Showing papers on "Methylglyoxal published in 2009"
TL;DR: Addition of dihydroxyacetone to clover honey followed by incubation resulted in methylglyoxal levels similar to those found in manuka honey, which was freshly produced by bees, and storage of these honeys at 37 degrees C led to a decrease in the dihydroxacetone content and a related increase in methyl glyoxal.
271 citations
TL;DR: Measurements of evaporation fractions can be used to estimate the global aerosol formation potential of glyoxal and methylglyoxal via self-reactions at 1 and 1.6 Tg C yr(-1), respectively, which is a factor of 4 less than the SOA formed by these compounds if their uptake is assumed to be irreversible.
Abstract: Glyoxal and methylglyoxal are scavenged by clouds, where a fraction of these compounds are oxidized during the lifetime of the droplet. As a cloud droplet evaporates, the remaining glyoxal and methylglyoxal must either form low-volatility compounds such as oligomers and remain in the aerosol phase, or transfer back to the gas phase. A series of experiments on evaporating aqueous aerosol droplets indicates that over the atmospherically relevant concentration range for clouds and fog (4-1000 microM), 33 +/- 11% of glyoxal and 19 +/- 13% of methylglyoxal remains in the aerosol phase while the remainder evaporates. Measurements of aerosol density and time-dependent AMS signal changes are consistent with the formation of oligomers by each compound during the drying process. Unlike glyoxal, which forms acetal oligomers, exact mass AMS data indicates that the majority of methylglyoxal oligomers are formed by aldol condensation reactions, likely catalyzed by pyruvic acid, formed from methylglyoxal disproportionation. Our measurements of evaporation fractions can be used to estimate the global aerosol formation potential of glyoxal and methylglyoxal via self-reactions at 1 and 1.6 Tg C yr(-1), respectively. This is a factor of 4 less than the SOA formed by these compounds if their uptake is assumed to be irreversible. However, these estimates are likely lower limits for their total aerosol formation potential because oxidants and amines will also react with glyoxal and methylglyoxal to form additional low-volatility products.
180 citations
TL;DR: The present study concentrates on the evaluation of the anti-glycation effect of some bioactive substances present in yerba maté (Ilex paraguariensis): 5-caffeoylquinic acid, caffeic acid and a sapogenin (oleanolic acid).
177 citations
Journal Article•
TL;DR: The stress-induced increases in methylglyoxal level, glyoxalase I activity and Gly I transcript found in the present study suggest an important role of glyoxAlase I in conferring tolerance to plants under stress conditions and show that the gly oxalase pathway is the main detoxification pathway of methylgly oxal in plants.
Abstract: Abiotic stresses cause extensive losses to agricultural production worldwide. In this study, the effects of various abiotic stresses on the upregulation of methylglyoxal levels and glyoxalase I activities in pumpkin seedlings (Cucurbita maxima Duch.) were investigated. Most of the stresses caused significant increases in methylglyoxal level and glyoxalase I activity, white light causing the highest induction followed by salinity, chemical, drought, and heavy metal stresses. We showed that accumulation of methylglyoxal in plants under various stressful conditions is a common phenomenon, and methylglyoxal could therefore act as a signal for plants to respond to stress. The stress-induced increases in methylglyoxal level, glyoxalase I activity and Gly I transcript found in the present study suggest an important role of glyoxalase I in conferring tolerance to plants under stress conditions and showed that the glyoxalase pathway is the main detoxification pathway of methylglyoxal in plants. The multistress response of glyoxalase I gene indicates its future utility in developing tolerance to various stresses in crop plants. A cDNA encoding glyoxalase I has been isolated, subcloned and nucleotide sequence was determined. The pumpkin glyoxalase I cDNA consists of 975-bp nucleotides encoding a polypeptide of 185 amino acids having a predicted molecular weight of 20,772.14 Da. Based on the number of amino acids, it is categorized as short-type glyoxalase I and the nucleotide sequence of pumpkin glyoxalase I showed significant homology with other known glyoxalase I sequences of plants.
174 citations
TL;DR: Early glycation and AGE residue content of endoneurial ECM proteins increase markedly in STZ-induced diabetes, which may provide a mechanism for the failure of collateral sprouting and axonal regeneration in diabetic neuropathy.
