Evidence Against a Role for the Parkinsonism-associated Protein DJ-1 in Methylglyoxal Detoxification.

Methylglyoxal (MG) is a reactive metabolite that forms adducts on cysteine, lysine and arginine residues of proteins, thereby affecting their function. Methylglyoxal is detoxified by the Glyoxalase system, consisting of two enzymes, Glo1 and Glo2, that act sequentially to convert MG into d-lactate. Recently, the Parkinsonism-associated protein DJ-1 was described in vitro to have glyoxalase activity, thereby detoxifying the MG metabolite, or deglycase activity, thereby removing the adduct formed by MG on proteins. Since Drosophila is an established model system to study signaling, neurodegeneration, and metabolic regulation in vivo, we asked whether DJ-1 contributes to MG detoxification in vivo Using both DJ-1 knockdown in Drosophila cells in culture, and DJ-1ß knock-out flies, we could detect no contribution of DJ-1 to survival to MG challenge or to accumulation of MG protein adducts. Furthermore, we provide data suggesting that the previously reported deglycation activity of DJ-1 can be ascribed to a TRIS buffer artifact.

Advanced glycation end products and diabetic nephropathy: a comparative study using diabetic and normal rats with methylglyoxal-induced glycation

Hyperglycemia-related advanced glycation end product (AGE) formation is a key mechanism in diabetic nephropathy. Since methylglyoxal (MG) is a potent AGE precursor, we aimed to assess the role of MG-related AGE formation in the progression of renal damages. A comparative study between Wistar (W, normal) and Goto-Kakizaki (GK, nonobese type 2 diabetic) rats was performed at 6 and 14 months old and after 14 weeks of MG administration to 6-month-old rats. Diabetic rats showed progressive structural, biochemical, and functional alterations, including AGE, albuminuria, and tissue hypoxia, which were partially mimicked by MG administration to young GK rats. Aged Wistar rats had an impairment of some parameters, whereas MG administration caused a phenotype similar to young GK rats, including oxidative stress, impaired apoptotic and angiogenic markers, and structural lesions. MG accumulation specifically impaired several of the renal disease markers progressively observed in diabetic rats, and thus, it contributes to the progression of diabetic nephropathy

Glyoxalase 1 increases anxiety by reducing GABAA receptor agonist methylglyoxal.

Glyoxalase 1 (Glo1) expression has previously been associated with anxiety in mice; however, its role in anxiety is controversial, and the underlying mechanism is unknown. Here, we demonstrate that GLO1 increases anxiety by reducing levels of methylglyoxal (MG), a GABAA receptor agonist. Mice overexpressing Glo1 on a Tg bacterial artificial chromosome displayed increased anxiety-like behavior and reduced brain MG concentrations. Treatment with low doses of MG reduced anxiety-like behavior, while higher doses caused locomotor depression, ataxia, and hypothermia, which are characteristic effects of GABAA receptor activation. Consistent with these data, we found that physiological concentrations of MG selectively activated GABAA receptors in primary neurons. These data indicate that GLO1 increases anxiety by reducing levels of MG, thereby decreasing GABAA receptor activation. More broadly, our findings potentially link metabolic state, neuronal inhibitory tone, and behavior. Finally, we demonstrated that pharmacological inhibition of GLO1 reduced anxiety, suggesting that GLO1 is a possible target for the treatment of anxiety disorders.

Characteristic effects of methylglyoxal and its degraded product formate on viability of human histiocytes: a possible detoxification pathway of methylglyoxal.

Methylglyoxal (MGO) is a toxic and highly reactive alpha-oxoaldehyde, elevated in the states of various diseases underlying enhanced oxidative stress. Furthermore, MGO has been reported to generate another aldehyde, formic acid (FA). In this sense, investigating the biological property of FA is crucially important. The present study examined the effects of MGO and FA on cell viability using the U937 human histiocytic cell line. FA showed a dose-dependent increase in cell viability at the concentrations of MGO in which cell viability decreased. The mechanism of the increase by FA involved the presence of endogenous hydrogen peroxide (H2O2) and tetrahydrofolate in the folate pathway, whereas that of the decrease in cell viability by MGO involved interaction with H2O2 and oxidative damage. These findings suggest that FA production by MGO degradation may play a role in attenuation of oxidative cellular injury caused by MGO. We hypothesize that FA generation pathway constitutes a detoxification system for MGO.

Glycobiology of the Leishmania parasite and emerging targets for antileishmanial drug discovery.

