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Stephen M. Beverley - One of the best experts on this subject based on the ideXlab platform.

  • SM: Pteridine salvage throughout the Leishmania infectious cycle: implications for antifolate chemotherapy
    2020
    Co-Authors: Mark L Cunningham, Stephen M. Beverley
    Abstract:

    Abstract Protozoan parasites of the trypanosomatid genus Leishmania are pteridine auxotrophs, and have evolved an elaborate and versatile pteridine salvage network capable of accumulating and reducing Pteridines. This includes biopterin and folate transporters (BT1 and FT1), pteridine reductase (PTR1), and dihydrofolate reductase-thymidylate synthase (DHFR-TS). Notably, PTR1 is a novel alternative pteridine reductase whose activity is resistant to inhibition by standard antifolates. In cultured promastigote parasites, PTR1 can function as a metabolic by-pass under conditions of DHFR inhibition and thus reduce the efficacy of chemotherapy. To test whether pteridine salvage occurred in the infectious stage of the parasite, we examined several pathogenic species of Leishmania and the disease-causing amastigote stage that resides within human macrophages. To accomplish this we developed a new sensitive HPLC-based assay for PTR1 activity. These studies established the existence of the pteridine salvage pathway throughout the infectious cycle of Leishmania, including amastigotes. In general, activities were not well correlated with RNA transcript levels, suggesting the occurrence of at least two different modes of post-transcriptional regulation. Thus, pteridine salvage by amastigotes may account for the clinical inefficacy of antifolates against leishmaniasis, and ultimately provide insights into how this may be overcome in the future

  • PTR1-dependent synthesis of tetrahydrobiopterin contributes to oxidant susceptibility in the trypanosomatid protozoan parasite Leishmania major
    Current Genetics, 2009
    Co-Authors: Bakela Nare, Levi A. Garraway, Tim J. Vickers, Stephen M. Beverley
    Abstract:

    Leishmania must survive oxidative stress, but lack many classical antioxidant enzymes and rely heavily on trypanothione-dependent pathways. We used forward genetic screens to recover loci mediating oxidant resistance via overexpression in Leishmania major , which identified pteridine reductase 1 (PTR1). Comparisons of isogenic lines showed ptr1 ^− null mutants were 18-fold more sensitive to H_2O_2 than PTR1-overproducing lines, and significant three- to fivefold differences were seen with a broad panel of oxidant-inducing agents. The toxicities of simple nitric oxide generators and other drug classes (except antifolates) were unaffected by PTR1 levels. H_2O_2 susceptibility could be modulated by exogenous biopterin but not folate, in a PTR1- but not dihydrofolate reductase-dependent manner, implicating H_4B metabolism specifically. Neither H_2O_2 consumption nor the level of intracellular oxidative stress was affected by PTR1 levels. Coupled with the fact that reduced Pteridines are at least 100-fold less abundant than cellular thiols, these data argue strongly that reduced Pteridines act through a mechanism other than scavenging. The ability of unconjugated Pteridines to counter oxidative stress has implications to infectivity and response to chemotherapy. Since the intracellular pteridine levels of Leishmania can be readily manipulated, these organisms offer a powerful setting for the dissection of pteridine-dependent oxidant susceptibility in higher eukaryotes.

  • characterization of quinonoid dihydropteridine reductase qdpr from the lower eukaryote leishmania major
    Journal of Biological Chemistry, 2002
    Co-Authors: Lonfye Lye, Mark Cunningham, Stephen M. Beverley
    Abstract:

    Biopterin is required for growth of the protozoan parasite Leishmania and is salvaged from the host through the activities of a novel biopterin transporter (BT1) and broad-spectrum pteridine reductase (PTR1). Here we characterize Leishmania major quinonoid-dihydropteridine reductase (LmQDPR), the key enzyme required for regeneration and maintenance of H4biopterin pools. LmQDPR shows good homology to metazoan quinonoiddihydropteridine reductase and conservation of domains implicated in catalysis and regulation. Unlike other organisms, LmQDPR is encoded by a tandemly repeated array of 8 –9 copies containing LmQDPR plus two other genes. QDPR mRNA and enzymatic activity were expressed at similar levels throughout the infectious cycle. The pH optima, kinetic properties, and substrate specificity of purified LmQDPR were found to be similar to that of other qDPRs, although it lacked significant activity for non-quinonoid Pteridines. These and other data suggest that LmQDPR is unlikely to encode the dihydrobiopterin reductase activity (PTR2) described previously. Similarly LmQDPR is not inhibited by a series of antifolates showing anti-leishmanial activity beyond that attributable to dihydrofolate reductase or PTR1 inhibition. qDPR activity was found in crude lysates of Trypanosoma brucei and Trypanosoma cruzi, further emphasizing the importance of H4biopterin throughout this family of human parasites.

