by surface-enhanced laser desorption ionization (SELDI) mass spectrometry of the fractions. The mass spectra of KD and FC plasma samples were analyzed for peaks that were statistically significantly different.A mass spectrometry peak with a mass of 7,860 Da had high intensity in acute KD subjects compared to subacute KD (p = 0.0003) and FC (p = 7.9 x 10-10) subjects. We identified this peak as a novel truncated form of serum amyloid A with N-terminal at Lys-34 of the circulating form and validated its identity using a hybrid mass spectrum immunoassay technique. The truncated form of serum amyloid A was present in plasma of KD subjects when blood was collected in tubes containing protease inhibitors. This peak disappeared when the patients were examined after their symptoms resolved. Intensities of this peptide did not correlate with KD-associated laboratory values or with other mass spectrum peaks from the plasma of these KD subjects.Using SELDI mass spectrometry, we have discovered a novel truncated form of serum amyloid A that is elevated in the plasma of KD when compared with FC subjects. Future studies will evaluate its relevance as a diagnostic biomarker and its potential role in the pathophysiology of KD.
View details for DOI 10.1371/journal.pone.0157024
View details for Web of Science ID 000377560200029
View details for PubMedID 27271757
View details for PubMedCentralID PMC4894573
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Cerebrospinal fluid protein dynamic driver network: At the crossroads of brain tumorigenesis
METHODS
2015; 83: 36-43
Abstract
To get a better understanding of the ongoing in situ environmental changes preceding the brain tumorigenesis, we assessed cerebrospinal fluid (CSF) proteome profile changes in a glioma rat model in which brain tumor invariably developed after a single in utero exposure to the neurocarcinogen ethylnitrosourea (ENU). Computationally, the CSF proteome profile dynamics during the tumorigenesis can be modeled as non-smooth or even abrupt state changes. Such brain tumor environment transition analysis, correlating the CSF composition changes with the development of early cellular hyperplasia, can reveal the pathogenesis process at network level during a time before the image detection of the tumors. In our controlled rat model study, matched ENU- and saline-exposed rats' CSF proteomics changes were quantified at approximately 30, 60, 90, 120, 150days of age (P30, P60, P90, P120, P150). We applied our transition-based network entropy (TNE) method to compute the CSF proteome changes in the ENU rat model and test the hypothesis of the critical transition state prior to impending hyperplasia. Our analysis identified a dynamic driver network (DDN) of CSF proteins related with the emerging tumorigenesis progressing from the non-hyperplasia state. The DDN associated leading network CSF proteins can allow the early detection of such dynamics before the catastrophic shift to the clear clinical landmarks in gliomas. Future characterization of the critical transition state (P60) during the brain tumor progression may reveal the underlying pathophysiology to device novel therapeutics preventing tumor formation. More detailed method and information are accessible through our website at http://translationalmedicine.stanford.edu.
View details for DOI 10.1016/j.ymeth.2015.05.004
View details for Web of Science ID 000358755100005
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Cerebrospinal fluid protein dynamic driver network: At the crossroads of brain tumorigenesis.
Methods (San Diego, Calif.)
2015; 83: 36-43
Abstract
To get a better understanding of the ongoing in situ environmental changes preceding the brain tumorigenesis, we assessed cerebrospinal fluid (CSF) proteome profile changes in a glioma rat model in which brain tumor invariably developed after a single in utero exposure to the neurocarcinogen ethylnitrosourea (ENU). Computationally, the CSF proteome profile dynamics during the tumorigenesis can be modeled as non-smooth or even abrupt state changes. Such brain tumor environment transition analysis, correlating the CSF composition changes with the development of early cellular hyperplasia, can reveal the pathogenesis process at network level during a time before the image detection of the tumors. In our controlled rat model study, matched ENU- and saline-exposed rats' CSF proteomics changes were quantified at approximately 30, 60, 90, 120, 150days of age (P30, P60, P90, P120, P150). We applied our transition-based network entropy (TNE) method to compute the CSF proteome changes in the ENU rat model and test the hypothesis of the critical transition state prior to impending hyperplasia. Our analysis identified a dynamic driver network (DDN) of CSF proteins related with the emerging tumorigenesis progressing from the non-hyperplasia state. The DDN associated leading network CSF proteins can allow the early detection of such dynamics before the catastrophic shift to the clear clinical landmarks in gliomas. Future characterization of the critical transition state (P60) during the brain tumor progression may reveal the underlying pathophysiology to device novel therapeutics preventing tumor formation. More detailed method and information are accessible through our website at http://translationalmedicine.stanford.edu.
View details for DOI 10.1016/j.ymeth.2015.05.004
View details for PubMedID 25982164
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CSF protein dynamic driver network: at the crossroads of brain tumorigenesis
IEEE. 2014
View details for Web of Science ID 000377412300186
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Identifying technical aliases in SELDI mass spectra of complex mixtures of proteins.
BMC research notes
2013; 6: 358-?
Abstract
Biomarker discovery datasets created using mass spectrum protein profiling of complex mixtures of proteins contain many peaks that represent the same protein with different charge states. Correlated variables such as these can confound the statistical analyses of proteomic data. Previously we developed an algorithm that clustered mass spectrum peaks that were biologically or technically correlated. Here we demonstrate an algorithm that clusters correlated technical aliases only.In this paper, we propose a preprocessing algorithm that can be used for grouping technical aliases in mass spectrometry protein profiling data. The stringency of the variance allowed for clustering is customizable, thereby affecting the number of peaks that are clustered. Subsequent analysis of the clusters, instead of individual peaks, helps reduce difficulties associated with technically-correlated data, and can aid more efficient biomarker identification.This software can be used to pre-process and thereby decrease the complexity of protein profiling proteomics data, thus simplifying the subsequent analysis of biomarkers by decreasing the number of tests. The software is also a practical tool for identifying which features to investigate further by purification, identification and confirmation.
View details for DOI 10.1186/1756-0500-6-358
View details for PubMedID 24010718
View details for PubMedCentralID PMC3847147
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Cloud-based solution to identify statistically significant MS peaks differentiating sample categories.
BMC research notes
2013; 6: 109-?
Abstract
Mass spectrometry (MS) has evolved to become the primary high throughput tool for proteomics based biomarker discovery. Until now, multiple challenges in protein MS data analysis remain: large-scale and complex data set management; MS peak identification, indexing; and high dimensional peak differential analysis with the concurrent statistical tests based false discovery rate (FDR). "Turnkey" solutions are needed for biomarker investigations to rapidly process MS data sets to identify statistically significant peaks for subsequent validation.Here we present an efficient and effective solution, which provides experimental biologists easy access to "cloud" computing capabilities to analyze MS data. The web portal can be accessed at http://transmed.stanford.edu/ssa/.Presented web application supplies large scale MS data online uploading and analysis with a simple user interface. This bioinformatic tool will facilitate the discovery of the potential protein biomarkers using MS.
View details for DOI 10.1186/1756-0500-6-109
View details for PubMedID 23522030
View details for PubMedCentralID PMC3621609
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Alterations in Cerebrospinal Fluid Proteins in a Presymptomatic Primary Glioma Model
PLOS ONE
2012; 7 (11)
Abstract
Understanding the early relationship between brain tumor cells and their environment could lead to more sensitive biomarkers and new therapeutic strategies. We have been using a rodent model of neurocarcinogenesis in which all animals develop brain tumors by six months of age to establish two early landmarks in glioma development: the appearance of a nestin(+) cell at thirty days of age and the appearance of cellular hyperplasia between 60 and 120 days of age. We now report an assessment of the CSF proteome to determine the changes in protein composition that occur during this period.Nestin(+) cell clusters and microtumors were assessed in 63 ethylnitrosourea-exposed rats on 30, 60, and 90 days of age. CSF was obtained from the cisterna magna from 101 exposed and control rats at 30, 60, and 90 days and then analyzed using mass spectrometry. Differentially expressed peaks were isolated and identified.Nestin(+) cells were noted in all ethylnitrosourea-exposed rats assessed pathologically. Small microtumors were noted in 0%, 18%, and 67% of 30-, 60-, and 90-day old rats, respectively (p<0.05, Chi square). False Discovery Rate analysis of peak intensities showed that the number of true discoveries with p<0.05 increased markedly with increasing age. Isolation and identification of highly differentially detected proteins at 90 days of age revealed increases in albumin and a fragment of α1 macroglobulin and alterations in glutathionylated transthyretin.The presence of increased albumin, fragments of cerebrospinal fluid proteins, and glutathione breakdown in temporal association with the development of cellular hyperplasia, suggests that, similar to many other systemic cancers, inflammation and oxidative stress is playing an important early role in the host's response to brain tumor development and may be involved in affecting the early growth of brain tumor.
View details for DOI 10.1371/journal.pone.0049724
View details for Web of Science ID 000311333800047
View details for PubMedID 23185417
View details for PubMedCentralID PMC3501526
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Identification of protein marker in vaginal wall tissues of women with stress urinary incontinence by protein chip array
JOURNAL OF OBSTETRICS AND GYNAECOLOGY RESEARCH
2012; 38 (1): 89-96
Abstract
We sought to investigate protein biomarkers for stress urinary incontinence (SUI) in vaginal tissues using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) and examine if this is a reliable methodology to examine proteins in small tissue specimens. We compared protein expression profile of vaginal tissue from women with SUI and continent controls. A 22.6kDa peak was identified by subsequent weak cation-exchange, reverse-phase fractionation, gel electrophoresis, and trypsin digestion, then analyzed by matrix assisted laser desorption/ionization mass spectrometry (MALDI MS) and MALDI MS-MS. Biomarker identity and expression level were confirmed by Western-blotting and immunohistochemistry.Expression of the 22.6kDa protein, identified as SM-22α, was significantly higher in women with SUI versus controls. A 3×3-mm tissue sample was sufficient for identification. Western-blot/immunohistochemistry confirmed the SELDI-TOS MS findings.SM-22α, a marker for myofibroblasts, was identified as a biomarker of SUI. Differential protein profiling by SELDI-TOF MS is a powerful and reliable tool for urogynecological research as it allows us to study an array of proteins simultaneously using small tissue samples.
View details for DOI 10.1111/j.1447-0756.2011.01690.x
View details for Web of Science ID 000298882800014
View details for PubMedID 22136672
View details for PubMedCentralID PMC3253185
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Plasma proteomic profiles in preterm labor (PTL) and preterm premature rupture of membranes (PPROM)
32nd Annual Pregnancy Meeting of the Society-for-Maternal-Fetal-Medicine (SMFM)
MOSBY-ELSEVIER. 2012: S224–S224
View details for Web of Science ID 000298889900491
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A diagnostic algorithm combining clinical and molecular data distinguishes Kawasaki disease from other febrile illnesses
BMC MEDICINE
2011; 9
Abstract
Kawasaki 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.Urine 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.Comparative 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.A 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.