Abstract: OBJECTIVE: The goal of this study was to characterize glycation adducts formed in both in vivo extracellular matrix (ECM) proteins of endoneurium from streptozotocin (STZ)-induced diabetic rats and in vitro by glycation of laminin and fibronectin with methylglyoxal and glucose. We also investigated the impact of advanced glycation end product (AGE) residue content of ECM on neurite outgrowth from sensory neurons. RESEARCH DESIGN AND METHODS: Glycation, oxidation, and nitration adducts of ECM proteins extracted from the endoneurium of control and STZ-induced diabetic rat sciatic nerve (3-24 weeks post-STZ) and of laminin and fibronectin that had been glycated using glucose or methylglyoxal were examined by liquid chromatography with tandem mass spectrometry. Methylglyoxal-glycated or unmodified ECM proteins were used as substrata for dissociated rat sensory neurons as in vitro models of regeneration. RESULTS: STZ-induced diabetes produced a significant increase in early glycation N(epsilon)-fructosyl-lysine and AGE residue contents of endoneurial ECM. Glycation of laminin and fibronectin by methylglyoxal and glucose increased glycation adduct residue contents with methylglyoxal-derived hydroimidazolone and N(epsilon)-fructosyl-lysine, respectively, of greatest quantitative importance. Glycation of laminin caused a significant decrease in both neurotrophin-stimulated and preconditioned sensory neurite outgrowth. This decrease was prevented by aminoguanidine. Glycation of fibronectin also decreased preconditioned neurite outgrowth, which was prevented by aminoguanidine and nerve growth factor. CONCLUSIONS: Early glycation and AGE residue content of endoneurial ECM proteins increase markedly in STZ-induced diabetes. Glycation of laminin and fibronectin causes a reduction in neurotrophin-stimulated neurite outgrowth and preconditioned neurite outgrowth. This may provide a mechanism for the failure of collateral sprouting and axonal regeneration in diabetic neuropathy.
164 citations
TL;DR: The use of lignocellulose as a source of sugars for bioproducts requires the development of biocatalysts that maximize product yields by fermenting mixtures of hexose and pentose sugars to completion.
Abstract: The use of lignocellulose as a source of sugars for bioproducts requires the development of biocatalysts that maximize product yields by fermenting mixtures of hexose and pentose sugars to completion. In this study, we implicate mgsA encoding methylglyoxal synthase (and methylglyoxal) in the modulation of sugar metabolism. Deletion of this gene (strain LY168) resulted in the co-metabolism of glucose and xylose, and accelerated the metabolism of a 5-sugar mixture (mannose, glucose, arabinose, xylose and galactose) to ethanol.
120 citations
TL;DR: The Arabidopsis thaliana locus At5g06580 encodes an ortholog to Saccharomyces cerevisiae d-lactate dehydrogenase (AtD-LDH), andGene coexpression analysis indicated that AtD-2HGDH is in the same network as several genes involved in β-oxidation and degradation of branched-chain amino acids and chlorophyll.
119 citations
TL;DR: The data demonstrated that mitochondrial function is under the control of MG, and discoveries will help unmask molecular mechanisms for various MG-induced mitochondrial dysfunction-related cellular disorders.
103 citations
TL;DR: Aldose reductase–catalyzed reduction is an important pathway in the endothelial and cardiac metabolism of AGE precursors, and it prevents AGE accumulation and atherosclerotic lesion formation.
