Showing papers on "Methylglyoxal published in 1998"

Overexpression of glyoxalase-I in bovine endothelial cells inhibits intracellular advanced glycation endproduct formation and prevents hyperglycemia-induced increases in macromolecular endocytosis.

Abstract: Methylglyoxal (MG), a dicarbonyl compound produced by the fragmentation of triose phosphates, forms advanced glycation endproducts (AGEs) in vitro. Glyoxalase-I catalyzes the conversion of MG to S-D-lactoylglutathione, which in turn is converted to D-lactate by glyoxalase-II. To evaluate directly the effect of glyoxalase-I activity on intracellular AGE formation, GM7373 endothelial cells that stably express human glyoxalase-I were generated. Glyoxalase-I activity in these cells was increased 28-fold compared to neo-transfected control cells (21.80+/-0.1 vs. 0. 76+/-0.02 micromol/min/mg protein, n = 3, P 10-fold (18.9+/-3.2 vs. 18.4+/- 5.8, n = 3, P = NS, and 107.1+/-9.0 vs. 9.4+/-0 pmol/10(6) cells, n = 3, P < 0.001, respectively). After exposure to 30 mM glucose, intracellular AGE formation in neo cells was increased 13.6-fold (2.58+/-0.15 vs. 0.19+/-0.03 total absorbance units, n = 3, P < 0.001). Concomitant with increased intracellular AGEs, macromolecular endocytosis by these cells was increased 2.2-fold. Overexpression of glyoxalase-I completely prevented both hyperglycemia-induced AGE formation and increased macromolecular endocytosis.

Chemical modification of proteins by methylglyoxal

Abstract: Methylglyoxal is formed in vivo by spontaneous decomposition of triose phosphate intermediates in aerobic glycolysis. It may also be formed during oxidative degradation of both carbohydrates (pentoses and ascorbate) and lipids (arachidonate). In addition to reaction with arginine residues to form imidazolone adducts, methylglyoxal reacts with lysine residues in protein to form N(epsilon)-(carboxyethyl)lysine (CEL) and the imidazolium crosslink, methylglyoxal-lysine dimer (MOLD). Like the glycoxidation products, N(epsilon)-(carboxymethyl)lysine (CML) and glyoxal-lysine dimer (GOLD) which are formed on reaction of glyoxal with protein, CEL and MOLD increase in lens proteins and skin collagen with age. CML and CEL also increase in skin collagen in diabetes, while all four compounds increase in plasma proteins in uremia. Overall, CML, CEL, GOLD and MOLD are quantitatively the major biomarkers of the Maillard reaction in tissue proteins. GOLD and MOLD, in particular, are present at 10-50 fold higher concentrations than the fluorescent crosslink, pentosidine. Together, these dicarbonyl-derived advanced glycation endproducts (AGEs) represent the major chemical modifications that accumulate in tissue proteins with age and in chronic diseases such as diabetes and atherosclerosis.

Methylglyoxal production in bacteria: suicide or survival?

Abstract: Methylglyoxal is a toxic electrophile. In Escherichia coli cells, the principal route of methylglyoxal production is from dihydroxyacetone phosphate by the action of methylglyoxal synthase. The toxicity of methylglyoxal is believed to be due to its ability to interact with the nucleophilic centres of macromolecules such as DNA. Bacteria possess an array of detoxification pathways for methylglyoxal. In E. coli, glutathione-based detoxification is central to survival of exposure to methylglyoxal. The glutathione-dependent glyoxalase I-II pathway is the primary route of methylglyoxal detoxification, and the glutathione conjugates formed can activate the KefB and KefC potassium channels. The activation of these channels leads to a lowering of the intracellular pH of the bacterial cell, which protects against the toxic effects of electrophiles. In addition to the KefB and KefC systems, E. coli cells are equipped with a number of independent protective mechanisms whose purpose appears to be directed at ensuring the integrity of the DNA. A model of how these protective mechanisms function will be presented. The production of methylglyoxal by cells is a paradox that can be resolved by assigning an important role in adaptation to conditions of nutrient imbalance. Analysis of a methylglyoxal synthase-deficient mutant provides evidence that methylglyoxal production is required to allow growth under certain environmental conditions. The production of methylglyoxal may represent a high-risk strategy that facilitates adaptation, but which on failure leads to cell death. New strategies for antibacterial therapy may be based on undermining the detoxification and defence mechanisms coupled with deregulation of methylglyoxal synthesis.