View details for DOI 10.1186/1741-7015-9-130
View details for Web of Science ID 000298862200001
View details for PubMedID 22145762
View details for PubMedCentralID PMC3251532
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Extracellular glutathione peroxidase (Gpx3) binds specifically to basement membranes of mouse renal cortex tubule cells
AMERICAN JOURNAL OF PHYSIOLOGY-RENAL PHYSIOLOGY
2010; 298 (5): F1244-F1253
Abstract
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.
View details for DOI 10.1152/ajprenal.00662.2009
View details for Web of Science ID 000276870800021
View details for PubMedID 20015939
View details for PubMedCentralID PMC2867408
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Identification of Protein Marker in Vaginal Wall Tissues of Women with Stress Urinary Incontinence by Protein Chip Array
57th Annual Meeting of the Society-for-Gynecologic-Investigation
SAGE PUBLICATIONS INC. 2010: 275A–275A
View details for Web of Science ID 000275558601205
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Effects of moderate versus deep hypothermic circulatory arrest and selective cerebral perfusion on cerebrospinal fluid proteomic profiles in a piglet model of cardiopulmonary bypass
JOURNAL OF THORACIC AND CARDIOVASCULAR SURGERY
2009; 138 (6): 1290-1296
Abstract
Our objective was to compare protein profiles of cerebrospinal fluid between control animals and those subjected to cardiopulmonary bypass after moderate versus deep hypothermic circulatory arrest with selective cerebral perfusion.Immature Yorkshire piglets were assigned to one of four study groups: (1) deep hypothermic circulatory arrest at 18 degrees C, (2) deep hypothermic circulatory arrest at 18 degrees C with selective cerebral perfusion, (3) moderate hypothermic circulatory arrest at 25 degrees C with selective cerebral perfusion, or (4) age-matched control animals without surgery. Animals undergoing cardiopulmonary bypass were cooled to their assigned group temperature and exposed to 1 hour of hypothermic circulatory arrest. After arrest, animals were rewarmed, weaned off bypass, and allowed to recover for 4 hours. Cerebrospinal fluid collected from surgical animals after the recovery period was compared with cerebrospinal fluid from controls by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry. Protein spectra were analyzed for differences between groups by Mann-Whitney U test and false discovery rate analysis.Baseline and postbypass physiologic parameters were similar in all surgical groups. A total of 194 protein peaks were detected. Compared with controls, groups 1, 2, and 3 had 64, 100, and 13 peaks that were significantly different, respectively (P < .05). Three of these peaks were present in all three groups. Cerebrospinal fluid protein profiles in animals undergoing cardiopulmonary bypass with moderate hypothermic circulatory arrest (group 3) were more similar to controls than either of the groups subjected to deep hypothermia.The mass spectra of cerebrospinal fluid proteins are altered in piglets exposed to cardiopulmonary bypass and hypothermic circulatory arrest. Moderate hypothermic circulatory arrest (25 degrees C) with selective cerebral perfusion compared with deep hypothermic circulatory arrest (18 degrees C) is associated with fewer changes in cerebrospinal fluid proteins, when compared with nonbypass controls.
View details for DOI 10.1016/j.jtcvs.2009.06.001
View details for Web of Science ID 000272029800004
View details for PubMedID 19660276
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Microfluidic Device for Coupling Capillary Electrophoresis and Matrix-Assisted Laser Desorption Ionization-Mass Spectrometry
JALA
2009; 14 (5): 252-261
View details for DOI 10.1016/j.jala.2009.02.005
View details for Web of Science ID 000285163200003
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Plasma Biomarkers in a Mouse Model of Preterm Labor
PEDIATRIC RESEARCH
2009; 66 (1): 11-16
Abstract
Preterm labor (PTL) is frequently associated with inflammation. We hypothesized that biomarkers during pregnancy can identify pregnancies most at risk for development of PTL. An inflammation-induced mouse model of PTL was used. Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry was used to analyze and compare the plasma protein (PP) profile between CD-1 mice injected intrauterine with either lipopolysaccharide (LPS) or PBS on d 14.5 of gestation. The median differences of normalized PP peaks between the two groups were determined using the Mann-Whitney U test and the false discovery rate. In a second series of experiments, both groups of mice were injected with a lower dose of LPS. A total of 1665 peaks were detected. Thirty peaks were highly differentially expressed (p < 0.0001) between the groups. Two 11 kDa protein peaks were identified by MALDI-TOF/TOF-MS and confirmed to be mouse serum amyloid A (SAA) 1 and 2. Plasma SAA2 levels were increased in LPS-treated animals compared with controls and in LPS-treated animals that delivered preterm vs. those that delivered at term. SAA2 has the potential to be a plasma biomarker that can identify pregnancies at risk for development of PTL.
View details for Web of Science ID 000267249300004
View details for PubMedID 19287348
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BIOMARKERS OF CEREBRAL INJURY IN A NEONATAL PIG MODEL OF CARDIOPULMONARY BYPASS AND CIRCULATORY ARREST AT 18 degrees C and 25 degrees C WITH AND WITHOUT SELECTIVE CEREBRAL PERFUSION
LIPPINCOTT WILLIAMS & WILKINS. 2009: 413
View details for Web of Science ID 000270092400930
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Capture of phosphopeptides using alpha-zirconium phosphate nanoplatelets
ANALYTICAL CHEMISTRY
2008; 80 (14): 5542-5549
Abstract
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.
View details for DOI 10.1021/ac800577z
View details for Web of Science ID 000257598100036
View details for PubMedID 18522436
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Sulfur X-ray absorption Spectroscopy of living mammalian cells: An enabling tool for sulfur metabolomics. In situ observation of uptake of taurine into MDCK cells
BIOCHEMISTRY
2007; 46 (51): 14735-14741
Abstract
Sulfur is essential for life, with important roles in biological structure and function. However, because of a lack of suitable biophysical techniques, in situ information about sulfur biochemistry is generally difficult to obtain. Here, we present an in situ sulfur X-ray absorption spectroscopy (S-XAS) study of living cell cultures of the mammalian renal epithelial MDCK cell line. A great deal of information is retrieved from a characteristic sulfonate feature in the X-ray absorption spectrum of the cell cultures, which can be related to the amino acid taurine. We followed the time and dose dependence of uptake of taurine into MDCK cell monolayers. The corresponding uptake curves showed a typical saturation behavior with considerable levels of taurine accumulation inside the cells (as much as 40% of total cellular sulfur). We also investigated the polarity of uptake of taurine into MDCK cells, and our results confirmed that uptake in situ is predominantly a function of the basolateral cell surface.
View details for DOI 10.1021/bi701979h
View details for PubMedID 18044978
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A potential biomarker in the cord blood of preterm infants who develop retinopathy of prematurity
PEDIATRIC RESEARCH
2007; 61 (2): 215-221
Abstract
Preterm infants are at risk of developing sepsis, necrotizing enterocolitis (NEC), chronic lung disease (CLD), and retinopathy of prematurity (ROP). We used high-throughput mass spectrometry to investigate whether cord blood proteins can be used to predict development of these morbidities. Cord blood plasma from 44 infants with a birth weight of <1500 g was analyzed by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF). Six infants developed ROP >or=stage II, 10 CLD, three sepsis, and one NEC. We detected 814 protein signals representing 330 distinct protein species. Nineteen biomarkers were associated with development of >or=stage II ROP [false-discovery rate (FDR) <5%] and none with CLD. Several proteins with molecular weight (Mr) 15-16 kD and pI 4-5 were detected with increased abundance in infants with ROP, while similar Mr proteins with pI 7-9 were less abundant in these patients. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and sequence analysis identified these proteins as alpha-, beta-, and gamma-globin chains. Partial deamidation of Asn139 in beta-globin chains was observed only in the pI 4-5 proteins. We conclude that there are several promising biomarkers for the risk of ROP. Deamidation of globin chains is especially promising and may indicate underlying prenatal pathologic mechanisms in ROP. Validation studies will be undertaken to determine their clinical utility.
View details for DOI 10.1203/pdr.0b013e31802d776d
View details for Web of Science ID 000243714700016
View details for PubMedID 17237725
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Sulfur x-ray absorption spectroscopy of living mammalian cells: taurine uptake/release and oxidative stress in renal epithelial cell cultures
BIOPHYSICAL SOCIETY. 2007: 336A
View details for Web of Science ID 000243972402122
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Deletion of GPX3 affects glutathione metabolism
FEDERATION AMER SOC EXP BIOL. 2006: A1068
View details for Web of Science ID 000236326202130
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Improving feature detection and analysis of surface-enhanced laser desorption/ionization-time of flight mass spectra
PROTEOMICS
2005; 5 (11): 2778-2788
Abstract
Discovering valid biological information from surface-enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF MS) depends on clear experimental design, meticulous sample handling, and sophisticated data processing. Most published literature deals with the biological aspects of these experiments, or with computer-learning algorithms to locate sets of classifying biomarkers. The process of locating and measuring proteins across spectra has received less attention. This process should be tunable between sensitivity and false-discovery, and should guarantee that features are biologically meaningful in that they represent chemical species that can be identified and investigated. Existing feature detection in SELDI-TOF MS is not optimal for acquiring biologically relevant data. Most methods have so many user-defined settings that reproducibility and comparability among studies suffer considerably. To address these issues, we have developed an approach, called simultaneous spectrum analysis (SSA), which (i) locates proteins across spectra, (ii) measures their abundance, (iii) subtracts baseline, (iv) excludes irreproducible measurements, and (v) computes normalization factors for comparing spectra. SSA uses only two key parameters for feature detection and one parameter each for quality thresholds on spectra and peaks. The effectiveness of SSA is demonstrated by identifying proteins differentially expressed in SELDI-TOF spectra from plasma of wild-type and knockout mice for plasma glutathione peroxidase. Comparing analyses by SSA and CiphergenExpress Data Manager 2.1 finds similar results for large signal peaks, but SSA improves the number and quality of differences betweens groups among lower signal peaks. SSA is also less likely to introduce systematic bias when normalizing spectra.
View details for DOI 10.1002/pmic.200401184
View details for Web of Science ID 000231036100008
View details for PubMedID 15986333
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Intracellular mediators of granulysin-induced cell death
JOURNAL OF IMMUNOLOGY
2003; 171 (5): 2556-2562
Abstract
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.