Abstract: Objective: To examine the role of aldo-keto reductases (AKRs) in the cardiovascular metabolism of the precursors of advanced glycation end products (AGEs). Research Design and Methods: Steady-state kinetic parameters of AKRs with AGE precursors were determined using recombinant proteins expressed in bacteria. Metabolism of methylglyoxal and AGE accumulation were studied in human umbilical vein endothelial cells (HUVECs), C57-wild-type, akr1b3 (aldose reductase, AR)-null and cardiospecific akr1b4 (rat AR) and akr1b8 -(FR-1) transgenic mice. AGE accumulation and atherosclerotic lesions were studied 12 weeks after streptozotocin-treatment of C57, akr1b3 -null, and apoE - and akr1b3 - apoE -null mice. Results: Higher levels of AGEs were generated in the cytosol than at the external surface of HUVECs cultured in high glucose, indicating that intracellular metabolism may be an important regulator of AGE accumulation and toxicity. In vitro , AKR 1A and 1B catalyzed the reduction of AGE precursors, whereas AKR1C, AKR6, and AKR7 were relatively ineffective. Highest catalytic efficiency was observed with AKR1B1. Acetol formation in methylglyoxal-treated HUVECs was prevented by the AR inhibitor sorbinil. Acetol was generated in hearts perfused with methylglyoxal and its formation was increased in akr1b4 - or akr1b8 -transgenic mice. Reduction of AGE precursors was diminished in hearts from akr1b3 -null mice. Diabetic akr1b3 -null mice accumulated more AGEs in the plasma and the heart than WT mice and deletion of akr1b3 increased AGE accumulation and atherosclerotic lesion formation in apoE -null mice. Conclusion: Aldose reductase-catalyzed reduction is an important pathway in the endothelial and cardiac metabolism of AGE precursors and it prevents AGE accumulation and atherosclerotic lesion formation.
99 citations
TL;DR: This work tested several protocols on different biological samples, which resulted in significant differences in MG values measured in a given sample, and recommended protocols that provide consistent values of MG in biological samples are recommended.
92 citations
TL;DR: Evidence is provided for methylglyoxal as one of the causative factors in the pathogenesis of insulin resistance and salt-sensitive hypertension by increasing oxidative stress and/or AGEs formation in Sprague–Dawley rats.
Abstract: ObjectivesMethylglyoxal, a metabolite of the glycolysis pathway, may play an important role in the development of diabetes and hypertension, but the exact mechanism has not been fully elucidated. The present study was designed to investigate whether methylglyoxal could directly induce insulin resist
TL;DR: Glyoxalase I exerts renoprotective effects in renal I/R injury via a reduction in MG accumulation in tubular cells, and is significantly ameliorated in association with a decrease in intracellular MG adduct accumulation, oxidative stress, and tubular cell apoptosis.
Abstract: Methylglyoxal (MG), a highly reactive carbonyl compound generated by carbohydrate oxidation and glycolysis, is the major precursor of protein glycation and induces cytotoxicity leading to apoptosis...
TL;DR: The toxicity of fructose towards hepatocytes is assessed and the molecular cytotoxic mechanisms involved are investigated and it is proposed that the highly potent Fenton derived ROS catalyse the oxidation of fructose and particularly its carbonyl metabolites glycolaldehyde, dihydroxyacetone, glyceraldehyde.
TL;DR: It is suggested that proline and betaine might provide protection to cold stress in tea by regulating MG and lipid peroxidation formation as well as by activating or protecting some of antioxidant and glyoxalase pathway enzymes.
Abstract: The aim of this study was to monitor the influence of proline and betaine exposure on antioxidant and methylglyoxal (MG) detoxification system during cold stress in Camellia sinensis (L.) O. Kuntze. Cold stress enhanced MG and lipid peroxidation levels in tea bud (youngest topmost leaf). This increase was resisted upon the exposure of tea bud to proline and betaine. Exposure of tea bud with proline and betaine also help in maintaining thiol/disulfide ratio during cold stress. Proline exposure enhanced glutathione-S-transferase and glutathione reductase (GR) activity, while betaine exposure increased only GR activity during cold stress. Furthermore, effect of proline/betaine was studied on glyoxalase pathway enzymes that are involved in MG detoxification and comprise of two enzymes glyoxalase I and glyoxalase II. Both proline and betaine showed protective effect on glyoxalase I and activating effect on glyoxalase II during cold stress in tea bud. This investigation, therefore, suggest that proline and betaine might provide protection to cold stress in tea by regulating MG and lipid peroxidation formation as well as by activating or protecting some of antioxidant and glyoxalase pathway enzymes.
TL;DR: The results suggest that secondary oxidation processes involving dienedial and epoxide primary products are likely responsible for previous observations of glyoxal and methylglyoxal products from toluene oxidation.