  • Pteridine salvage throughout the Leishmania infectious cycle: implications for antifolate chemotherapy.
    Molecular and biochemical parasitology, 2001
    Co-Authors: Mark Cunningham, Stephen M. Beverley
    Abstract:

    Protozoan parasites of the trypanosomatid genus Leishmania are pteridine auxotrophs, and have evolved an elaborate and versatile pteridine salvage network capable of accumulating and reducing Pteridines. This includes biopterin and folate transporters (BT1 and FT1), pteridine reductase (PTR1), and dihydrofolate reductase‐thymidylate synthase (DHFR-TS). Notably, PTR1 is a novel alternative pteridine reductase whose activity is resistant to inhibition by standard antifolates. In cultured promastigote parasites, PTR1 can function as a metabolic by-pass under conditions of DHFR inhibition and thus reduce the efficacy of chemotherapy. To test whether pteridine salvage occurred in the infectious stage of the parasite, we examined several pathogenic species of Leishmania and the disease-causing amastigote stage that resides within human macrophages. To accomplish this we developed a new sensitive HPLC-based assay for PTR1 activity. These studies established the existence of the pteridine salvage pathway throughout the infectious cycle of Leishmania, including amastigotes. In general, activities were not well correlated with RNA transcript levels, suggesting the occurrence of at least two different modes of post-transcriptional regulation. Thus, pteridine salvage by amastigotes may account for the clinical inefficacy of antifolates against leishmaniasis, and ultimately provide insights into how this may be overcome in the future. © 2001 Elsevier Science B.V. All rights reserved.

  • new approaches to leishmania chemotherapy pteridine reductase 1 ptr1 as a target and modulator of antifolate sensitivity
    Parasitology, 1997
    Co-Authors: Bakela Nare, Larry W. Hardy, James Luba, Stephen M. Beverley
    Abstract:

    summary Leishmania and other trypanosomatid protozoa require reduced Pteridines (pterins and folates) for growth, suggesting that inhibition of these pathways could be targeted for eective chemotherapy. This goal has not yet been realized, indicating that pteridine metabolism may be unusual in this lower eukaryote. We have investigated this possibility using both wild type and laboratory-selected antifolate-resistant strains, and with defined genetic knockouts of several pteridine metabolic genes. In Leishmania, resistance to the antifolate methotrexate is mediated through several mechanisms singly or in combination, including alterations in transport leading to reduced drug influx, overproduction (R-region amplification) or point mutation of dihydrofolate reductase-thymidylate synthase (DHFR-TS), and amplification of a novel pteridine reductase (PTR 1, encoded by the H-region). All of the proteins involved are potential targets for antifolate chemotherapy. Notably, parasites in which the gene encoding dihydrofolate reductase (DHFR) has been deleted (dhfr-ts’ knockouts) do not survive in animal models, validating this enzyme as a target for eective chemotherapy. However, the properties of pteridine reductase 1 (PTR1) suggest a reason why antifolate chemotherapy has so far not been successful in trypanosomatids. PTR1, by its ability to provide reduced pterins and folates, has the potential to act as a by-pass and}or modulator of DHFR inhibition under physiological conditions. Moreover, PTR1 is less sensitive to many antifolates targeted primarily against DHFR. These findings suggest that successful antifolate chemotherapy in Leishmania will have to target simultaneously both DHFR and PTR1.

Casey Burton - One of the best experts on this subject based on the ideXlab platform.