View details for Web of Science ID 000184970900047
View details for PubMedID 12928406
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Selenomethionine does not affect PSA secretion independent of its effect on LNCaP cell growth
PROSTATE
2003; 54 (4): 315-321
Abstract
Individuals supplemented with selenium have reduced incidence of prostate cancer. This study determines whether selenomethionine specifically affects the secretion of prostate specific antigen (PSA) in vitro.LNCaP cells were supplemented with selenomethionine for 7 days. PSA secretion was determined by ELISA. Cell proliferation was assessed by enumeration of trypan blue excluding cells. Colony formation was determined in soft agar. Cell cycle distribution was determined by FACS analysis of propidium iodide stained cells.Selenomethionine at > or = 70 microM inhibited LNCaP cell growth and colony formation. 0-100 microM selenomethionine did not affect the secretion of PSA by LNCaP cells in cell culture supernatants when normalized to the number of cells in culture. At supra-nutritional concentrations of selenomethionine, LNCaP cells had longer G(0)/G(1) phase in agreement with the inhibitory effects on cell growth.PSA secretion is not specifically inhibited by concentrations of selenomethionine corresponding to plasma selenium concentrations found in individuals supplemented with chemopreventive concentrations of selenized yeast. These data suggest that changes in serum PSA levels in individual patients during selenium supplementation is not an effect specific for PSA secretion, but rather may be a useful indicator for changes in disease progression in individual patients.
View details for DOI 10.1002/pros.10184
View details for Web of Science ID 000181056800009
View details for PubMedID 12539231
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Extracellular glutathione peroxidase is secreted basolaterally by human renal proximal tubule cells
AMERICAN JOURNAL OF PHYSIOLOGY-RENAL PHYSIOLOGY
2002; 283 (1): F20-F28
Abstract
Extracellular glutathione peroxidase (eGPx) is a secreted selenoenzyme with GPx activity. eGPx protein and activity are found in blood plasma and other extracellular fluids. eGPx in plasma is predominantly derived from the proximal tubules of kidneys in humans. Two types of human proximal tubule cells were cultured on semipermeable polycarbonate membranes to determine whether these cells secrete eGPx in a polarized direction. Immortalized human proximal tubule HK-2 cells and primary human proximal tubule cells formed confluent monolayers when cultured on these membrane inserts in culture dishes, as evidenced by transepithelial resistance. Both cell lines also constituted a barrier to diffusion of a fluoresceinated dextran of 75 kDa, a size similar to eGPx homotetramers. In both cell lines, 6- to 12-fold more 35S-methionine-labeled eGPx was immunoprecipitated from the basolateral media than from the apical media, indicating basolateral secretion of eGPx. eGPx was immunolocalized to the extracellular fluid at the basolateral surface of proximal tubules in human kidney. These data support the conclusion that eGPx is secreted through the basolateral membrane of human kidney proximal tubule cells into the extracellular fluid of the kidney, and from there enters blood plasma.
View details for DOI 10.1152/ajprenal.00014.2001
View details for Web of Science ID 000176138800003
View details for PubMedID 12060583
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Increased expression of extracellular glutathione peroxidase in mice with dextran sodium sulfate-induced experimental colitis
PEDIATRIC RESEARCH
2002; 51 (5): 641-646
Abstract
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.
View details for Web of Science ID 000175171600016
View details for PubMedID 11978890
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Ozone exposure and the production of reactive oxygen species by bronchoalveolar cells in humans
INHALATION TOXICOLOGY
2001; 13 (6): 465-483
Abstract
Exposure to ozone injures respiratory epithelium, and the mechanisms may involve the generation of reactive oxygen species (ROS). This study tested the hypothesis that ozone exposure increases the airway burden of ROS to a greater degree in smokers than nonsmokers, and that this effect is independent of ozone-induced changes in spirometry. Healthy subjects were selected as either responders (decrement in FEV1 > 15%) or nonresponders (decrement in FEV1 < 5%) to ozone; each underwent 2 exposures to ozone and 1 to air, with bronchoalveolar lavage (BAL) performed 30 min (early) and 18 h (late) after exposure. Release of superoxide anion (O2(-)) was used as a measure of ROS release by all BAL cells, and flow cytometry was used to detect ROS production in alveolar macrophages (AM) only. Recovery of AM was approximately threefold greater in smokers than nonsmokers. Unstimulated, but not stimulated, cells obtained by BAL from smokers released approximately twofold greater amounts of O2(-) than cells from nonsmokers, both early and late after ozone exposure (p =.012 and p =.046, respectively). Stimulated, but not unstimulated, ROS generation by AM from smokers increased following ozone exposure, but the ozone effect was not significant. ROS production by AM decreased in nonsmokers (air vs. ozone late, p =.014). Total protein, albumin, and immunoglobulin M (IgM) increased in BAL fluid, consistent with an increase in epithelial permeability. In addition, the concentration of alpha2-macroglobulin increased approximately threefold 18 h after exposure in nonsmokers (p <.001). No relationship was found between measures of ROS production and lung function responsiveness to ozone. These studies suggest the airways of smokers experience a greater burden of ROS than those of nonsmokers following ozone exposure.
View details for Web of Science ID 000168753300001
View details for PubMedID 11445887
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Intracellular reduction of selenite into glutathione peroxidase. Evidence for involvement of NADPH and not glutathione as the reductant
MOLECULAR AND CELLULAR BIOCHEMISTRY
2000; 211 (1-2): 9-17
Abstract
Selenium (Se) in selenite is present in an oxidized state, and must be reduced for it to be incorporated as selenocysteine into selenoenzymes such as glutathione peroxidase (GPx). In vitro, Se, as in selenite, can be reduced utilizing glutathione (GSH) and glutathione reductase (GRed). We determined the effects of decreasing GSH levels, inhibiting GRed activity, and decreasing cellular NADPH on the selenite-dependent rate of GPx synthesis in cultured cells: PC3, CHO, and the E89 glucose-6-phosphate dehydrogenase (G-6-PD)-deficient cell line. A novel statistical analysis method was developed (using Box Cox transformed regression and a bootstrap method) in order to assess the effects of these manipulations singly and in combinations. Buthionine sulfoximine (BSO) was used to decrease GSH levels, 1,3 bis-(2 chloroethyl)-1 -nitrosourea (BCNU) was used to inhibit GRed activity and methylene blue (MB) was used to decrease cellular NADPH levels. This statistical method evaluates the effects of BSO, BCNU, MB and selenite alone and in combinations on GPx activity. Decreasing the GSH level (< 5% of control) did not have an effect on the selenite-dependent rate of GPx synthesis in PC3 or CHO cells, but did have a small inhibitory effect on the rate of GPx synthesis in E89 cells. Inhibiting GRed activity was also associated with either no effect (CHO, E89) or a small effect (PC3) on GPx activity. In contrast, decreasing NADPH levels in cells treated with MB was associated with a large decrease in the selenite-dependent rate of GPx synthesis to 36, 34 and 25% of control in PC3, CHO, and E89 cells, respectively. The effects of BSO plus BCNU were not synergistic in any of the cell lines. The effects of BSO plus MB were synergistic in G-6-PD-deficient E89 cells, but not in PC3 or CHO cells. We therefore conclude that under normal culture conditions, NADPH, and not glutathione, is the primary reductant of Se in selenite to forms that are eventually incorporated into GPx. For cells with abnormal ability to generate NADPH, lowering the GSH levels had a small effect on selenite-dependent GPx synthesis. GRed activity is not required for the selenite-dependent synthesis of GPx.
View details for Web of Science ID 000089137800002
View details for PubMedID 11055542
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Increase in extracellular glutathione peroxidase in plasma and lungs of mice exposed to hyperoxia
PEDIATRIC RESEARCH
1999; 46 (6): 715-721
Abstract
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.
View details for Web of Science ID 000083870200013
View details for PubMedID 10590029
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Expression of extracellular glutathione peroxidase in human and mouse gastrointestinal tract
AMERICAN JOURNAL OF PHYSIOLOGY-GASTROINTESTINAL AND LIVER PHYSIOLOGY
1998; 275 (6): G1463-G1471
Abstract
Extracellular glutathione peroxidase (EGPx) is a glycosylated selenoprotein capable of reducing hydrogen peroxide, organic hydroperoxides, free fatty acid hydroperoxides, and phosphatidylcholine hydroperoxides. We found that human large intestinal explant cultures synthesize EGPx and cellular glutathione peroxidase (CGPx) and secrete EGPx. The level of EGPx mRNA expression relative to alpha-tubulin was similar throughout the mouse gastrointestinal tract. EGPx mRNA transcripts have been localized to mature absorptive epithelial cells in human and mouse large intestine. Western blot analysis of mouse intestinal protein has demonstrated the presence of EGPx protein in the small intestine, cecum, and large intestine, with the highest protein levels found in the cecum. Immunohistochemistry studies of human large intestine and mouse small and large intestine sections demonstrated the presence of EGPx protein within mature absorptive epithelial cells. In human large intestine and mouse small intestine, EGPx protein is also present in the extracellular milieu. These results suggest a role for EGPx in protection of the intestinal tract from peroxidative damage and/or in intercellular metabolism of peroxides.
View details for Web of Science ID 000077746400031
View details for PubMedID 9843785
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Expression of extracellular glutathione peroxidase in human and mouse gastrointestinal tract.
American journal of physiology. Gastrointestinal and liver physiology
1998; 275 (6): G1463–G1471
Abstract
Extracellular glutathione peroxidase (EGPx) is a glycosylated selenoprotein capable of reducing hydrogen peroxide, organic hydroperoxides, free fatty acid hydroperoxides, and phosphatidylcholine hydroperoxides. We found that human large intestinal explant cultures synthesize EGPx and cellular glutathione peroxidase (CGPx) and secrete EGPx. The level of EGPx mRNA expression relative to alpha-tubulin was similar throughout the mouse gastrointestinal tract. EGPx mRNA transcripts have been localized to mature absorptive epithelial cells in human and mouse large intestine. Western blot analysis of mouse intestinal protein has demonstrated the presence of EGPx protein in the small intestine, cecum, and large intestine, with the highest protein levels found in the cecum. Immunohistochemistry studies of human large intestine and mouse small and large intestine sections demonstrated the presence of EGPx protein within mature absorptive epithelial cells. In human large intestine and mouse small intestine, EGPx protein is also present in the extracellular milieu. These results suggest a role for EGPx in protection of the intestinal tract from peroxidative damage and/or in intercellular metabolism of peroxides.