Evidence of high levels of methylglyoxal in cultured Chinese hamster ovary cells

Abstract: Methylglyoxal is an alpha-ketoaldehyde and dicarbonyl formed in cells as a side product of normal metabolism. Endogenously produced dicarbonyls, such as methylglyoxal, are involved in numerous pathogenic processes in vivo, including carcinogenesis and advanced glycation end-product formation; advanced glycation end-products are contributors to the pathophysiology of aging and chronic diabetes. Despite recent advances in understanding of the systemic effects of methylglyoxal, the full significance of this compound remains unknown. Herein we provide evidence that the majority of the methylglyoxal present in vivo is bound to biological ligands. The basis for our finding is an experimental approach that provides a measure of the bound methylglyoxal present in living systems, in this instance Chinese hamster ovary cells; with our approach, as much as 310 microM methylglyoxal was detected, 100- to 1,000-fold more than observed previously in biological systems. Several artifacts were considered before concluding that the methylglyoxal was associated with cellular structures, including phosphate elimination from triose phosphates, carbohydrate degradation under the assay conditions, and interference from the derivatizing agent used as part of the assay procedure. A major source of the recovered methylglyoxal is most probably modified cellular proteins. With methylglyoxal at about 300 microM, 0.02% of cellular amino acid residues could be modified. As few as one or two "hits" with methylglyoxal per protein molecule have previously been reported to be sufficient to cause protein endocytosis and subsequent degradation. Thus, 5-10% of cellular proteins may be modified to physiologically significant levels.

Immunohistochemical and ELISA Assays for Biomarkers of Oxidative Stress in Aging and Diseasea

Abstract: Oxidative stress is apparent in pathology associated with aging and many age-related, chronic diseases, including atherosclerosis, diabetes mellitus, rheumatoid arthritis, and neurodegenerative diseases. Although it cannot be measured directly in biological systems, several biomarkers have been identified that provide a measure of oxidative damage to biomolecules. These include amino acid oxidation products (methionine sulfoxide, ortho-tyrosine (o-tyr) and dityrosine, chlorotyrosine and nitrotyrosine), as well as chemical modifications of protein following carbohydrate or lipid oxidation, such as N epsilon-(carboxymethyl)lysine and N epsilon-(carboxyethyl)lysine, and malondialdehyde and 4-hydroxynonenal adducts to amino acids. Other biomarkers include the amino acid cross-link pentosidine, the imidazolone adducts formed by reaction of 3-deoxyglucosone or methylglyoxal with arginine, and the imidazolium cross-links formed by the reaction of glyoxal and methylglyoxal with lysine residues in protein. These compounds have been measured in short-lived intracellular proteins, plasma proteins, long-lived extracellular proteins, and in urine, making them valuable tools for monitoring tissue-specific and systemic chemical and oxidative damage to proteins in biological systems. They are normally measured by sensitive high-performance liquid chromatography or gas chromatography-mass spectrometry methods, requiring both complex analytical instrumentation and derivatization procedures. However, sensitive immunohistochemical and ELISA assays are now available for many of these biomarkers. Immunochemical assays should facilitate studies on the role of oxidative stress in aging and chronic disease and simplify the evaluation of therapeutic approaches for limiting oxidative damage in tissues and treating pathologies associated with aging and disease. In this article we summarize recent data and conclusions based on immunohistochemical and ELISA assays, emphasizing the strengths and limitations of the techniques.