  • The role of urinary Pteridines as disease biomarkers
    Pteridines, 2017
    Co-Authors: Casey Burton
    Abstract:

    AbstractPteridines and their derivatives function as intermediates in the metabolism of several vitamins and cofactors, and their relevance to disease has inspired new efforts to study their roles as disease biomarkers. Recent analytical advances, such as the emergence of sensitive mass spectrometry techniques, new workflows for measuring pteridine derivatives in their native oxidation states and increased multiplexing capacities for the simultaneous determination of many pteridine derivatives, have enabled researchers to explore the roles of urinary Pteridines as disease biomarkers at much lower levels with greater accuracy than with previous technologies or methods. As a result, urinary Pteridines are being increasingly studied as putative cancer biomarkers with promising results being reported from exploratory studies. In addition, the role of urinary neopterin as a universal biomarker for immune system activation is being investigated in new diseases where it is anticipated to become a useful supplementary marker in clinical diagnostic settings. In summary, this review provides an overview of recent developments in the clinical study of urinary Pteridines as disease biomarkers, covers the most promising aspects of advanced analytical techniques being developed for the determination of urinary Pteridines and discusses the major challenges associated with implementing pteridine biomarkers in clinical laboratory settings.

  • Development of a high-performance liquid chromatography - Tandem mass spectrometry urinary pterinomics workflow.
    Analytica chimica acta, 2016
    Co-Authors: Casey Burton, Honglan Shi
    Abstract:

    Abstract Pteridines have evoked considerable interest from the scientific community owing to their prominent roles in human health and disease. The availability of analytical methodologies suitable for comprehensive pteridine profiling, termed here as “pterinomics”, has been limited by inconsistent sample preparation and the exclusion of lesser studied pteridine derivatives. In response, the present study describes a new pterinomics workflow using a high-performance liquid chromatography – tandem mass spectrometry (HPLC-MS/MS) methodology for the simultaneous analysis of 15 pteridine derivatives including four structural isomers, marking the largest quantitative pteridine panel that has been studied to-date. The validated method possessed excellent sensitivity with method detection limits (0.025 μg L−1 to 0.5 μg L−1) that were comparable or superior to existing techniques. Spiked recovery studies demonstrated the technique was both accurate (88–112%) and precise (RSD: 0–6%). A comparative study of commonly used oxidative pretreatments, including triiodide, permanganate, and manganese dioxide, revealed that the oxidative mechanisms were inefficient, complex, and concentration dependent. Finally, 50 clinical urine specimens were examined with the new technique wherein 10 pteridine derivatives were quantified and population ranges have been given. This technique can be used to examine pteridine molecular epidemiology and biochemistry to support related research applications, and may further be readily extended to include additional pteridine derivatives and biological matrices for specific applications.

  • Daily variation and effect of dietary folate on urinary Pteridines
    Metabolomics, 2016
    Co-Authors: Casey Burton, Yinfa Ma
    Abstract:

    Introduction Urinary Pteridines are putative molecular biomarkers for noninvasive cancer screening and prognostication. Central to their translational biomarker development is the need to understand the sources and extent of their non-epidemiological variation. Objectives This study was designed to characterize the two primary sources of urinary pteridine variance: daily variation and the effect of dietary folate. Methods Daily variation was studied by collecting urine specimens ( n  = 81) three times daily for 3 days. The effect of dietary folate was investigated in a treatment study in which urine specimens ( n  = 168) were collected daily during a control week and a treatment week during which participants received dietary folate supplements. Measurements of six urinary Pteridines were made using high-performance liquid chromatography–tandem mass spectrometry. Coefficients of variation were calculated to characterize daily variance between and within subjects, while nearest neighbor non-parametric analyses were used to identify diurnal patterns and measure dietary folate effects. Results Daily variance was approximately 35 % RSD for both within-day and between-day periods for most Pteridines. Diurnal patterns in response to circadian rhythms were similarly observed for urinary Pteridines. Folate supplementation was shown to alter urinary pteridine profiles in a pathway dependent manner, suggesting that dietary folate may regulate endogenous neopterin and biopterin biosynthesis. Conclusions Urinary pteridine levels were found to be responsive to both daily variation and folate supplementation. These findings provide new insights into pteridine biosynthesis and regulation as well as useful information for the design of future clinical translational research.