View details for DOI 10.1152/ajpgi.1998.275.6.G1463
View details for PubMedID 29585770
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Plasma glutathione peroxidase and its relationship to renal proximal tubule function
MOLECULAR GENETICS AND METABOLISM
1998; 65 (3): 238-245
Abstract
Selenium-dependent extracellular glutathione peroxidase (E-GPx) is found in plasma and other extracellular fluids. Previous studies have indicated that patients with chronic renal failure on dialysis have low plasma GPx activity. In this study, dialysis patients had approximately 40% of control plasma GPx activity, while anephric individuals had lowest plasma GPx activities ranging from 2 to 22% of control. The residual plasma GPx activity in anephric individuals could be completely precipitated by anti-E-GPx antibodies, indicating that all plasma GPx activity can be attributed to E-GPx in both normal and anephric individuals. Plasma GPx activity rises rapidly following kidney transplantation, often reaching normal values within 10 days. The plasma GPx activity in some transplanted patients rises to levels higher than the normal range, followed by a return to the normal range. Since E-GPx in the kidney is primarily synthesized in the proximal tubules, we investigated whether nephrotoxic agents known to disrupt proximal tubule function also affected plasma GPx activity. The beta-lactam antibiotic cephaloglycin rapidly caused a decrease in plasma GPx activity in rabbits. In addition, the chemotherapeutic agent ifosfamide caused a decrease in plasma GPx activity in pediatric osteosarcoma patients. Fanconi syndrome associated with either ifosfamide therapy or valproic acid therapy also caused a decrease in plasma GPx activity. Thus plasma GPx activity is related to kidney function and is decreased in certain situations where nephrotoxic drugs are administered. Monitoring plasma GPx activity may have predictive value in evaluating the function of transplanted kidneys or in predicting those patients particularly at risk of nephrotoxic injury associated with certain medications.
View details for Web of Science ID 000077722800008
View details for PubMedID 9851889
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Developmental expression of extracellular glutathione peroxidase suggests antioxidant roles in deciduum, visceral yolk sac, and skin
MOLECULAR REPRODUCTION AND DEVELOPMENT
1998; 49 (4): 343-355
Abstract
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 antioxidant role than previously recognized in the interstitium of multiple localized tissues, particularly those associated with the active transport of lipids.
View details for Web of Science ID 000072245700001
View details for PubMedID 9508085
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Selenite can increase selenium-dependent glutathione peroxidase activity in glutathione and glutathione reductase-deficient cells.
FEDERATION AMER SOC EXP BIOL. 1998: A824
View details for Web of Science ID 000076006501158
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Airway inflammation in smokers and nonsmokers with varying responsiveness to ozone
AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE
1997; 156 (3): 728-736
Abstract
Exposure to ozone causes symptoms, changes in lung function, and airway inflammation. We studied whether individuals who differ in lung-function responsiveness to ozone, or in smoking status, also differ in susceptibility to airway inflammation. Healthy subjects were selected on the basis of responsiveness to a classifying exposure to 0.22 ppm ozone for 4 h with exercise (responders, with a decrease in FEV1 > 15%; and non-responders, with a decrease in FEV1 < 5%). Three groups were studied: nonsmoker-nonresponders (n = 12), nonsmoker-responders (n = 13), and smokers (n = 13, 11 nonresponders and two responders). Each subject underwent two exposures to ozone and one to air, separated by at least 3 wk; bronchoalveolar and nasal lavages were performed on three occasions: immediately (early) and 18 h (late) after ozone exposure, and either early or late after air exposure. Recovery of polymorphonuclear leukocytes (PMN) increased progressively in all groups, and by up to 6-fold late after ozone exposure. Interleukin-6 (IL-6) and IL-8 increased early (by up to 10-fold and up to 2-fold, respectively), and correlated with the late increase in PMN. Lymphocytes, mast cells, and eosinophils also increased late after exposure. We conclude that ozone-induced airway inflammation is independent of smoking status or airway responsiveness to ozone.
View details for Web of Science ID A1997XX17700008
View details for PubMedID 9309986
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Selenium concentration controls cellular glutathione peroxidase (CGPx) synthesis by translational mechanisms without changing polysome patterns
FEDERATION AMER SOC EXP BIOL. 1997: A1406
View details for Web of Science ID 000073305603701
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Standardization of an opsonophagocytic assay for the measurement of functional antibody activity against Streptococcus pneumoniae using differentiated HL-60 cells
CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY
1997; 4 (4): 415-422
Abstract
Host protection against pneumococcal disease i primarily mediated by phagocytosis. We developed and standardized an opsonophagocytic assay using HL-60 cells (human promyelocytic leukemia cells). Fifty-five serum samples were analyzed for the presence of functional antibody against seven pneumococcal serogroups or serotypes (4, 6B, 9V, 14, 18C, 19F, and 23F) by using differentiated HL-60 cells (granulocytes) and peripheral blood leukocytes (PBLs). Six of the 55 serum samples were from unvaccinated adult volunteers, 31 serum samples were from adults who received one dose of the 14-valent or the 23-valent polysaccharide vaccine, and 18 serum samples were from 16-month-old infants who received four doses of an investigational 7-valent polysaccharide-protein conjugate vaccine. The results of an opsonophagocytic assay with HL-60 cells correlated highly with those of an assay with PBLs as effector cells (median r for seven serotypes = 0.87: P < 0.01). Opsonophagocytic titers were compared with the immunoglobulin G antibody concentrations determined by enzyme-linked immunosorbent assay (ELISA). The r values for serogroups or serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F were 0.61, 0.60, 0.67 0.90, 0.61, 0.39, and 0.57, respectively, when HL-60 cells were used as effector cells and 0.56, 0.47, 0.61, 0.90, 0.71, 0.31, and 0.62, respectively, when PBLs were used. The assay requires small amounts of serum (40 microliters per serotype), making this test suitable for assaying infant sera. Culturable cells aid in assay standardization and likely reduce donor-to-donor variability. This standardized assay, in combination with the standardized ELISA, can be used to evaluate current and developing pneumococcal vaccines, in which functional opsonophagocytic antibody activity may correlate with protection against pneumococcal disease.
View details for Web of Science ID A1997XH99800005
View details for PubMedID 9220157
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Effects of ozone on normal and potentially sensitive human subjects. Part II: Airway inflammation and responsiveness to ozone in nonsmokers and smokers.
Research report (Health Effects Institute)
1997: 39-72
Abstract
Exposure to ozone at levels near the National Ambient Air Quality Standard causes respiratory symptoms, changes in lung function, and airway inflammation. Although ozone-induced changes in lung function have been well characterized in healthy individuals, the relationship between airway inflammation and changes in pulmonary function have not been prospectively examined. The purpose of this study was to determine whether individuals who differ in, lung function responsiveness to ozone also differ in susceptibility to airway inflammation and injury. A secondary goal was to determine whether ozone exposure induces airway inflammation in smokers, a population known to have airway inflammation and an increased burden of toxic oxygen species. Healthy nonsmokers (n = 56) and smokers (n = 34) were exposed to 0.22 parts per million (ppm)* ozone for 4 hours, with intermittent exercise, for the purpose of selecting ozone "responders" (decrement in forced expiratory volume in 1 second [FEV1] > 15%) and "nonresponders" (decrement in FEV1 < 5%). Selected subjects then were exposed twice to ozone (0.22 ppm for 4 hours with exercise) and once to air (with the same exposure protocol), each pair of exposures separated by at least 3 weeks, in a randomized, double-blind fashion. Nasal lavage (NL) and bronchoalveolar lavage (BAL) were performed immediately after one ozone exposure and 18 hours after the other, and either immediately or 18 hours after the air exposure. Indicators of airway effects in lavage fluid included changes in inflammatory cells, proinflammatory cytokines, protein markers of epithelial injury and repair, and generation of toxic oxygen species. In the classification exposure, fewer smokers than nonsmokers were responsive to ozone (11.8% vs. 28.6%, respectively); an insufficient number of smoker-responders were identified to study as a separate group. In the BAL study, all groups developed a similar degree of airway inflammation, consisting of increases in interleukins 6 and 8 (maximal immediately after exposure), and increases in polymorphonuclear leukocytes (PMNs), lymphocytes, and mast cells (maximal 18 hours after exposure). The increase in PMNs was inversely correlated with age (p = 0.013), but gender, nonspecific airway responsiveness, and allergy history were not predictive of inflammation. Alveolar macrophage production of toxic oxygen species decreased after ozone exposure in nonsmokers; however, not in smokers. Findings from nasal lavage did not mirror lower airway inflammatory responses in these studies. We conclude that, in response to ozone exposure, smokers experienced smaller decrements in lung function and fewer symptoms than nonsmokers; however, the intensity of the airway inflammatory response was independent of smoking status or airway responsiveness to ozone. Furthermore, the burden of toxic oxygen species following ozone exposure was greater for smokers than for nonsmokers. Subjects were young, healthy, and able to sustain exercise; the results may not be representative of nonsmokers or smokers in general. Nevertheless, the findings indicate that measuring symptoms and spirometric changes is not sufficient to assess the potential risks associated with ozone exposure.
View details for PubMedID 9387196
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Production of reactive oxygen intermediates following exposure to ozone. Relative contribution of alveolar macrophages.
Chest
1996; 109 (3): 8S-?
View details for PubMedID 8598168
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Production of reactive oxygen intermediates following exposure to ozone - Relative contribution of alveolar macrophages
Symposium on Environmental Lung Disease in Exposures and Mechanisms, at the Thomas-L-Petty-Aspen-Lung-Conference 38th Annual Meeting
AMER COLL CHEST PHYSICIANS. 1996: S8–S8
View details for Web of Science ID A1996UA47200005
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COMPARATIVE PHARMACOKINETIC STUDIES OF 3 ASPARAGINASE PREPARATIONS
JOURNAL OF CLINICAL ONCOLOGY
1993; 11 (9): 1780-1786
Abstract
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.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).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).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.