Abstract: The products of the primary OH-initiated oxidation of toluene were investigated using the turbulent flow chemical ionization mass spectrometry technique at temperatures ranging from 228 to 298 K. A major dienedial-producing pathway was detected for the first time for toluene oxidation, and glyoxal and methylglyoxal were found to be minor primary oxidation products. The results suggest that secondary oxidation processes involving dienedial and epoxide primary products are likely responsible for previous observations of glyoxal and methylglyoxal products from toluene oxidation. Because the dienedial-producing pathway is a null cycle for tropospheric ozone production and glyoxal and methylglyoxal are important secondary organic aerosol precursors, these new findings have important implications for the modeling of toluene oxidation in the atmosphere.
TL;DR: It is proposed that the predominant mechanism for methylglyoxal detoxification in the African trypanosome is via the methyl Glyoxal reductase pathway to l‐lactate.
Abstract: The glyoxalase system, comprising the metalloenzymes glyoxalase I (GLO1) and glyoxalase II (GLO2), is an almost universal metabolic pathway involved in the detoxification of the glycolytic byproduct methylglyoxal to d-lactate. In contrast to the situation with the trypanosomatid parasites Leishmania major and Trypanosoma cruzi, this trypanothione-dependent pathway is less well understood in the African trypanosome, Trypanosoma brucei. Although this organism possesses a functional GLO2, no apparent GLO1 gene could be identified in the T. brucei genome. The absence of GLO1 in T. brucei was confirmed by the lack of GLO1 activity in whole cell extracts, failure to detect a GLO1-like protein on immunoblots of cell lysates, and lack of d-lactate formation from methylglyoxal as compared to L. major and T. cruzi. T. brucei procyclics were found to be 2.4-fold and 5.7-fold more sensitive to methylglyoxal toxicity than T. cruzi and L. major, respectively. T. brucei also proved to be the least adept of the 'Tritryp' parasites in metabolizing methylglyoxal, producing l-lactate rather than d-lactate. Restoration of a functional glyoxalase system by expression of T. cruzi GLO1 in T. brucei resulted in increased resistance to methylglyoxal and increased conversion of methylglyoxal to d-lactate, demonstrating that GLO2 is functional in vivo. Procyclic forms of T. brucei possess NADPH-dependent methylglyoxal reductase and NAD(+)-dependent l-lactaldehyde dehydrogenase activities sufficient to account for all of the methylglyoxal metabolized by these cells. We propose that the predominant mechanism for methylglyoxal detoxification in the African trypanosome is via the methylglyoxal reductase pathway to l-lactate.
TL;DR: It is concluded that MG is a potent suppressor of myeloid and T‐cell immune function and may be a major player in diabetes‐associated susceptibility to infection.
Abstract: Increased methylglyoxal (MG) concentrations and formation of advanced glycation end-products (AGEs) are major pathways of glycaemic damage in diabetes, leading to vascular and neuronal complications. Diabetes patients also suffer increased susceptibility to many common infections, the underlying causes of which remain elusive. We hypothesized that immune glycation damage may account for this increased susceptibility. We previously showed that the reaction mixture (RM) for MG glycation of peptide blocks up regulation of CD83 in myeloid cells and inhibits primary stimulation of T cells. Here, we continue to investigate immune glycation damage, assessing surface and intracellular cytokine protein expression by flow cytometry, T-cell proliferation using a carboxyfluorescein succinimidyl ester assay, and mRNA levels by RT-PCR. We show that the immunomodulatory component of this RM was MG itself, with MG alone causing equivalent block of CD83 and loss of primary stimulation. Block of CD83 expression could be reversed by MG scavenger N-acetyl cysteine. Further, MG within RM inhibited stimulated production of interleukin (IL)-10 protein from myeloid cells plus interferon (IFN)-γ and tumour necrosis factor (TNF)-α from T cells. Loss of IL-10 and IFN-γ was confirmed by RT-PCR analysis of mRNA, while TNF-α message was raised. Loss of TNF-α protein was also shown by ELISA of culture supernatants. In addition, MG reduced major histocompatibility complex (MHC) class I expression on the surface of myeloid cells and increased their propensity to apoptose. We conclude that MG is a potent suppressor of myeloid and T-cell immune function and may be a major player in diabetes-associated susceptibility to infection.
TL;DR: This experimental model of accelerated cellular aging in vitro can be useful for studies on testing the effects of various physical, chemical and biological conditions, including natural and synthetic molecules, for the modulation of aging.