From famine to feast: the role of methylglyoxal production in Escherichia coli

Abstract: The enzyme methylglyoxal synthase (MGS) was partially purified from Escherichia coli extracts, and the amino-terminal sequence of candidate proteins was determined, based on the native protein being a tetramer of about 69 kDa. Database analysis identified an open reading frame in the E. coli genome, YccG, corresponding to a protein of 16.9 kDa. When amplified and expressed from a controlled promoter, it yielded extracts that contained high levels of MGS activity. MGS expressed from the trc promoter accumulated to approximately 20% of total cell protein, representing approximately 900-fold enhanced expression. This caused no detriment during growth on glucose, and the level of methylglyoxal (MG) in the medium rose to only 0.08 mM. High-level expression of MGS severely compromised growth on xylose, arabinose and glycerol. A mutant lacking MGS was constructed, and it grew normally on a range of carbon sources and on low-phosphate medium. However, the mutant failed to produce MG during growth on xylose in the presence of cAMP, and growth was inhibited.

The role of glyoxalase I in the detoxification of methylglyoxal and in the activation of the KefB K+ efflux system in Escherichia coli

Abstract: The glyoxalase I gene (gloA) of Escherichia coli has been cloned and used to create a null mutant. Cells overexpressing glyoxalase I exhibit enhanced tolerance of methylglyoxal (MG) and exhibit elevated rates of detoxification, although the increase is not stoichiometric with the change in enzyme activity. Potassium efflux via KefB is also enhanced in the overexpressing strain. Analysis of the physiology of the mutant has revealed that growth and viability are quite normal, unless the cell is challenged with MG either added exogenously or synthesized by the cells. The mutant strain has a low rate of detoxification of MG, and cells rapidly lose viability when exposed to this electrophile. Activation of KefB and KefC is diminished in the absence of functional glyoxalase I. These data suggest that the glutathione-dependent glyoxalase I is the dominant detoxification pathway for MG in E. coli and that the product of glyoxalase I activity, S-lactoylglutathione, is the activator of KefB and KefC.

Importance of Glutathione for Growth and Survival of Escherichia coli Cells: Detoxification of Methylglyoxal and Maintenance of Intracellular K+

Abstract: The role of the tripeptide glutathione in the growth and survival of Escherichia coli cells has been investigated. Glutathione-deficient mutants leak potassium and have a reduced cytoplasmic pH. These mutants are more sensitive to methylglyoxal than the parent strain, indicating that in the absence of glutathione-dependent detoxification, acidification of the cytoplasm cannot fully protect cells. However, increasing the intracellular pH of the glutathione-deficient strain resulted in enhanced sensitivity to methylglyoxal. This suggests that acidification of the cytoplasm can provide some protection to E. coli cells in the absence of glutathione. In the presence of the Kdp system, glutathione-deficient mutants are highly sensitive to methylglyoxal. This is due to the higher intracellular pH in these cells. In the absence of methylglyoxal, the presence of the Kdp system in a glutathione-deficient strain also leads to an extended lag upon dilution into fresh medium. These data highlight the importance of glutathione for the regulation of the K+ pool and survival of exposure to methylglyoxal.

Incidence and potential implications of the toxic metabolite methylglyoxal in cell culture: A review.

Abstract: Methylglyoxal is a toxic metabolite unavoidably produced in mammalian systems as a by-product of glycolysis. Detoxification of this compound occurs principally through the glyoxalase pathway, which consists of glyoxalase I and glyoxalase II, and requires reduced glutathione as a co-enzyme. Recently, it has been demonstrated that variations in glucose, glutamine and fetal bovine serum levels can cause significant changes in the intracellular concentration of methylglyoxal. More importantly, comparative studies involving wild-type Chinese hamster ovary cells and clones overexpressing glyoxalase I indicate that glucose and glutamine, within the range normally found in cell culture media, can cause decreased cell viability mediated solely through increased production of methylglyoxal. In addition, endogenously produced methylglyoxal has been shown to cause apoptosis in cultured HL60 cells. While the exact mechanism of the impact of methylglyoxal on cultured cells is unknown, methylglyoxal is a potent protein and nucleic acid modifying agent at physiological concentrations and under physiological conditions. Protein modification occurs mainly at arginine, lysine and cysteine residues and is believed to be an important signal for the degradation of senescent proteins. Modification of arginine and lysine results in the irreversible formation of advanced glycation endproducts, whereas modification of cysteine results in the formation of a highly reversible hemithioacetal. Methylglyoxal also forms adducts with nucleic acids, principally with guanyl residues. At high extracellular concentrations, it is genotoxic to cells grown in culture. Even at physiological concentrations (100 nM free methylglyoxal), methylglyoxal can modify unprotected plasmid DNA and cause gene mutation and abnormal gene expression.