View details for Web of Science ID A1993LW36900021
View details for PubMedID 8355045
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ROLE OF ANTIOXIDANT ENZYMES IN THE INDUCTION OF INCREASED EXPERIMENTAL METASTASIS BY HYDROXYUREA
JOURNAL OF THE NATIONAL CANCER INSTITUTE
1993; 85 (9): 711-721
Abstract
Treatment of tumor cells with hydroxyurea and other DNA-damaging agents has been shown to increase the experimental metastatic potential of these cells.We sought to elucidate some of the biochemical and genetic changes that promote tumor cell metastasis in hydroxyurea-treated cells. We hypothesized that drug treatment induces resistance to oxidative damage and that elimination of this resistance reverses the drug-induced experimental metastatic capabilities of tumor cells.We examined the effect of hydroxyurea treatment on B16 melanoma cells with respect to experimental metastatic potential, resistance to hydrogen peroxide (H2O2), glutathione peroxidase activity and messenger RNA (mRNA) level, glutathione reductase activity, glutathione levels, glutathione-S-transferase activity, and catalase activity and mRNA level.Hydroxyurea-treated cells were transiently more metastatic following intravenous injection in syngeneic mice and transiently more resistant than untreated cells to exogenous H2O2. Hydroxyurea-induced experimental metastases and H2O2 resistance were eliminated by depletion of intracellular glutathione with buthionine sulfoximine. Glutathione peroxidase activity and mRNA level, glutathione reductase activity, and reduced glutathione levels were all transiently increased in hydroxyurea-treated cells, whereas the increase in glutathione-S-transferase activity was sustained. Catalase activity was modestly increased with no increase in its mRNA levels.In B16 melanoma cells, experimental metastasis induced by hydroxyurea appears to depend on a process that requires glutathione. Hydroxyurea treatment also induces resistance to exogenous H2O2, which may be due to induction of glutathione and antioxidant enzyme activity.The role of antioxidants in B16 melanoma cells offers new insights into the metastatic process and the cellular response to chemotherapy.
View details for Web of Science ID A1993LA67200011
View details for PubMedID 8478957
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MODULATION OF THE RESPIRATORY BURST IN HUMAN NEUTROPHILS BY ISOPROTERENOL AND DIBUTYRYL-CYCLIC-AMP
JOURNAL OF NEUROIMMUNOLOGY
1993; 43 (1-2): 59-68
Abstract
The effects of a beta-adrenergic agonist and a cyclic AMP analogue on activation, activity, and termination of FMLP-stimulated superoxide anion production were investigated. Incubation with isoproterenol resulted in a 50% reduction in the maximal rate of superoxide production and a 3-4-fold increase in the rate of termination of superoxide production. Exposure to 1 mM dibutyryl cyclic AMP resulted in a 40% decrease in the maximal rate and a 3-fold increase in the rate of termination of FMLP-induced superoxide production. Neither agent had a significant effect on the lag time prior to superoxide anion generation.
View details for Web of Science ID A1993KV48500008
View details for PubMedID 8384637
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PROPHYLACTIC EFFECTS OF DEXAMETHASONE IN LUNG INJURY CAUSED BY HYPEROXIA AND HYPERVENTILATION
JOURNAL OF APPLIED PHYSIOLOGY
1992; 72 (4): 1320-1325
Abstract
To determine if prophylactic corticosteroids would prevent acute lung injury caused by hyperoxia and barotrauma, 29 piglets (1.2 +/- 0.3 kg, 1-2 days of age) were studied. Ten piglets were hyperventilated [arterial PCO2 (PaCO2) 15-20 Torr] with 100% O2 for 48 h and compared with 10 piglets treated with the identical management but given 0.7 mg/kg of dexamethasone at time 0 and every 12 h for the 48-h study. Six piglets were normally ventilated (PaCO2 40-45 Torr) for 48 h with 21% O2 as an additional control group. Pulmonary function and tracheal aspirates were examined at time 0 and every 24 h. Bronchoalveolar lavage was performed for surfactant analyses at the conclusion of the study. In animals treated with hyperoxia and hyperventilation, lung compliance decreased 32% and tracheal aspirate polymorphonuclear leukocyte (PMN) chemotactic activity increased by 51%, cell counts by 204%, number of PMNs by 277%, elastase activity by 111%, and albumin concentration by 328% over 48 h (P less than 0.05). In contrast, dexamethasone-treated piglets had increases in only tracheal aspirate albumin concentration (206%) over the 48-h study. All cellular and biochemical variables were lower in dexamethasone-treated compared with hyperoxic hyperventilated piglets. Room air normal ventilation controls had only a 108% increase in tracheal aspirate albumin concentration noted. Despite quantitative differences in surfactant among the three groups, activity was unaffected. Results indicate that hyperoxia and hyperventilation for 48 h causes significant inflammatory changes and acute lung injury and that prophylactic high-dose dexamethasone significantly ameliorates this lung damage.
View details for Web of Science ID A1992HP08600014
View details for PubMedID 1592721
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MEASUREMENT OF SERUM L-ASPARAGINE IN THE PRESENCE OF L-ASPARAGINASE REQUIRES THE PRESENCE OF AN L-ASPARAGINASE INHIBITOR
CANCER RESEARCH
1991; 51 (24): 6568-6573
Abstract
The antileukemic activity of L-asparaginase (ASNase), an important component of therapy for acute lymphoblastic leukemia, is thought to result from depletion of serum L-asparagine (Asn). In studies of the pharmacological effects of ASNase, investigators have reported prolonged reduction in the serum concentration of Asn after the administration of ASNase. Such measurements may not be valid because ASNase present in the blood sample may hydrolyze Asn before its determination. We examined recovery of [U-14C]Asn from blood samples with and without various concentrations of added ASNase. In the presence of greater than or equal to 0.01 IU/ml of ASNase, the amount of [U-14C]Asn recovered was less than 15% of that without ASNase. Utilizing this assay, we studied the effect of 2 known inhibitors of ASNase in an attempt to improve Asn recovery. In the presence of aspartic beta semialdehyde (ASA), or 5-diazo-4-oxo-L-norvaline (DONV), and up to 1.0 IU/ml ASNase, Asn levels remained at greater than 90% of control. ASA prevented the hydrolysis of exogenous Asn in blood samples drawn from patients after ASNase injection. We also developed a method to determine Asn in serum utilizing high pressure liquid chromatography. Using this method, we found that the Asn level was greater than 90% of a normal level in the presence of 40 mM DONV and 1.0 IU/ml ASNase. Examination of serum from 4 patients treated with ASNase showed that Asn is detectable 7-19 days sooner when DONV is present in the blood collection system than in its absence. We conclude that: (a) as little as 0.01 IU/ml ASNase can hydrolyze Asn added to blood; (b) continued hydrolysis of Asn by ASNase ex vivo can result in falsely low serum Asn measurements; (c) ASA or DONV present in the collection tubes obviates the problem of continued ASNase activity; and (d) the degree and duration of Asn depletion after ASNase therapy is much less than previously believed. Thus, for accurate measurements of the duration and degree of Asn depletion by ASNase, an ASNase inhibitor such as ASA or DONV should be present in the blood collection system.
View details for Web of Science ID A1991GU41600012
View details for PubMedID 1742729
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SIGNAL TRANSDUCTION IN N-FORMYL-METHIONYL-LEUCYL-PHENYLALANINE AND CONCANAVALIN-A STIMULATED HUMAN NEUTROPHILS - SUPEROXIDE PRODUCTION WITHOUT A RISE IN INTRACELLULAR FREE CALCIUM
JOURNAL OF CELLULAR PHYSIOLOGY
1990; 145 (2): 295-302
Abstract
Changes in intracellular ionized free calcium ([Ca]i), inositol triphosphate (IP3), and sn-1,2-diacylglycerol (DAG) were determined in relation to agonist-induced human neutrophil superoxide (O2-) production. With 0.1 microM N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulation, generation of IP3 and a peak rise in [Cai] occurred at 30 sec, preceding maximal O2- production (1.5 min) and the maximal rise in DAG mass (4 min). FMLP-induced O2- production was inhibited by pertussis toxin. In cytochalasin B-primed, concanavalin A (Con A) stimulated neutrophils, a peak rise in [Ca]i but not IP3 proceeded O2- production, and pertussis toxin did not inhibit O2- production. EGTA inhibited the cytochalasin B/fMLP-induced increment in [Ca]i and O2- production by 75% and 50%, respectively, and completely ablated the response to cytochalasin B/Con A, suggesting a role for extracellular as well as intracellular calcium in the respiratory burst. However, three types of experiments indicate that an increase in [Ca]i is neither sufficient nor always required for O2- production. First, treatment with ionomycin resulted in a marked increase in [Ca]i but did not cause O2- production. Second, pertussis toxin inhibited both fMLP-induced IP3 generation and O2- production but did not inhibit the rise in [Ca]i. Third, following neutrophil priming with dioctanoylglycerol (diC8), maximal O2- production occurred in response to 0.015 microM fMLP or Con A without a rise in [Ca]i, and diC8/fMLP-induced O2- production was not inhibited by EGTA. Taken together, these data suggest that 1) an increment in [Ca]i is not strictly essential for neutrophil O2- production, 2) unlike fMLP, Con A-induced O2- production does not proceed through a pathway involving the pertussis toxin-sensitive G protein, and 3) regulation of neutrophil [Ca]i involves mechanisms independent of IP3 concentration.
View details for Web of Science ID A1990EJ64600013
View details for PubMedID 2174064
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PLASMA SELENIUM-DEPENDENT GLUTATHIONE-PEROXIDASE - CELL OF ORIGIN AND SECRETION
JOURNAL OF BIOLOGICAL CHEMISTRY
1989; 264 (27): 15850-15855
Abstract
Human plasma glutathione peroxidase (GSHPx) has been shown to be a glycosylated selenoprotein distinct enzymatically, structurally, and antigenically from known cellular glutathione peroxidases. The extracellular location of the enzyme and the fact that it is glycosylated suggested that it is a secreted protein. Utilizing mutually non-cross-reactive antibodies to human cellular and plasma GSHPx, we conducted a search to determine the tissue of origin for plasma GSHPx. The cells screened were endothelial cells because they are the main source of extracellular superoxide dismutase, HL-60 cells (myeloid cell line) because they are the main source of extracellular H2O2, and Hep G2 cells (hepatic cell line) because they are the source of many plasma proteins. Human umbilical vein endothelial cells were metabolically labeled with either [35S]methionine or [75Se]selenious acid, and HL-60 cells and Hep G2 cells were metabolically labeled with [75Se]selenious acid. Proteins were immunopurified from the labeled cells and their media with either anti-red blood cell (RBC) GSHPx IgG or with anti-plasma GSHPx IgG. Utilizing anti-RBC GSHPx IgG, only the cellular form of the enzyme was precipitated from all the cells tested but not from their media. When anti-plasma GSHPx IgG was applied to the cells and their media, a selenoprotein was precipitated only from the media of Hep G2 cells. When Hep G2 cells were incubated in the presence of the carboxylic ionophore monensin, an intracellular selenoprotein could be detected using anti-plasma GSHPx IgG. The precipitation of the cellular form from all three cell types was partially inhibited by preincubation of the anti-RBC GSHPx IgG with purified RBC GSHPx while the precipitation of the selenoprotein from the medium of Hep G2 cells by anti-plasma GSHPx IgG was prevented by preincubation of the antibody with purified plasma GSHPx. We suggest that plasma GSHPx is synthesized by and secreted from hepatic cells. This is, to the best of our knowledge, the only known selenoprotein with a defined function that has been shown to be synthesized for secretion by mammalian cells.