Abstract: Dicarbonyls glyoxal (GO) and methylglyoxal (MGO) produced during the autoxidation of reducing sugars are a source of macromolecular damage in cells. Since an accumulation of damaged macromolecules is a universal characteristic of aging, we have tested whether GO and MGO which cause oxidative damage to proteins and other macromolecules can bring about accelerated aging in normal human skin fibroblasts in vitro. A treatment of cells with 1.0 mM GO or 400 microM MGO leads to the appearance of senescent phenotype within 3 days, as judged by the following criteria: morphological phenotype, irreversible growth arrest and G2 arrest, increased senescence-associated beta-galactosidase (SABG) activity, increased H2O2 level, increased Nxi-(carboxymethyl)-lysine (CML) protein level, and altered activities of superoxide dismutase and catalase antioxidant enzymes. This experimental model of accelerated cellular aging in vitro can be useful for studies on testing the effects of various physical, chemical and biological conditions, including natural and synthetic molecules, for the modulation of aging.
TL;DR: The findings strongly implicate dicarbonyl/metal catalyzed oxidation of lysine to allysine, whereby low GSH combined with ascorbate-derived H2O2 likely contributes toward 2-AAA formation, since virtually no 2- AAA formed in the presence of methylglyoxal instead of ascorBate.
TL;DR: The potential beneficial effects of methylglyoxal far outweigh its possible toxic role in vivo, and it should be utilized for the benefit of suffering humanity.
Abstract: In various organisms, an array of enzymes is involved in the synthesis and breakdown of methylglyoxal. Through these enzymes, it is intimately linked to several other physiologically important metabolites, suggesting that methylglyoxal has some important role to play in the host organism. Several in vitro and in vivo studies showed that methylglyoxal acts specifically against different types of malignant cells. These studies culminated in a recent investigation to evaluate a methylglyoxal-based formulation in treating a small group of cancer patients, and the results were promising. Methylglyoxal acts against a number of pathogenic microorganisms. However, recent literature abounds with the toxic effects of methylglyoxal, which are supposed to be mediated through methylglyoxal-derived advanced glycation end products (AGE). Many diseases such as diabetes, cataract formation, hypertension, and uremia are proposed to be intimately linked with methylglyoxal-derived AGE. However methylglyoxal-derived AGE formation and subsequent pathogenesis might be a very minor event because AGE are nonspecific reaction products that are derived through the reactions of carbonyl groups of reducing sugars with amino groups present in the side chains of lysine and arginine and in terminal amino groups of proteins. Moreover, the results of some in vitro experiments with methylglyoxal under non-physiological conditions were extrapolated to the in vivo situation. Some experiments even showed contradictory results and were differently interpreted. For this reason conclusions about the potential beneficial effects of methylglyoxal have often been neglected, thus hindering the advancement of medical science and causing some confusion in fundamental understanding. Overall, the potential beneficial effects of methylglyoxal far outweigh its possible toxic role in vivo, and it should be utilized for the benefit of suffering humanity.
TL;DR: It is proposed that diabetic ECM affects mesangial cell functions via two distinct mechanisms: modification of arginine residues by MGO inhibits cell adhesion, whereas oxidative modification of lysine residuesBy glucose inhibits cell proliferation and increases collagen IV production.
Abstract: Diabetic nephropathy (DN) affects both glomerular cells and the extracellular matrix (ECM), yet the pathogenic mechanisms involving cell-matrix interactions are poorly understood. Glycation alters integrin-dependent cell-ECM interactions, and perturbation of these interactions results in severe renal pathology in diabetic animals. Here, we investigated how chemical modifications of the ECM by hyperglycemia and carbonyl stress, two major features of the diabetic milieu, affect mesangial cell functions. Incubation of collagen IV with pathophysiological levels of either the carbonyl compound methylglyoxal (MGO) or glucose resulted in modification of arginine or lysine residues, respectively. Mouse mesangial cells plated on MGO-modified collagen IV showed decreased adhesion and migration. Cells plated on glucose-modified collagen IV showed reduced proliferation and migration and increased collagen IV production. Inhibiting glucose-mediated oxidative modification of collagen IV lysine residues rescued the alterations in cell growth, migration, and collagen synthesis. We propose that diabetic ECM affects mesangial cell functions via two distinct mechanisms: modification of arginine residues by MGO inhibits cell adhesion, whereas oxidative modification of lysine residues by glucose inhibits cell proliferation and increases collagen IV production. These mechanisms may contribute to mesangial cell hypertrophy and matrix expansion in DN.