  • normalization of urinary Pteridines by urine specific gravity for early cancer detection
    Clinica Chimica Acta, 2014
    Co-Authors: Casey Burton
    Abstract:

    Abstract Background Urinary biomarkers, such as Pteridines, require normalization with respect to an individual's hydration status and time since last urination. Conventional creatinine-based corrections are affected by a multitude of patient factors whereas urine specific gravity (USG) is a bulk specimen property that may better resist those same factors. We examined the performance of traditional creatinine adjustments relative to USG to six urinary Pteridines in aggressive and benign breast cancers. Methods 6-Biopterin, neopterin, pterin, 6-hydroxymethylpterin, isoxanthopterin, xanthopterin, and creatinine were analyzed in 50 urine specimens with a previously developed liquid chromatography–tandem mass spectrometry technique. Creatinine and USG performance were evaluated with non-parametric Mann–Whitney hypothesis testing. Results USG and creatinine were moderately correlated (r = 0.857) with deviations occurring in dilute and concentrated specimens. In 48 aggressive and benign breast cancers, normalization by USG significantly outperformed creatinine adjustments which marginally outperformed uncorrected Pteridines in predicting pathological status. In addition, isoxanthopterin and xanthopterin were significantly higher in pathological specimens when normalized by USG. Conclusion USG, as a bulk property, can provide better performance over creatinine-based normalizations for urinary Pteridines in cancer detection applications.

Honglan Shi - One of the best experts on this subject based on the ideXlab platform.

  • Development of a high-performance liquid chromatography - Tandem mass spectrometry urinary pterinomics workflow.
    Analytica chimica acta, 2016
    Co-Authors: Casey Burton, Honglan Shi
    Abstract:

    Abstract Pteridines have evoked considerable interest from the scientific community owing to their prominent roles in human health and disease. The availability of analytical methodologies suitable for comprehensive pteridine profiling, termed here as “pterinomics”, has been limited by inconsistent sample preparation and the exclusion of lesser studied pteridine derivatives. In response, the present study describes a new pterinomics workflow using a high-performance liquid chromatography – tandem mass spectrometry (HPLC-MS/MS) methodology for the simultaneous analysis of 15 pteridine derivatives including four structural isomers, marking the largest quantitative pteridine panel that has been studied to-date. The validated method possessed excellent sensitivity with method detection limits (0.025 μg L−1 to 0.5 μg L−1) that were comparable or superior to existing techniques. Spiked recovery studies demonstrated the technique was both accurate (88–112%) and precise (RSD: 0–6%). A comparative study of commonly used oxidative pretreatments, including triiodide, permanganate, and manganese dioxide, revealed that the oxidative mechanisms were inefficient, complex, and concentration dependent. Finally, 50 clinical urine specimens were examined with the new technique wherein 10 pteridine derivatives were quantified and population ranges have been given. This technique can be used to examine pteridine molecular epidemiology and biochemistry to support related research applications, and may further be readily extended to include additional pteridine derivatives and biological matrices for specific applications.

  • pteridine analysis in urine by capillary electrophoresis using laser induced fluorescence detection
    Analytical Chemistry, 1999
    Co-Authors: Futian Han, Bryan H Huynh, Honglan Shi, Bingcheng Lin
    Abstract:

    Pteridines are a class of compounds excreted in urine, the levels of which are found to elevate significantly in tumor-related diseases. For the first time, we have developed a method, based on high-performance capillary electrophoresis (HPCE) and laser-induced fluorescence (LIF) detection, to monitor the pteridine levels in urine. HPCE provides better separation than high-performance liquid chromatography and the LIF detector enables us to detect minute amounts of Pteridines in body fluid. Eight different pteridine derivatives were well separated in 0.1 M Tris-0.1 M borate-2 mM EDTA buffer (pH 8.75) using a 60-cm fused-silica capillary (50-micron i.d., 35-cm effective length), six of which were detected and characterized in urine samples from normal persons and different cancer patients. The detection limits of these Pteridines are under 1 x 10(-10) M. The levels of neopterin, pterine, xanthopterin, and pterin-6-carboxylic acid were found to be significantly elevated in urine excreted by cancer patents, while the level of isoxanthopterin dropped in these patients. No significant change of biopterin level was found between healthy individuals and cancer patients. This method can be used in clinical laboratories either for cancer monitoring or for precancer screening.