View details for Web of Science ID A1989AQ95000021
View details for PubMedID 2777767
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ANTIHUMAN PLASMA GLUTATHIONE-PEROXIDASE ANTIBODIES - IMMUNOLOGICAL INVESTIGATIONS TO DETERMINE PLASMA GLUTATHIONE-PEROXIDASE PROTEIN AND SELENIUM CONTENT IN PLASMA
BLOOD
1989; 73 (1): 318-323
Abstract
Plasma glutathione peroxidase (GSHPx) (glutathione: H2O2 oxidoreductase) is a unique selenoglycoprotein. Treatment of this enzyme with glycopeptidase F partially deglycosylates it and establishes the presence of N-linked sugar moieties. Antibodies raised in a rabbit against the purified enzyme from plasma were found to be specific, noninhibitory, and capable of precipitating the enzymatic activity. The antibodies precipitated greater than 90% of the GSHPx activity of normal plasma, thus indicating that the selenoenzyme is the main if not the sole GSHPx activity of plasma. The antibodies did not precipitate RBC GSHPx. A slight cross-reactivity of the antibodies was found with rat plasma GSHPx. A GSHPx activity precipitation assay of normal plasma in the presence of selenium (Se)-deficient plasma indicates that no cross-reactive protein in the Se-deficient plasma interferes with the precipitation of the GSHPx activity from normal plasma. Thus, GSHPx protein as well as activity is deficient in plasma in the absence of Se. Antibodies against GSHPx either from RBCs or from plasma were used to specifically immunoprecipitate most of the GSHPx activity from RBCs or plasma, respectively, in healthy individuals to determine the amount of Se associated with the protein. GSHPx accounts for approximately 15% of the Se in RBCs and 12% of the Se in plasma. Thus, in normal individuals, these proteins account for only a fraction of plasma and RBC Se.
View details for Web of Science ID A1989R647600051
View details for PubMedID 2491950
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DISORDERS OF RESPIRATORY BURST TERMINATION
HEMATOLOGY-ONCOLOGY CLINICS OF NORTH AMERICA
1988; 2 (2): 289-299
Abstract
The biochemical mechanisms for the termination of the respiratory burst are likely to be multifactorial. Alterations in the endogenous oxidant-scavenging mechanisms can modulate the kinetics of the termination phase of neutrophil superoxide production. These are most apparent when those alterations are found in the neutrophils of certain patients. The use of inhibitors of the oxidant scavenging systems in normal neutrophils has not yielded results that exactly mimic the studies with neutrophils from the patients. For example, the glutathione reductase-inhibited neutrophils (from the action of BCNU) do not show the same degree of abnormality as do the neutrophils from the patient with glutathione reductase deficiency. Further investigations on the mechanisms of inactivation of the NADPH oxidase are warranted in order to gain a greater understanding of this important regulating mechanism.
View details for Web of Science ID A1988N647900010
View details for PubMedID 2839461
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PURIFICATION AND CHARACTERIZATION OF HUMAN-PLASMA GLUTATHIONE-PEROXIDASE - A SELENOGLYCOPROTEIN DISTINCT FROM THE KNOWN CELLULAR ENZYME
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
1987; 256 (2): 677-686
Abstract
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.
View details for Web of Science ID A1987J490300028
View details for PubMedID 3619451
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ACTIVATION OF NEUTROPHIL SUPEROXIDE PRODUCTION BY CONCANAVALIN-A CAN OCCUR AT LOW-LEVELS OF INTRACELLULAR IONIZED CALCIUM
BLOOD
1987; 69 (3): 762-768
Abstract
The effect of concanavalin A (Con A) on the concentration of ionized intracellular calcium [( Cai++]) in human granulocytes (PMN) was monitored using the fluorescent calcium indicator and chelator, Quin2. The addition of Con A to PMN resulted in a rise in [Cai++] that was markedly enhanced by the presence of Ca++ in the external buffer. The onset of the increment in [Cai++] preceded the onset of O2- production. The rise in [Cai++] induced by Con A is not transient, with a new, higher steady-state level of [Cai++] being attained within five minutes. The addition of alpha-methylmannoside (alpha-MM) one minute after Con A resulted in the return of [Cai++] to the original baseline level and the cessation of O2- production. The addition of a second stimulus (such as arachidonic acid) to these cells resulted in a second increment in [Cai++] and the return of O2- production. Thus the rise in [Cai++] induced by Con A is tightly coupled to the activation, inactivation, and reactivation of the O2- generating system by Con A. Further experiments were undertaken to assess the possible requirement for the rise in [Cai++] in the activation of PMN by Con A. PMN could be depleted of Cai++ by loading with Quin2 in the absence of extracellular Ca++. These Ca++- depleted PMN can be induced to produce O2- after treatment with PMA but not with Con A. The addition of Ca++ to Ca++ -depleted PMN results in a return of [Cai++] to the normal resting level of Ca++ -replete PMN. The time required to return to baseline is a function of the concentration of intracellular Quin2. The addition of Ca++ to Ca++ -depleted, Con A-treated PMN results in the elevation of [Cai++] and the production of O2-. Over a tenfold range of intracellular Quin2 concentration, the onset of O2- production always occurred at [Cai++] that were less than the normal resting level. Thus, activation of the O2- generating system by Con A can occur at [Cai++] which are much lower than the incremental level induced by Con A in Ca++ -replete PMN. Supporting this is the observation that only a very small increment in [Cai++] is induced by Con A in PMN cytoplasts, even though Con A could induce O2- production in the Quin2-loaded cytoplasts.(ABSTRACT TRUNCATED AT 400 WORDS)
View details for Web of Science ID A1987G302900009
View details for PubMedID 3028535
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THE ROLE OF GLUTATHIONE-REDUCTASE IN MAINTAINING HUMAN GRANULOCYTE FUNCTION AND SENSITIVITY TO EXOGENOUS H2O2
BLOOD
1987; 69 (2): 493-500
Abstract
Human granulocytes (polymorphonuclear leukocytes, PMN) produce H2O2 and other reactive oxygen species while undergoing phagocytosis. To examine the role of the glutathione cycle in metabolizing H2O2, we incubated PMN with 1,3-bis (2-chloroethyl) nitrosourea (BCNU). Incubation of PMN with BCNU results in a dose-dependent inhibition of PMN glutathione reductase (GRED), with 50% inhibition occurring at approximately 2 micrograms/mL BCNU. PMN hexose monophosphate shunt activity stimulated with an exogenous H2O2-generating system was inhibited only when the GRED activity was reduced to less than 30% of control. BCNU-treated cells contained lower levels of reduced sulfhydryls and reduced glutathione, which decreased even more in the presence of an exogenous H2O2-generating system. The effect of BCNU and exogenous H2O2 on various aspects of phagocytosis were examined. Exposure of BCNU-treated PMN to an H2O2-generating system resulted in an inhibition of chemotactic peptide-induced shape changes and degranulation. The ability of BCNU-treated cells to produce O2- was diminished only when the PMN were incubated with an H2O2-generating system in the presence of cyanide. Ingestion of opsonized bacteria by BCNU-treated PMN was unaffected by incubation in an H2O2-generating system even in the presence of cyanide. We conclude that PMN GRED is inhibited by BCNU, the ability of PMN to metabolize H2O2 is affected only when GRED is reduced more than 70%, this inhibition affects the glutathione content of these cells, and some, but not all of the phagocytic functions of GRED-inhibited PMN are inhibited after exposure to an H2O2-generating system.
View details for Web of Science ID A1987F919300019
View details for PubMedID 3801665
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LYMPHOMA MODELS FOR B-CELL ACTIVATION AND TOLERANCE .5. ANTI-IG MEDIATED GROWTH-INHIBITION IS REVERSED BY PHORBOL-MYRISTATE ACETATE BUT DOES NOT INVOLVE CHANGES IN CYTOSOLIC FREE CALCIUM
JOURNAL OF MOLECULAR AND CELLULAR IMMUNOLOGY
1987; 3 (2): 109-120
Abstract
B cell lymphomas which can be growth inhibited by crosslinking their surface IgM receptors by anti-Ig reagents provide models for normal B cell regulation and tolerance. WEHI-231 and CH31 are two independently derived lines that are exquisitely sensitive to negative signalling by antibodies specific for mu or kappa chains, but are unaffected by antibodies against MHC class 1 or 2 antigens. In order to determine the mechanism of this growth inhibition as a model for tolerance, we have examined the roles played by protein kinase C activation and calcium mobilization/influx during negative signalling in these cells. We found that growth inhibition caused by anti-mu crosslinking was reversed in the presence of either phorbol myristate acetate (PMA) or by lipopolysaccharide (LPS) from E. coli. The effect of PMA on negative signalling was a true reversal since phorbol esters could be added after anti-mu treatment, thus allowing nearly normal cellular progression into the S phase of the cell cycle. In contrast, pretreatment with PMA did not provide protection against the growth inhibition from anti-mu. Indeed, a "desensitization" protocol demonstrated that PMA pretreatment actually decreased reversal by both PMA and LPS of the effects of anti-mu on B lymphoma growth. These studies suggest that both LPS and PMA act via at least one common intermediate, which is assumed to involve activation and translocation of protein kinase C. Analysis of changes in calcium ion concentration after treatment with anti-Ig reagents showed both mobilization from internal stores and influx via calcium channels in WEHI-231, as has been reported for normal B cells. However, these changes did not correlate with negative signalling for the several reasons. Firstly, anti-mu inhibition of the growth of WEHI-231 could be induced in the relative absence of extracellular Ca++ or in quin-2 loaded (buffered) cells. Secondly, pretreatment with high concentrations of PMA ablated calcium mobilization, yet failed to modulate growth inhibition in WEHI-231 cells. Moreover, LPS provided protection from the effects of anti-mu yet did not alter cellular [Cai++]. In addition, PMA posttreatment (under conditions causing a reversal of the effects of anti-mu) can be applied as long as four hours after the initial exposure to anti-mu and the rapid measurable changes in calcium flux. Indeed, such changes in intracellular free calcium occurred in elutriated WEHI-231 lymphoma cells at all phases of the cell cycle, although we have previously identified early G1 as the only critical period in which negative signalling can be delivered.(ABSTRACT TRUNCATED AT 400 WORDS)
View details for Web of Science ID A1987H561900005
View details for PubMedID 3509921
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Monoclonal antibodies to Fc receptors for IgG on human mononuclear phagocytes. Antibody characterization and induction of superoxide production in a monocyte cell line.
journal of biological chemistry
1986; 261 (27): 12856-12864
Abstract
We have utilized monoclonal antibodies against the two IgG Fc receptors (p40 and p72) of U937 cells to stimulate the release of superoxide. The monoclonal antibody (mAb) specific for p40 (IV3) has been described elsewhere. A murine IgG1 mAb specific for the high affinity p72 Fc receptor (designated mAb FcR32 or simply mAb 32) bound to the same p72 precipitated by Sepharose-human IgG as shown by preclearing experiments and by identical isoelectric focussing patterns. Binding of mAb 32 to p72 was independent of the Fc region of the antibody since Fab' fragments of mAb 32 affinity adsorbed p72. The binding of both mAb 32 and human IgG1 to the intact U937 cell was not reciprocally inhibitory, indicating that mAb 32 does not interfere with the ligand binding site of p72. mAb 32 bound to human monocytes, U937, and HL60 cells, but not to granulocytes or lymphocytes. U937 cells cultured in gamma-interferon and 1,25-dihydroxycholecalciferol generated superoxide when incubated with mAb 32 or IV3 followed by cross-linking with F(ab')2 anti-murine Ig. Incubation with mAb 32 or IV3 alone or with 3 of 5 other anti-U937 mAbs cross-linked with anti-murine Ig did not result in superoxide generation. Immune complex-mediated superoxide production was inhibited 80% by IgG, but not by mAb 32 or IV3.