TL;DR: Investigating the effect of 7-O-galloyl-D-sedoheptulose against diabetic oxidative stress and AGE formation demonstrated that 20 d of treatment with GS had beneficial effects on hypoglycemic and renal metabolic abnormalities, including renal glucose, oxidative stress, and A GE formation.
Abstract: Diabetes is the leading cause of end-stage renal failure, since glucose-dependent metabolic factors are synergistically activated within the diabetic kidney. Accordingly, in Japan, there is much debate over the health benefits of natural therapies to reduce these risk factors. In our previous study, we reported that Cornus officinalis SIEB. et ZUCC. possessed an antidiabetic effect via ameliorating glucose-mediated metabolic disorders as well as aminoguanidine, an inhibitor of advanced glycation endproduct (AGE) formation, with a renoprotective effect. The aim of the present study was to investigate the effect of 7-O-galloyl-D-sedoheptulose (GS) against diabetic oxidative stress and AGE formation. Streptozotocin-induced diabetic rats were orally administered GS for 20 d, and the changes in serum glucose levels, as well as those of body weight every 10 d were evaluated. In addition, glucose, fluorescent AGE, methylglyoxal, glycolaldehyde (GA), and immunoblotting analyses for heme oxygenase-1, receptor for AGE, N(epsilon)-(carboxymethyl)lysine, N(epsilon)-(carboxyethyl)lysine, and GA-pyridine were performed in the kidney at the end of the experiment. The results obtained in this study demonstrated that 20 d of treatment with GS had beneficial effects on hypoglycemic and renal metabolic abnormalities, including renal glucose, oxidative stress, and AGE formation. Together, our data help to elucidate its potential therapeutic value against diabetic kidney disease.
TL;DR: To determine the mechanism by which carbonyl scavenging drugs prevent glyoxal toxicity in a cell-free system as well as in isolated rat hepatocytes, the ability of the drugs was measured by following Glyoxal disappearance using Girard's Reagent T.
TL;DR: The increased levels of free MG-H observed in individuals with TIDM are not merely the result of short term changes in glucose or methylglyoxal, but may reflect long-term alterations to tissue proteins.
TL;DR: It is suggested that MG could be a missing link between hyperglycaemia and immune suppression in diabetes and the effects of MG on components of the immune system.
Abstract: No matter the cause of diabetes, the result is always hyperglycaemia. This excess glucose metabolism drives several damage pathways and raises concentrations of the reactive dicarbonyl, methylglyoxal (MG). MG can modify the structure and function of target molecules by forming advanced glycation end-products (AGEs) that act through their receptor (RAGE) to perpetuate vascular and neuronal injury responsible for long-term complications of diabetes. Diabetes patients also suffer lower resistance to many common infections, although the cause(s) for this lower resistance remains elusive. Here, we review recent evidence concerning immune suppression in diabetes and discuss the effects of MG on components of the immune system. We suggest that MG could be a missing link between hyperglycaemia and immune suppression in diabetes.
TL;DR: D density functional theory method is used to investigate the reaction mechanism of Glyoxalase II and it is found that the bridging hydroxide is capable of performing nucleophilic attack at the substrate carbonyl to form a tetrahedral intermediate.
TL;DR: It is shown that trypanothione-thioesters can be generated from the respective coenzyme A derivative by transesterification and obtained by this spontaneous reaction proved to be excellent substrates of T. brucei glyoxalase II.
TL;DR: Porcine cornea showed a nonlinear reduction in solute permeability and specific hydraulic conductivity with increasing cross-link density, which suggests that age-related nonenzymatic cross- link accumulation can have a substantial impact on corneal permeability.