IMPORTANCE OF THE FIELD: Parasitic diseases that pose a threat to human life include leishmaniasis - caused by protozoa of Leishmania species. Existing drugs have limitations due to deleterious side effects like teratogenicity and factors like cost and drug resistance, thus furthering the need to develop this area of research. AREAS COVERED IN THIS REVIEW: We came across drug targets, very recently characterised, cloned and validated by genomics and bioinformatics. We bring these promising drug targets into focus so that they can be explored to their fullest. WHAT THE READER WILL GAIN: In an effort to bridge the gaps between existing knowledge and future prospects of drug discovery, we found interesting studies validating drug targets and paving the way for better experiments to be designed. In a few cases, novel pathways have been characterized, while in others, well established pathways when probed further, led to the discovery of new drug targets. TAKE HOME MESSAGE: The review constitutes a comprehensive report on upcoming drug targets, with emphasis on glycosylphosphatidylinositol (GPI)-anchored glycoconjugates along with related biochemistry of enolase, glycosome and purine salvage pathways, as we strive to bring ourselves a step closer to being able to combat this deadly disease.

Kinetic characterization and comparison of various protein crosslinking reagents for matrix modification.

We have characterized the relative efficacies of a number of protein crosslinking agents that have the potential for use in the crosslinking of proteinaceous matrices both in vitro and in vivo. The crosslinkers tested were; L: -threose (LT), Genipin (GP), Methylglyoxal (MG), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), proanthrocyanidin (PA) and glutaraldehyde (GA). The relative effectiveness of the crosslinkers with regard to their saturating concentrations was: GA > PA > EDC > MG = GP >> LT. Most of the crosslinkers displayed a pH dependence and were more effective at more alkaline pH. At optimal pH and saturating conditions, the relative reaction rates of the crosslinkers were: PA = GA > EDC > GP > MG >> LT.

Effects of advanced glycation end product modification on proximal tubule epithelial cell processing of albumin.

AIM: The goal of this work is to understand the cellular effects of advanced glycation end product (AGE)-modified protein on renal proximal tubule cells. BACKGROUND: A major function of the proximal tubule is to reabsorb and process filtered proteins. Diabetes is characterized by increased quantities of tissue and circulating proteins modified by AGEs. Therefore in diabetes, plasma proteins filtered at the glomerulus and presented to the renal proximal tubule are likely to be highly modified by AGEs. METHODS: The model system was electrically resistant polarized renal proximal tubular epithelial cells in monolayer culture. The model proteins comprise a well-characterized AGE, methylglyoxal-modified bovine serum albumin (MGO-BSA), and unmodified BSA. RESULTS: Renal proximal tubular cells handle MGO-BSA and native BSA in markedly disparate ways, including differences in: (1) kinetics of binding, uptake, and intracellular accumulation, (2) processing and fragmentation, and (3) patterns of electrical conductance paralleling temporal changes in binding, uptake and processing. CONCLUSION: These differences support the idea that abnormal protein processing by the renal tubule can be caused by abnormal proteins, thereby forging a conceptual link between the pathogenic role of AGEs and early changes in tubular function that can lead to hypertrophy and nephropathy in diabetes.

Methylglyoxal in food and living organisms.

Methylglyoxal (MG) is a highly reactive alpha-oxoaldehyde formed endogenously in numerous enzymatic and nonenzymatic reactions. It modifies arginine and lysine residues in proteins forming advanced glycation end-products such as N(delta)-(5-methyl-4-imidazolon-2-yl)-L-ornithine (MG-H1), 2-amino-5-(2-amino-5-hydro-5-methyl-4-imidazolon-1-yl)pentanoic acid (MG-H2), 2-amino-5-(2-amino-4-hydro-4-methyl-5-imidazolon-1-yl)pentanoic acid (MG-H3), argpyrimidine, N(delta)-(4-carboxy-4,6-dimethyl-5,6-dihydroxy-1,4,5,6-tetrahydropyrimidine-2-yl)-L-ornithine (THP), N(epsilon)-(1-carboxyethyl)lysine (CEL), MG-derived lysine dimer (MOLD), and 2-ammonio-6-({2-[4-ammonio-5-oxido-5-oxopently)amino]-4-methyl-4,5-dihydro-1H-imidazol-5-ylidene}amino)hexanoate (MODIC), which have been identified in vivo and are associated with complications of diabetes and some neurodegenerative diseases. In foodstuffs and beverages, MG is formed during processing, cooking, and prolonged storage. Fasting and metabolic disorders and/or defects in MG detoxification processes cause accumulation of this reactive dicarbonyl in vivo. In addition, the intake of low doses of MG over a prolonged period of time can cause degenerative changes in different tissues, and can also exert anticancer activity. MG in biological samples can be quantified by HPLC or GC methods with preliminary derivatization into more stable chromophores and/or fluorophores, or derivatives suitable for determination by MS by use of diamino derivatives of benzene and naphthalene, 6-hydroxy-2,4,5-triaminopyrimidine, cysteamine, and o-(2,3,4,5,6-pentafluorobenzyl) hydroxylamine. The methods include three basic steps: deproteinization, incubation with derivatization agent, and chromatographic analysis with or without preliminary extraction of the formed products.