View details for PubMedID 3017990
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ACTIVATION OF HUMAN-GRANULOCYTES BY ARACHIDONIC-ACID - ITS USE AND LIMITATIONS FOR INVESTIGATING GRANULOCYTE FUNCTIONS
BLOOD
1986; 67 (4): 1103-1109
Abstract
The effects of arachidonic acid on human granulocytes (polymorphonuclear neutrophil leukocytes, PMN) were examined with respect to early events associated with activation of the superoxide (O2-)-generating system and its reactivation with other stimuli after the addition of fatty acid-free bovine serum albumin. As with other stimuli, O2- production occurred after a lag that was dependent on the amount of arachidonic acid used. Although the lag, rate, and extent of O2- production were dose dependent, at all concentrations used, the duration of O2- production was four to six minutes. As previously described, when fatty acid-free albumin was added one minute after stimulation of PMN by arachidonic acid, O2- production ceased immediately. The O2(-)-generating system could then be reactivated by the addition of either phorbol myristate acetate (PMA), concanavalin A, or serum-treated zymosan. In previously activated cells, the lag time for reactivation was shorter and the rate of O2- production greater with PMA. PMN membrane potential was depolarized by arachidonic acid. This depolarization was not reversed with the addition of albumin. PMN cytoplasts were also capable of reversible activation by arachidonic acid in a manner identical to whole cells. Divalent cations were found to be necessary for the activation of PMN by arachidonic acid. In the absence of calcium, arachidonic acid caused rapid lysis of the cells, as manifested by the release of lactic acid dehydrogenase. We were unable to measure later events in PMN stimulated by arachidonic acid since even in the presence of divalent cations lactic acid dehydrogenase was released from the cells after five minutes of incubation. We conclude that, in addition to its ability to activate PMN, arachidonic acid is toxic to the cells and cannot be used to study late events in granulocyte activity, and, in the absence of calcium, it may be difficult to interpret its action as a stimulant of the oxidase.
View details for Web of Science ID A1986A807600041
View details for PubMedID 3006830
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MEMBRANE FLUIDITY AND PLATELET FIBRINOGEN RECEPTOR EXPOSURE BY PROTEOLYTIC-ENZYMES
THROMBOSIS RESEARCH
1985; 39 (1): 91-96
Abstract
Incubation of platelets with pronase or chymotrypsin results in the exposure of fibrinogen receptors. We determined that these enzymes did not affect the membrane fluidity as evaluated by the depolarization of the fluorescence of 1,6-diphenyl-1,3,5 hexatriene (DPH). There was no significant difference in either the depolarization or in its temperature dependence for control, pronase or chymotrypsin-treated platelets. Thus, it can be concluded that the exposure of fibrinogen receptors on the platelet surface by proteolytic enzymes does not depend on the changes of membrane fluidity. We also propose that the proteolytic enzymes do not cause a major alteration in the extent of protein chains embedded in the lipid layers of the platelet membranes.
View details for Web of Science ID A1985AKY2200009
View details for PubMedID 2994250
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DISSOCIATION BETWEEN AGGREGATION AND SUPEROXIDE PRODUCTION IN HUMAN-GRANULOCYTES
JOURNAL OF IMMUNOLOGY
1985; 134 (2): 1206-1211
Abstract
Aggregation and the activation of the granulocyte (PMN) superoxide (O2-) generating system occur when certain stimuli are added to resting cells. It had previously been postulated that PMN aggregation is essential for maximal O2- production. This study was undertaken to test the hypothesis that PMN aggregation is required for full expression of PMN O2- production. We examined aggregation and O2- production induced by four stimuli; concanavalin A (Con A), phorbol myristate acetate (PMA), N-formylmethionyl-leucyl-phenylalanine (FMLP), and ionophore A23187. Cytochalasin B enhanced aggregation by all four stimuli but only enhanced the rate of O2- production by Con A; 2-deoxyglucose inhibited aggregation by all stimuli. Dissociation of PMN aggregation and O2- production was achieved by using NEM, TPCK, and divalent cations. NEM and TPCK prevent Con A-induced O2- production but have no effect on Con A-induced aggregation. PMA-stimulated PMN generate O2- in the presence or absence of Ca++ and Mg++. In contrast, PMA stimulated maximum PMN aggregation only in the presence of both Ca++ and Mg++. Thus PMN can generate O2- without aggregating, and PMN can aggregate without producing O2-. PMN from patients with chronic granulomatous disease do not generate O2- or undergo membrane potential depolarization in response to PMA. These PMN aggregated when stimulated with PMA, providing evidence that depolarization is not required for PMN aggregation. We conclude that aggregation and the activation of the O2- generating system, though temporally related, are not necessarily causally related.
View details for Web of Science ID A1985AAL9200089
View details for PubMedID 2981262
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GRADED RESPONSES OF HUMAN-NEUTROPHILS INDUCED BY SERUM-TREATED ZYMOSAN
BLOOD
1985; 66 (5): 1182-1188
Abstract
A modified zymosan preparation was used to probe the interaction of particulate stimuli with human neutrophils (PMNs). After extraction with alkali and detergent, the zymosan particles retained their ability to be opsonized in serum and to stimulate PMNs. Serum-treated zymosan (STZ) induced dose-dependent superoxide (O2-) production and membrane potential depolarization in the range of 1 to 10 mg/mL of STZ. The rate and extent of secretion of lysozyme and beta-glucuronidase were also dose-dependent in the range of 1 to 10 mg/mL of STZ. Cytochemical studies using nitroblue tetrazolium, however, showed that 92% of PMNs were stimulated to produce O2- at 0.1 mg/mL of STZ. The dose response of O2- production induced by STZ is therefore due to increasing O2- production by individual PMNs and not to the stimulation of more PMNs to produce O2-. Evidence for O2- production was found only in the area of PMN-zymosan contact, suggesting a mechanism for the graded responses of PMNs treated with particulate stimuli. In order to determine the nature of the dose dependence of depolarization (a measure of PMN activation), PMNs equilibrated with the fluorescent probe 3,3'-dipentyloxacarbocyanine were analyzed by flow cytometry. The results demonstrate that STZ induces a dose-dependent depolarization of the membrane potential of individual PMNs. These results also demonstrate that increasing concentrations of STZ can induce increasing PMN responses even when all of the PMNs have been activated. These results are consistent with the hypothesis that receptor-mediated particulate stimulation of PMNs is a phenomenon that results in graded PMN responses.
View details for Web of Science ID A1985AUP7600028
View details for PubMedID 2996662
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DEVELOPMENT OF THE SUPEROXIDE-GENERATING SYSTEM DURING DIFFERENTIATION OF THE HL-60 HUMAN PROMYELOCYTIC LEUKEMIA-CELL LINE
JOURNAL OF BIOLOGICAL CHEMISTRY
1984; 259 (6): 3771-3776
Abstract
Utilizing the induced differentiation of HL-60 promyelocytic leukemia cells as a model of myeloid maturation, we examined the development of the superoxide-generating system, focusing on NADPH oxidase activity, membrane depolarization, and cytochrome b content. NADPH oxidase activity, measured as NADPH-dependent superoxide production, increased with both spontaneous and N,N-dimethylformamide-induced differentiation. Activity in particulate fractions from induced HL-60 cells and human peripheral blood polymorphonuclear leukocytes was proportional to their relative rates of superoxide production, but activity from uninduced cells was surprisingly high: one-third that from induced cells, despite only 7% their rate of superoxide generation. NADPH oxidase activities in phagocytic vesicles from induced HL-60 cells and polymorphonuclear leukocytes were equal, indicating the equivalence of the enzyme system in active portions of their cell membranes. Separation by centrifugal elutriation of the HL-60 cell population into fractions of varying maturity confirmed the relationship of NADPH oxidase activity to advancing differentiation in both dimethylformamide-induced and spontaneously maturing cells. Membrane potential change, an early event related to activation of the oxidase, was followed by 3,3'-dipropylthiodicarbocyanine dye fluorescence. The depolarization response increased dramatically in both magnitude and initial rate of change during differentiation. The cells' cytochrome b content increased 3-fold with induction of differentiation, in proportion to the change in NADPH oxidase activity.
View details for Web of Science ID A1984SJ39800060
View details for PubMedID 6323439
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IS ACTIVATION OF THE GRANULOCYTE BY CONCANAVALIN-A A REVERSIBLE PROCESS
BLOOD
1984; 63 (1): 114-120
Abstract
The stimulation of granulocyte O2- production by concanavalin-A can be reversed with alpha-methylmannoside. Such cells can be reactivated to generate O2- by adding phorbol myristate acetate or N-formyl-methionyl-leucyl-phenylalanine. Opsonized zymosan, however, is not an effective stimulant to these cells. alpha-Methylmannoside prevents, but does not reverse, depolarization of granulocytes by concanavalin-A. Previously activated cells have a shorter lag time for reactivation by phorbol myristate acetate. Incubation in 2-deoxyglucose of cells previously treated with concanavalin-A and alpha-methylmannoside prevents reactivation. EGTA prevents concanavalin-A-stimulated O2- production only when added prior to the stimulant. EGTA has only a slight effect on reactivation. alpha-Methylmannoside prevents concanavalin-A-stimulated release of lysozyme only when added prior to the stimulant. Prior treatment of cells with concanavalin-A and alpha-methylmannoside inhibits subsequent ingestion of complement-coated particles. We conclude that although the O2--generating system can be reversibly activated with concanavalin-A followed by alpha-methylmannoside, these cells are different from untreated cells. Cells treated in such a way do not respond to all stimuli, remain depolarized, have shortened lag times, no longer require calcium for activation, continue to degranulate, and do not ingest well. Thus, although some changes that accompany the interaction of stimuli with granulocytes are reversible, some are not, and the previously activated cell does not return to a true resting state.