Ma Yinfa - One of the best experts on this subject based on the ideXlab platform.

  • High-throughput intracellular pteridinic profiling by liquid chromatography-quadrupole time-of-flight mass spectrometry
    analytica chimica acta, 2015
    Co-Authors: Burton Casey, Weng Rui, Li Yang, Yu Bai, Liu Huwei, Ma Yinfa
    Abstract:

    Pteridines are a diverse family of endogenous metabolites that may serve as useful diagnostic biomarkers for disease. While many preparative and analytical techniques have been described for analysis of selected Pteridines in biological fluids, broad intracellular pteridine detection remains a significant analytical challenge. In this study, a novel, specific and sensitive extraction and high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (HPLC-QTOF MS) method was developed to simultaneously quantify seven intracellular Pteridines and monitor 18 additional, naturallyoccurring intracellular Pteridines. The newly developed method was validated through evaluation of spiked recoveries (84.5-109.4%), reprodu cibility (2.1-5.4% RSD), method detection limits (0.1-3.0 mu g L-1) and limits of quantitation (0.1-1 mu g L-1), and finally application to non-small cell lung cancer A549 cells. Twenty-three pteridine derivatives were successfully detected from cell lysates with an average RSD of 12% among culture replicates. Quantified intracellular pteridine levels ranged from 1 to 1000 nM in good agreement with previous studies. Finally, this technique may be applied to cellular studies to generate new biological hypotheses concerning pteridine physiological and pathological functions as well as to discovery new pteridine-based biomarkers. (C) 2014 Elsevier B.V. All rights reserved.Chemistry, AnalyticalSCI(E)0ARTICLEyu.bai@pku.edu.cn; yinfa@mst.edu442-45085

  • Simultaneous Detection of Six Urinary Pteridines and Creatinine by High-Performance Liquid Chromatography-Tandem Mass Spectrometry for Clinical Breast Cancer Detection
    'American Chemical Society (ACS)', 2013
    Co-Authors: Burton Casey, Shi Honglan, Ma Yinfa
    Abstract:

    Recent preliminary studies have implicated urinary Pteridines as candidate biomarkers in a growing number of malignancies including breast cancer. While the developments of capillary electrophoresis-laser induced fluorescence (CE-LIF), high performance liquid chromatography (HPLC), and liquid chromatography-mass spectroscopy (LC-MS) pteridine urinalyses among others have helped to enable these findings, limitations including poor pteridine specificity, asynchronous or nonexistent renal dilution normalization, and a lack of information regarding adduct formation in mass spectrometry techniques utilizing electrospray ionization (ESI) have prevented application of these techniques to a larger clinical setting. In this study, a simple, rapid, specific, and sensitive high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method has been developed and optimized for simultaneous detection of six Pteridines previously implicated in breast cancer and creatinine as a renal dilution factor in urine. In addition, this study reports cationic adduct formation of urinary Pteridines under ESI-positive ionization for the first time. This newly developed technique separates and detects the following six urinary Pteridines: 6-biopterin, 6- hydroxymethylpterin, d-neopterin, pterin, isoxanthopterin, and xanthopterin, as well as creatinine. The method detection limit for the Pteridines is between 0.025 and 0.5 µg/L, and for creatinine, it is 0.15 µg/L. The method was also validated by spiked recoveries (81-105%), reproducibility (RSD: 1-6%), and application to 25 real urine samples from breast cancer positive and negative samples through a double-blind study. The proposed technique was finally compared directly with a previously reported CE-LIF technique, concluding that additional or alternative renal dilution factors are needed for proper investigation of urinary Pteridines as breast cancer biomarkers

  • Early cancer screening with P-scan technology
    2010
    Co-Authors: Ma Yinfa
    Abstract:

    Advances in Biomaterials and Medical Devices PanelEarly detection and treatment of cancers continue to be the best line of defense to prevent fatalities as a result of developing cancer. Several tests have been developed, such as mammograms for breast cancer and colonoscopies for colon cancer, but most of these tests are invasive, uncomfortable and are not always able to detect cancer earlier enough to provide a winning scenario for patients. Some cancers are still very difficult to diagnose in the early stage, such as ovarian cancer, colorectal cancer, and some liver cancers. In our study, we have developed a P-scan instrument to successfully identify and quantify six Pteridines in urine samples. The data demonstrated a great correlation between pteridine levels and cancer development. The detailed experimental conditions will be presented at the life science summit. After analysis of over 60 urine samples from cancer patients, we also discovered that two chemical components exist only in the urine of cancer patients but are not present in individuals who do not have cancers

Burton Casey - One of the best experts on this subject based on the ideXlab platform.

  • High-throughput intracellular pteridinic profiling by liquid chromatography-quadrupole time-of-flight mass spectrometry
    analytica chimica acta, 2015
    Co-Authors: Burton Casey, Weng Rui, Li Yang, Yu Bai, Liu Huwei, Ma Yinfa
    Abstract:

    Pteridines are a diverse family of endogenous metabolites that may serve as useful diagnostic biomarkers for disease. While many preparative and analytical techniques have been described for analysis of selected Pteridines in biological fluids, broad intracellular pteridine detection remains a significant analytical challenge. In this study, a novel, specific and sensitive extraction and high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (HPLC-QTOF MS) method was developed to simultaneously quantify seven intracellular Pteridines and monitor 18 additional, naturallyoccurring intracellular Pteridines. The newly developed method was validated through evaluation of spiked recoveries (84.5-109.4%), reprodu cibility (2.1-5.4% RSD), method detection limits (0.1-3.0 mu g L-1) and limits of quantitation (0.1-1 mu g L-1), and finally application to non-small cell lung cancer A549 cells. Twenty-three pteridine derivatives were successfully detected from cell lysates with an average RSD of 12% among culture replicates. Quantified intracellular pteridine levels ranged from 1 to 1000 nM in good agreement with previous studies. Finally, this technique may be applied to cellular studies to generate new biological hypotheses concerning pteridine physiological and pathological functions as well as to discovery new pteridine-based biomarkers. (C) 2014 Elsevier B.V. All rights reserved.Chemistry, AnalyticalSCI(E)0ARTICLEyu.bai@pku.edu.cn; yinfa@mst.edu442-45085

  • Simultaneous Detection of Six Urinary Pteridines and Creatinine by High-Performance Liquid Chromatography-Tandem Mass Spectrometry for Clinical Breast Cancer Detection
    'American Chemical Society (ACS)', 2013
    Co-Authors: Burton Casey, Shi Honglan, Ma Yinfa
    Abstract:

    Recent preliminary studies have implicated urinary Pteridines as candidate biomarkers in a growing number of malignancies including breast cancer. While the developments of capillary electrophoresis-laser induced fluorescence (CE-LIF), high performance liquid chromatography (HPLC), and liquid chromatography-mass spectroscopy (LC-MS) pteridine urinalyses among others have helped to enable these findings, limitations including poor pteridine specificity, asynchronous or nonexistent renal dilution normalization, and a lack of information regarding adduct formation in mass spectrometry techniques utilizing electrospray ionization (ESI) have prevented application of these techniques to a larger clinical setting. In this study, a simple, rapid, specific, and sensitive high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method has been developed and optimized for simultaneous detection of six Pteridines previously implicated in breast cancer and creatinine as a renal dilution factor in urine. In addition, this study reports cationic adduct formation of urinary Pteridines under ESI-positive ionization for the first time. This newly developed technique separates and detects the following six urinary Pteridines: 6-biopterin, 6- hydroxymethylpterin, d-neopterin, pterin, isoxanthopterin, and xanthopterin, as well as creatinine. The method detection limit for the Pteridines is between 0.025 and 0.5 µg/L, and for creatinine, it is 0.15 µg/L. The method was also validated by spiked recoveries (81-105%), reproducibility (RSD: 1-6%), and application to 25 real urine samples from breast cancer positive and negative samples through a double-blind study. The proposed technique was finally compared directly with a previously reported CE-LIF technique, concluding that additional or alternative renal dilution factors are needed for proper investigation of urinary Pteridines as breast cancer biomarkers