Abstract: Purpose To investigate the relationship between corneal permeability and nonenzymatic cross-link density. Methods Corneas were dissected from 90 cadaveric porcine eyes. Samples were incubated for 24 hours with control solution or methylglyoxal at concentrations of 0.01%, 0.10%, and 1.00%. Nonenzymatic cross-link density in treated and control groups was quantified by papain digest and fluorescence spectrophotometry. Control and treated corneas were mounted in a customized Ussing-type chamber connected to vertical tubing, and specific hydraulic conductivity was determined according to the descent of a column of degassed saline at room temperature. Permeability to diffusion of fluorescein in a static chamber was determined for a similar set of corneal samples. Results Methylglyoxal treatment effectively increased nonenzymatic cross-link content, as indicated by the average fluorescence for each group. Specific hydraulic conductivity (m(2)) was reduced with increasing cross-link density. Similarly, the permeability coefficient for the fluorescein solute consistently decreased with increasing methylglyoxal concentration, indicating diffusion impedance resulting from the treatment. Conclusions Nonenzymatic cross-link density in the cornea can be significantly increased by treatment with methylglyoxal. Porcine cornea showed a nonlinear reduction in solute permeability and specific hydraulic conductivity with increasing cross-link density. This model suggests that age-related nonenzymatic cross-link accumulation can have a substantial impact on corneal permeability.
TL;DR: The present data supports that the GLOI plays an essential role in the survival of this pathogenic organism and that inhibition of the enzyme potentiates the toxicity of methylglyoxal.
Abstract: Background
Glyoxalase I is a metalloenzyme of the glyoxalase pathway that plays a central role in eliminating the toxic metabolite methyglyoxal. The protozoan parasite Leishmania donovani possesses a unique trypanothione dependent glyoxalase system.
Principal Findings
Analysis of the L. donovani GLOI sequence predicted a mitochondrial targeting sequence, suggesting that the enzyme is likely to be targeted to the mitochondria. In order to determine definitively the intracellular localization of GLOI in L. donovani, a full-length GLOI gene was fused to green fluorescent protein (GFP) gene to generate a chimeric construct. Confocal microscopy of L. donovani promastigotes carrying this chimeric construct and immunofluorescence microscopy using anti-GLOI antibodies demonstrated that GLOI is localized in the kinetoplast of the parasite apart from the cytosol. To study the physiological role of GLOI in Leishmania, we first created promastigote mutants heterozygous for GLOI by targeted gene replacement using either hygromycin or neomycin phosphotransferases as selectable markers. Heterozygous mutants of L. donovani display a slower growth rate, have lower glyoxalase I activity and have reduced ability to detoxify methylglyoxal in comparison to the wild-type parasites. Complementation of the heterozygous mutant with an episomal GLOI construct showed the restoration of heterozygous mutant phenotype nearly fully to that of the wild-type. Null mutants were obtained only after GLOI was expressed from an episome in heterozygous mutants.
Conclusions
We for the first time report localization of GLOI in L. donovani in the kinetoplast. To study the physiological role of GLOI in Leishmania, we have generated GLOI attenuated strains by targeted gene replacement and report that GLOI is likely to be an important gene since GLOI mutants in L. donovani showed altered phenotype. The present data supports that the GLOI plays an essential role in the survival of this pathogenic organism and that inhibition of the enzyme potentiates the toxicity of methylglyoxal.
TL;DR: Molecular electrostatic potential maps were calculated for three of the compounds tested and they provide suggestive evidence for the inhibitory regions of the molecules and help clarify the role of coumarin in the inhibition of glyoxalase I.
Abstract: Previous reports from these laboratories on the inhibition of glyoxalase I (S-lactoyl-glutathione methylglyoxal lyase, isomerizing; EC 4.4.1.5) (Glo I) have been presented for various flavones and other compounds. We report here the inhibition of Glo I by coumarin and various coumarin derivatives. Human red blood cell Glo I was purified 7000-fold and the concentration of various coumarins was determined for 50% inhibition (I50) of enzyme activity. These compounds resemble the transition state of the methylglyoxal hemimercaptal as previously reported. The I50 varies from 3.5 mM to 1.9 mM for the compounds tested with the parent compound coumarin having an I50 of 1.9 mM. The most inhibitory compounds had hydroxyls at various positions on the coumarin ring system and a phenyl or similar group at the 3 or 4 position on the pyrone ring. Molecular electrostatic potential maps were calculated for three of the compounds tested and they provide suggestive evidence for the inhibitory regions of the molecules.