View details for Web of Science ID A1984RZ10600014
View details for PubMedID 6317085
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IMPAIRED GRANULOCYTE SUPEROXIDE PRODUCTION AND PROLONGATION OF THE RESPIRATORY BURST DUE TO A LOW-AFFINITY NADPH-DEPENDENT OXIDASE
BLOOD
1983; 62 (3): 564-571
Abstract
A 7-yr-old girl with self-limited pulmonary aspergillosis was found to have a defect in granulocyte superoxide production. Her cells produced superoxide at 3% of control rates in response to phorbol myristate acetate (PMA) and opsonized zymosan. Lag times for O-2 production were normal with PMA, opsonized zymosan, and concanavalin-A stimulation. Her granulocyte membranes depolarized in response to all of these stimuli. Superoxide produced by podosomes and a particulate fraction demonstrated an enzyme activity with a normal maximal velocity but a decreased affinity for NADPH. NADH-dependent superoxide production by particles was similar with patient and control material. The duration of superoxide production was prolonged in the patient's intact granulocytes and in the particulate fractions from her cells. Bacterial killing by the patient's granulocytes was initially low, but approached control values after 90 min of incubation. These results are explained by an enzyme activity that has a decreased affinity for its substrate and a decreased rate of inactivation. Family studies indicate an autosomal recessive mode of inheritance.
View details for Web of Science ID A1983RG01000007
View details for PubMedID 6309280
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THE EFFECT OF VITAMIN-E-DEFICIENCY ON SOME PLATELET MEMBRANE-PROPERTIES
JOURNAL OF LIPID RESEARCH
1982; 23 (2): 276-282
Abstract
The effects of alpha-tocopherol (vitamin E) deficiency on membrane properties of platelets were studied to determine if vitamin E has a measureable stabilizing role in biological membranes. Three groups of rats and three of mice were studied: two groups consisted of Fisher strain rats and one of Sprague-Dawley rats fed a Draper corn oil diet with and without high levels of supplementary vitamin E. The mice were two groups of BALB/c animals maintained on an 8% hydrogenated coconut oil diet, and one group of CBA/J mice on an 8% lard diet, in each case either deficient in or supplemented with vitamin E. The relative content of fatty acids obtained from both rat platelets and erythrocytes was unchanged by vitamin E deficiency. Depletion of vitamin E had no effect on the degree of fluorescence polarization of 1,6-diphenyl-1,3,5,-hexatriene-labeled rat platelets. No changes in hematocrit values were seen in any of the studies. The platelet count of only the vitamin E-deficient Sprague-Dawley rats was elevated with respect to vitamin E-supplemented counterparts; the others remained constant. Platelet reactivities, as measured by ADP-and thrombin-induced platelet aggregation and by the thrombin-induced changes in platelet transmembrane potential, were unaffected by vitamin E deficiency in all three groups of rats. Our results indicate that a membrane stabilizing effect of vitamin E on rat platelet or erythrocyte membrane fatty acids or on platelet response to external stimuli could not be demonstrated, nor was elevation in platelet count a general phenomenon associated with vitamin E deficiency.-Whitin, J. C., R. K. Gordon, L. M. Corwin, and E. R. Simons. The effect of vitamin E deficiency on some platelet membrane properties.
View details for Web of Science ID A1982NF13700005
View details for PubMedID 7077141
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A VARIANT OF CHRONIC GRANULOMATOUS-DISEASE - DEFICIENT OXIDATIVE-METABOLISM DUE TO A LOW-AFFINITY NADPH OXIDASE
NEW ENGLAND JOURNAL OF MEDICINE
1981; 305 (22): 1329-1333
View details for Web of Science ID A1981MR01900007
View details for PubMedID 6270561
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OPSONIZED ZYMOSAN-STIMULATED GRANULOCYTES - ACTIVATION AND ACTIVITY OF THE SUPEROXIDE-GENERATING SYSTEM AND MEMBRANE-POTENTIAL CHANGES
BLOOD
1981; 58 (5): 975-982
Abstract
Phagocytic cells generate superoxide in response to stimulation by opsonized particles. A continuous assay for opsonized zymosan-stimulated granulocyte superoxide production shows that there is a lag time between the addition of particles and the onset of detectable superoxide production. Superoxide production is preceded by membrane potential depolarization. Neither superoxide production nor membrane depolarization occurs in granulocytes from patients with chronic granulomatous disease. The extent of activation by opsonized zymosan is affected by the dose of zymosan from 0.5 to 4.5 mg/ml, but the time necessary for activation (lag time) is not. Similarly, the extent of depolarization but not the time necessary for attaining maximum depolarization is concentration-dependent. Effects of temperature, divalent cations, 2-deoxyglucose, cyanide, and N-ethyl maleimide on superoxide production are similar for granulocytes treated with soluble stimuli and with opsonized zymosan. Thus, zymosan stimulates granulocytes to generate superoxide and undergo membrane depolarization in a manner similar to that elected by soluble stimuli.
View details for Web of Science ID A1981MQ07200019
View details for PubMedID 6271311
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EFFECTS OF THE MYELOPEROXIDASE SYSTEM ON FLUORESCENT-PROBES OF GRANULOCYTE MEMBRANE-POTENTIAL
JOURNAL OF BIOLOGICAL CHEMISTRY
1981; 256 (17): 8904-8906
Abstract
Activation of normal or myeloperoxidase-deficient human granulocytes by phorbol myristate acetate resulted in an initial membrane depolarization as indicated by an increase in fluorescence of the lipophilic cation probe of membrane potential, 3,3'-dipropylthiodicarbocyanine. A subsequent apparent hyperpolarization (decrease in fluorescence) was observed in normal but not myeloperoxidase-deficient cells. Addition of purified myeloperoxidase restored a normal pattern of fluorescence changes to the enzyme-deficient granulocytes. The secondary decrease in fluorescence in normal cells was markedly blunted by addition of azide, cyanide, or catalase. In a cell-free system, the fluorescence of 3,3'-dipropylthiodicarbocyanine, but not that of 3,3'-dipentyloxadicarbocyanine, was rapidly eliminated by myeloperoxidase in the presence of hydrogen peroxide and a halide; this loss of fluorescence was inhibited by azide, cyanide, or catalase. These findings indicate that secretion of myeloperoxidase and hydrogen peroxide by activated granulocytes results in decreased fluorescence of 3,3'-dipropylthiodicarbocyanine, probably by thioether oxidation. While the determination of initial rates of depolarization using this probe is unaffected by the myeloperoxidase system, measurement of extent of depolarization and any subsequent membrane potential changes requires the addition of inhibitors. In the absence of inhibitors, the secondary decrease in fluorescence can be used as an indicator of secretion of myeloperoxidase and hydrogen peroxide.
View details for Web of Science ID A1981MF70800013
View details for PubMedID 6267057
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CORRELATION BETWEEN MEMBRANE-POTENTIAL CHANGES AND SUPEROXIDE PRODUCTION IN HUMAN-GRANULOCYTES STIMULATED BY PHORBOL-MYRISTATE ACETATE - EVIDENCE FOR DEFECTIVE ACTIVATION IN CHRONIC GRANULOMATOUS-DISEASE
JOURNAL OF BIOLOGICAL CHEMISTRY
1980; 255 (5): 1874-1878
View details for Web of Science ID A1980JJ53800025
View details for PubMedID 6243652
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CIRCULAR-DICHROISM STUDIES OF CYANATE-INDUCED CONFORMATIONAL-CHANGES IN HEMOGLOBINS-A AND HEMOGLOBINS-S
BIOCHEMISTRY
1976; 15 (18): 4059-4064
Abstract
Circular dichroism and difference spectroscopy have been used to study dilute aqueous solutions of oxygenated, deoxygenated, and carbamoylated deoxygenated hemoglobins A and S (HbA and HbS, respectively). The spectra of HbA and HbS, in comparable state of oxygenation or carbamoylation, are identical, strongly implying identical conformations about the heme groups of the respective proteins. The spectra of the oxygenated forms change little upon addition of KCNO, which is known to carbamoylate the NH2 terminals of the individual chains (Cerami and Manning, 1971). The spectra of the deoxygenated forms, on the other hand, are markedly altered. The decreased magnitude of the 430-nm extremum with increased cyanate concentration can be used to calculate an addition curve which becomes asymptotic at a cyanate:heme molar ratio of approximately 10(3). This conformational change occurs in the absence of O2 and has been predicted (Njikam et al.,1973); it can also be demonstrated by difference spectroscopy techniques, whereby a comparable addition curve can be constructed from changes in the 555-nm absorption, while the 541-nm absorption remains invariant. The change described corresponds to the formation of a new conformation, corresponding to carbamoyldeoxyhemoglobin, carrying one carbamoyl group per chain. In the presence of a small quantity of oxygen, however, the above reported changes in CD are accompanied by a concomitant rise in the 415-nm peak-corresponding to the formation of oxyhemoglobin-while those in the difference spectra reflect not only a change in the 555-nm band but also a parallel one at 541 nm, confirming the formation of oxyhemoglobin. Thus the conformation achieved upon carbamoylation of deoxyhemoglobin has the higher oxygen affinity predicted by Nigen et al. (1974) for carbamoyldeoxyhemoglobin. Cyanate has been used (Cerami and Manning, 1971) as an antisickling reagent in vivo and in vitro, but, although it has been shown that it binds covalently to the NH2-terminal residues of hemoglobin (Lee and Manning, 1973), its effect on hemoglobin conformation has not been previously shown nor has its mechanism of action been fully clarified. The results presented here show that the effect of cyanate on hemoglobin is the formation of a new conformation with heightened oxygen affinity. Since oxyHbS does not aggregate while deoxyHbS does, in a temperature-dependent fashion, the formation of carbamoyldeoxyHbS interferes with such aggregation in vitro in deoxygenated samples. In vivo, where there are generally low residual concentrations of O2, the formation of oxyHb is favored by the higher O2 affinity of carbamoyldeoxyHbS, and aggregation with concomitant red cell sickling is therefore disfavored.
View details for Web of Science ID A1976CD09700022
View details for PubMedID 988840