Deamination of methylamine and angiopathy; toxicity of formaldehyde, oxidative stress and relevance to protein glycoxidation in diabetes

Abstract: Semicarbazide-sensitive amine oxidase (SSAO) is located in the vascular smooth muscles, retina, kidney and the cartilage tissues, and it circulates in the blood. The enzyme activity has been found to be significantly increased in blood and tissues in diabetic patients and animals. Methylamine and aminoacetone are endogenous substrates for SSAO. The deaminated products are formaldehyde and methylglyoxal respectively, as well as H2O2 and ammonia, which are all potentially cytotoxic. Formaldehyde and methylglyoxal are cytotoxic towards endothelial cells. Excessive SSAOmediated deamination may directly initiate endothelial injury and plaque formation, increase oxidative stress, which can potentiate oxidative glycation, and/or LDL oxidation and damage vascular systems. Formaldehyde is also capable of exacerbating advanced glycation, and thus increase the complexity of protein cross-linking. Uncontrolled SSAO-mediated deamination may be involved in the acceleration of the clinical complications in diabetes.

Overexpression of Aldehyde Reductase Protects PC12 Cells from the Cytotoxicity of Methylglyoxal or 3-Deoxyglucosone

Abstract: The glycation reaction (Maillard reaction) plays a major role in diabetic complications, since some reaction intermediates are responsible for the modification and cross-linking of long-lived proteins, resulting, in turn, in a deterioration of normal cell function. The reaction intermediates include methylglyoxal (MG) and 3-deoxyglucosone (3-DG), both of which are cytotoxic dicarbonyl compounds and are elevated during hyperglycemia. Aldehyde reductase (ALR) catalyzes the reduction of both compounds. To examine the intracellular role of ALR in the diabetic complications of neural cells, its gene was overexpressed in rat pheochromocytoma PC12 cells, which normally express a low level of ALR. Western blot analysis showed that ALR protein in the ALR gene-transfected cells was more than twice as much as in the control cells. In the parental cells, cytotoxicity, including apoptotic cell death, which was determined by fluorescent microscopy using the fluorescent DNA binding dye Hoechst 33258, was observed at 100 microM MG. In the ALR gene-transfected cells, the cytotoxicity of both MG and 3-DG and apoptotic cell death were decreased. This suggests that intracellular ALR protects neural cells from the cytotoxicity of 3-DG or MG, and that neural cells, which normally express a low level of ALR, might be susceptible to diabetic complications caused by intermediate products of the Maillard reaction, such as 3-DG and MG.

Aldehyde induced hypertension in rats: prevention by N-acetyl cysteine.

Abstract: Methylglyoxal, a highly reactive endogenous aldehyde is formed in the tissue of humans and animals as an intermediate of glucose and fructose metabolism. N-acetyl cysteine (NAC), an analogue of the dietary amino acid cysteine, binds aldehydes thus preventing their damaging effect on physiological proteins. We measured systolic blood pressure (SBP), platelet [Ca2+]i, circulating nitric oxide levels, tissue aldehyde conjugates and renal vascular changes in chronic methyglyoxal treated Wistar-Kyoto (WKY) rats and examined the effect of NAC in the diet on these parameters. Animals, age seven weeks, were divided into three groups of six animals each and were treated as follows: WKY-control (chow diet and normal drinking water); WKY-methylglyoxal (chow diet and methyglyoxal in drinking water); WKY-methyglyoxal + NAC (1.5% NAC in diet and methylglyoxal in drinking water) for the next 18 weeks. Methylgyoxal in drinking water was given at a concentration of 0.2% during weeks 0-5; 0.4%, weeks 6-10; and 0.8%, weeks 11-18. After 18 weeks systolic blood pressure, platelet [Ca2+]i and kidney aldehyde conjugates were significantly higher and serum nitric oxide levels lower in methylglyoxal treated rats. Methylglyoxal treated rats also showed smooth muscle cell hyperplasia in the small artery and arterioles of the kidney. N-acetyl cysteine, an aldehyde binding thiol compound, prevented these changes.