Methylglyoxal impairs the insulin signaling pathways independently of the formation of intracellular reactive oxygen species.

Nonenzymatic glycation is increased in diabetes and leads to elevated levels of advanced glycation end products (AGEs), which link hyperglycemia to the induction of insulin resistance. In hyperglycemic conditions, intracellularly formed alpha-ketoaldehydes, such as methylglyoxal, are an essential source of intracellular AGEs, and the abnormal accumulation of methylglyoxal is related to the development of diabetes complications in various tissues and organs. We have previously shown in skeletal muscle that AGEs induce insulin resistance at the level of metabolic responses. Therefore, it was important to extend our work to intermediates of the biosynthetic pathway leading to AGEs. Hence, we asked the question whether the reactive alpha-ketoaldehyde methylglyoxal has deleterious effects on insulin action similar to AGEs. We analyzed the impact of methylglyoxal on insulin-induced signaling in L6 muscle cells. We demonstrate that a short exposure to methylglyoxal induces an inhibition of insulin-stimulated phosphorylation of protein kinase B and extracellular-regulated kinase 1/2, without affecting insulin receptor tyrosine phosphorylation. Importantly, these deleterious effects of methylglyoxal are independent of reactive oxygen species produced by methylglyoxal but appear to be the direct consequence of an impairment of insulin-induced insulin receptor substrate-1 tyrosine phosphorylation subsequent to the binding of methylglyoxal to these proteins. Our data suggest that an increase in intracellular methylglyoxal content hampers a key molecule, thereby leading to inhibition of insulin-induced signaling. By such a mechanism, methylglyoxal may not only induce the debilitating complications of diabetes but may also contribute to the pathophysiology of diabetes in general.

In vivo assessment of toxicity and pharmacokinetics of methylglyoxal. Augmentation of the curative effect of methylglyoxal on cancer-bearing mice by ascorbic acid and creatine.

Previous in vivo studies from several laboratories had shown remarkable curative effect of methylglyoxal on cancer-bearing animals. In contrast, most of the recent in vitro studies have assigned a toxic role for methylglyoxal. The present study was initiated with the objective to resolve whether methylglyoxal is truly toxic in vivo and to reassess its therapeutic potential. Four species of animals, both rodent and non-rodent, were treated with different doses of methylglyoxal through oral, subcutaneous and intravenous routes. Acute (treatment for only 1 day) toxicity tests had been done with mouse and rat. These animals received 2, 1 and 0.3 g of methylglyoxal/kg of body weight in a day through oral, subcutaneous and intravenous routes respectively. Chronic (treatment for around a month) toxicity test had been done with mouse, rat, rabbit and dog. Mouse, rat and dog received 1, 0.3 and 0.1 g of methylglyoxal/kg of body weight in a day through oral, subcutaneous and intravenous routes respectively. Rabbit received 0.55, 0.3 and 0.1 g of methylglyoxal/kg of body weight in a day through oral, subcutaneous and intravenous routes respectively. It had been observed that methylglyoxal had no deleterious effect on the physical and behavioral pattern of the treated animals. Fertility and teratogenecity studies were done with rats that were subjected to chronic toxicity tests. It had been observed that these animals produced healthy litters indicating no damage of the reproductive systems as well as no deleterious effect on the offspring. Studies on several biochemical and hematological parameters of methylglyoxal-treated rats and dogs and histological studies of several organs of methylglyoxal-treated mouse were performed. These studies indicated that methylglyoxal had no apparent deleterious effect on some vital organs of these animals. A detailed pharmacokinetic study was done with mouse after oral administration of methylglyoxal. The effect of methylglyoxal alone and in combination with creatine and ascorbic acid on cancer-bearing animals had been investigated by measuring the increase in life span and tumor cell growth inhibition. The results indicated that anticancer effect of methylglyoxal was significantly augmented by ascorbic acid and further augmented by ascorbic acid and creatine. Nearly 80% of the animals treated with methylglyoxal plus ascorbic acid plus creatine were completely cured and devoid of any malignant cells within the peritoneal cavity.