Isolation and characterization of advanced glycation end products derived from the in vitro reaction of ribose and collagen.

Abstract: An amino acid component, NFC-1, when formed in vitro by the reaction of ribose and protein was shown to comprise a complex mixture of high and low molecular AGE compounds. Two low-molecular-weight components have been successfully isolated and their structure determined. These were alphaNFC-1 [Ndelta-(4-oxo-5-dihydroimidazol-2-yl)-l-ornithine] and betaNFC-1 a 4-imidazolon-2-yl derivative existing in three tautomeric forms. These imidazolone compounds have been shown to originate from the reaction of arginine with glyoxal and methylglyoxal, respectively. A third ninhydrin-positive AGE, gammaNFC-1, was shown to be composed of a number of chromatographically similar compounds which have not yet been characterized.

Methylglyoxal-derived modifications in lens aging and cataract formation

Abstract: PURPOSE. TO determine whether the Maillard reaction of methylglyoxal is associated with human lens aging and cataractogenesis and to investigate how glutathione depletion affects methylglyoxalderived modifications in organ-cultured lenses. METHODS. Antibodies against methylglyoxal-derived modifications were developed in rabbits and purified by immunoaffinity chromatography. A competitive enzyme-linked immunosorbent assay (ELISA) measured methylglyoxal-derived products in human lens proteins. Lenses of galactosemic rats grown in organ culture were used to assess the role of glutathione-dependent pathways in methylglyoxal metabolism and Maillard reactions. RESULTS. Methylglyoxal-derived modifications in the human lens were age dependent, and brunescent lenses had the highest levels of these modifications. Immunofluorescence staining identified antigens distributed throughout the lens, with higher levels in old lenses than in younger ones. Experiments with normal or galactosemic rat lenses grown in organ culture showed that lens proteins do not have an increase in methylglyoxal-modified proteins when cultured in medium containing 500 /xM methylglyoxal alone, but they accumulate modified proteins when cultured with DL-glyceraldehyde. Inclusion of 30 mM glucose in the medium marginally increased methylglyoxal-derived products, but there was no correlation between lens glutathione content and methylglyoxal-derived modifications. CONCLUSIONS. Methylglyoxal-mediated Maillard reactions that occur in the human lens may play a role in lens aging and cataract formation. Methylglyoxal is probably derived from metabolic pathways within the lens. Decreased glutathione in organ-cultured rat lenses does not significantly influence methylglyoxal-mediated Maillard reactions. (Invest Ophthalmol Vis Set. 1998;39: 2355-2364)

Methylglyoxal as substrate and inhibitor of human aldehyde dehydrogenase: Comparison of kinetic properties among the three isozymes

Abstract: Methylglyoxal was demonstrated to be a substrate for the isozymes E1, E2 and E3 of human aldehyde dehydrogenase. Pyruvate was the product from the oxidation of methylglyoxal by the three isozymes. At pH 7.4 and 25 degrees C, the major and minor components of the E3 isozyme catalyzed the reaction with Vmax of 1.1 and 0.8 mumol NADH min-1 mg-1 protein, respectively, compared to 0.067 and 0.060 mumol NADH min-1 mg-1 protein for the E1 and E2 isozymes, respectively. The E2 isozyme had a K(m) for methylglyoxal of 8.6 microM, the lowest compared to 46 microM for E1 and 586 and 552 microM for the major and minor components of the E3 isozyme, respectively. Both components of the E3 isozyme showed substrate inhibition by methylglyoxal, with Ki values of 2.0 mM for the major component and 12 mM for the minor component at pH 9.0. Substrate inhibition by methylglyoxal was not observed with the E1 and E2 isozymes. Methylglyoxal strongly inhibited the glycolaldehyde activity of the E1 and E2 isozymes. Mixed-type models of inhibition were employed as an approach to calculate the inhibition constants, 44 and 10.6 microM for E1 and E2 isozymes, respectively.