Glucación no enzimática de la antitrombina. Implicaciones en el riesgo trombótico de pacientes con diabetes mellitus / Non-enzymatic antithrombin glycation. Association with atherothrombotic risk in diabetic patients

Introducción/objetivos. La glucación no enzimática incorpora azúcares a los residuos lisina y arginina de proteínas, lo que puede alterar su función. La glucación de la antitrombina, un potente anticoagulante natural, podría asociarse con el riesgo trombótico observado en situaciones de hiperglucemia, como la diabetes mellitus. Nuestro objetivo fue estudiar el efecto de la glucación de la antitrombina y determinar si es relevante en las complicaciones trombóticas de la diabetes mellitus. Métodos. 1. Glucación no enzimática in vitro de antitrombina plasmática y purificada con metilglioxal y glucosa. 2. Se analizó el efecto de diferentes compuestos sobre la glucación no enzimática de la antitrombina in vitro. 3. Estudio de 101 pacientes diabéticos. En todas las muestras se analizaron los valores antigénicos, la actividad anti-FXa, las características conformacionales y la afinidad a la heparina de la antitrombina. Resultados. La glucación no enzimática in vitro de la antitrombina con metilglioxal o glucosa no ocasiona modificaciones conformacionales significativas en la molécula, pero induce su transformación a una forma con baja afinidad por la heparina, que explica la pérdida significativa de su actividad (valor < 40% del basal). Este efecto se previene con heparina, aminoguanidina y catequina. Los pacientes diabéticos muestran menores valores antigénicos y funcionales de antitrombina (80%) que los sujetos controles, pero esta disminución no se correlaciona con la glucemia ni con los valores de hemoglobina glucosilada. Conclusiones. La glucación de residuos lisina y arginina localizados en el sitio de unión a la heparina de la antitrombina reduce significativamente su actividad anticoagulante, aunque puede ser protegida por la heparina, la aminoguanidina y la catequina. Sin embargo, la relevancia de la glucación de la antitrombina en pacientes diabéticos es apenas perceptible debido a la lenta acción glucante de la glucosa, y a la reducida vida media de la antitrombina (AU)

Introduction/Aims. Non-enzymatic glycation of proteins can impair their function by incorporating sugars into their lysine and arginine residues. Glycation of antithrombin, a powerful anticoagulant, might be associated with the thrombotic risk observed in hyperglycemic conditions such as diabetes mellitus. Our aim was to study the effects of antithrombin glycation and determine its significance in the thrombotic complications observed in diabetes. Methods. 1) In vitro study of non-enzymatic glycation of purified and plasma antithrombin by their incubation with methylglyoxal and glucose. 2) The effect of different compounds on the in vitro glycation of antithrombin was analyzed. 3) We studied 101 diabetic patients. Antigen levels, anti-FXa activity, conformational features and antithrombin affinity to heparin were determined. Results. In vitro non-enzymatic glycation of antithrombin with methylglyoxal or glucose caused no significant conformational change in the molecule, but induced the transformation to a low heparin-affinity form, which explains the significant loss of activity observed (< 40% of basal). This effect was prevented by heparin, aminoguanidine and catechin. Diabetic patients presented lower antigenic and antithrombin functional levels (80%) than controls. However, no correlation between activity or antigen levels of antithrombin and glycemia or glycosylated hemoglobin was found in diabetic patients. Conclusions. In vitro glycation of lysine and arginine residues located in the heparin-binding site of antithrombin significantly reduces its anticoagulant activity. Interestingly, heparin, aminoguanidine and catechin prevented this effect. However, the non-enzymatic glycation of antithrombin in diabetic patients seems to be mild, since the action of glucose is very slow and the half life of antithrombin in plasma is short (AU)

Methylglyoxal, glyoxal, and their detoxification in Alzheimer's disease.

The accumulation of advanced glycation end products (AGEs) in brains with Alzheimer's disease (AD) has been implicated in the formation of insoluble deposits such as amyloid plaques and neurofibrillary tangles. AGEs are also known to activate glia, resulting in inflammation and neuronal dysfunction. As reactive intermediates of AGE formation, neurotoxic reactive dicarbonyl compounds such as glyoxal and methylglyoxal have been identified. One of the most effective detoxification systems for methylglyoxal and glyoxal is the glutathione-dependent glyoxalase system, consisting of glyoxalase I and glyoxalase II. In this study, we have determined the methylglyoxal and glyoxal levels in the cerebrospinal fluid of AD patients compared to healthy controls. Methylglyoxal levels in AD patients were twofold higher than in controls, but this difference was not significant due to the large intergroup variations and the small sample size. However, the concentrations of both compounds were five to seven times higher in CSF than in plasma. We also investigated the glyoxalase I level in AD and healthy control brains. The number of glyoxalase I- positive neurons were increased in AD brains compared to controls. Our findings suggest that glyoxalase I is upregulated in AD in a compensatory manner to maintain physiological methylglyoxal and glyoxal levels.

Renal clearance of glycolaldehyde- and methylglyoxal-modified proteins in mice is mediated by mesangial cells through a class A scavenger receptor (SR-A).

AIMS/HYPOTHESIS: Glomerular mesangial expansion is a characteristic feature of diabetic nephropathy, and the accumulation of AGE in the mesangial lesion has been implicated as one of its potential causes. However, the route for the AGE accumulation in mesangial lesions in diabetic patients is poorly established. METHODS: Glycolaldehyde-modified BSA (GA-BSA) and methylglyoxal-modified BSA (MG-BSA) were prepared as model AGE proteins, and their in vivo plasma clearance was examined in mice, and renal uptake by in vitro studies with isolated renal mesangial cells. RESULTS: Both (111)In-GA-BSA and (111)In-MG-BSA were rapidly cleared from the circulation mainly by both the liver and kidney. Immunohistochemical studies with an anti-GA-BSA antibody demonstrated that intravenously injected GA-BSA accumulated in mesangial cells, suggesting that such cells play an important role in the renal clearance of circulating AGE proteins. Binding experiments at 4 degrees C using mesangial cells isolated from mice showed that (125)I-GA-BSA and (125)I-MG-BSA exhibited specific and saturable binding. Upon incubation at 37 degrees C, (125)I-GA-BSA and (125)I-MG-BSA underwent endocytic degradation by these cells. The binding of the ligands to these cells was inhibited by several ligands for scavenger receptors. The endocytic degradation of GA-BSA by mesangial cells from class A scavenger receptor (SR-A) knock-out mice was reduced by 80% when compared with that of wild-type cells. The glomerular accumulation of GA-BSA after its intravenous administration was attenuated in SR-A knock-out mice, as evidenced by immunohistochemical observations. CONCLUSIONS/INTERPRETATION: These results raise the possibility that circulating AGE-modified proteins are subjected to renal clearance by mesangial cells, mainly via SR-A. This pathway may contribute to the pathogenesis of AGE-induced diabetic nephropathy.

Glyoxalase I in detoxification: studies using a glyoxalase I transfectant cell line.

The glyoxalase system (glyoxalase I, glyoxalase II and GSH as cofactor) is involved in the detoxification of methylglyoxal (a byproduct of the glycolytic pathway) and other alpha-oxoaldehydes. We have transfected a 622 bp cDNA encoding human glyoxalase I into murine NIH3T3 cells. The recipient cells were shown to express elevated transcript and protein levels and a 10-fold increase in glyoxalase I enzyme activity. This was accompanied by an increased tolerance for exogenous methylglyoxal and enhanced resistance to the cytotoxic effects of two glyoxalase I inhibitors (s-p-bromobenzylglutathione diethyl ester and s-p-bromobenzylglutathione dicyclopentyl ester), a glutathione analogue [gamma-glutamyl-(S)-(benzyl)cysteinyl-(R)-(-)-phenylglycine diethyl ester] and the anti-cancer drugs mitomycin C and adriamycin. Steady-state levels of GSH were significantly lower in the transfected cells, perhaps reflecting increased flux as a consequence of elevated glyoxalase activity. This decrease did not alter the sensitivity to the alkylating agent chlorambucil. Although transfection did not affect the growth or doubling time of the NIH3T3 cells, analysis of glyoxalase I activity showed a consistent increase in tumour tissue when compared with pair-matched controls. Thus increased glyoxalase I is associated with the malignant phenotype and may also contribute to protection against the cytotoxicity of certain anti-cancer drugs.