Showing papers on "Methylglyoxal published in 2012"

Molecular Mechanism of Heavy Metal Toxicity and Tolerance in Plants: Central Role of Glutathione in Detoxification of Reactive Oxygen Species and Methylglyoxal and in Heavy Metal Chelation

Abstract: Heavy metal (HM) toxicity is one of the major abiotic stresses leading to hazardous effects in plants. A common consequence of HM toxicity is the excessive accumulation of reactive oxygen species (ROS) and methylglyoxal (MG), both of which can cause peroxidation of lipids, oxidation of protein, inactivation of enzymes, DNA damage and/or interact with other vital constituents of plant cells. Higher plants have evolved a sophisticated antioxidant defense system and a glyoxalase system to scavenge ROS and MG. In addition, HMs that enter the cell may be sequestered by amino acids, organic acids, glutathione (GSH), or by specific metal-binding ligands. Being a central molecule of both the antioxidant defense system and the glyoxalase system, GSH is involved in both direct and indirect control of ROS and MG and their reaction products in plant cells, thus protecting the plant from HM-induced oxidative damage. Recent plant molecular studies have shown that GSH by itself and its metabolizing enzymes—notably glutathione S-transferase, glutathione peroxidase, dehydroascorbate reductase, glutathione reductase, glyoxalase I and glyoxalase II—act additively and coordinately for efficient protection against ROS- and MG-induced damage in addition to detoxification, complexation, chelation and compartmentation of HMs. The aim of this review is to integrate a recent understanding of physiological and biochemical mechanisms of HM-induced plant stress response and tolerance based on the findings of current plant molecular biology research.

Methylglyoxal modification of Na v 1.8 facilitates nociceptive neuron firing and causes hyperalgesia in diabetic neuropathy

Abstract: Glucose and its metabolic derivatives are increased the plasma of patients with diabetes. Peter Nawroth and colleagues demonstrate that one such metabolite, methylglyoxal, is increased in patients with painful diabetic neuropathy, and find that it acts by modifying the excitability characteristics of a sodium channel protein.

Methylglyoxal, glyoxalase 1 and the dicarbonyl proteome.

Abstract: Methylglyoxal (MG) is a potent protein glycating agent. Glycation is directed to guanidino groups of arginine residues forming mainly hydroimidazolone N δ-(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine (MG-H1) residues. MG-H1 formation is damaging to the proteome as modification is often directed to functionally important arginine residues. MG-H1 content of proteins is quantified by stable isotopic dilution analysis tandem mass spectrometry and also by immunoblotting with specific monoclonal antibodies. MG-glycated proteins undergo cellular proteolysis and release MG-H1 free adduct for excretion. MG-H1 residues have been found in proteins of animals, plants, bacteria, fungi and protoctista. MG-H1 is often the major advanced glycation endproduct in proteins of tissues and body fluids, increasing in diabetes and associated vascular complications, renal failure, cirrhosis, Alzheimer’s disease, arthritis, Parkinson’s disease and ageing. Glyoxalase 1 and aldo–keto reductase 1B1 metabolise >99% MG to innocuous products and thereby protect the proteome, providing an enzymatic defence against MG-mediated glycation. Proteins susceptible to MG modification with related functional impairment are called the “dicarbonyl proteome” (DCP). DCP includes albumin, haemoglobin, transcription factors, mitochondrial proteins, extracellular matrix proteins, lens crystallins and other proteins. DCP component proteins are linked to mitochondrial dysfunction in diabetes and ageing, oxidative stress, dyslipidemia, cell detachment and anoikis and apoptosis. Biochemical and physiological susceptibility of a protein to modification by MG and sensitivity of biochemical pathways and physiological systems to related functional impairment under challenge of physiologically relevant increases in MG exposure are key concepts. Improved understanding of the DCP will likely have profound importance for human health, longevity and treatment of disease.

Transcriptional control of glyoxalase 1 by Nrf2 provides a stress-responsive defence against dicarbonyl glycation.

Abstract: Abnormal cellular accumulation of the dicarbonyl metabolite MG (methylglyoxal) occurs on exposure to high glucose concentrations, inflammation, cell aging and senescence. It is associated with increased MG-adduct content of protein and DNA linked to increased DNA strand breaks and mutagenesis, mitochondrial dysfunction and ROS (reactive oxygen species) formation and cell detachment from the extracellular matrix. MG-mediated damage is countered by glutathione-dependent metabolism by Glo1 (glyoxalase 1). It is not known, however, whether Glo1 has stress-responsive up-regulation to counter periods of high MG concentration or dicarbonyl stress. We identified a functional ARE (antioxidant-response element) in the 5'-untranslated region of exon 1 of the mammalian Glo1 gene. Transcription factor Nrf2 (nuclear factor-erythroid 2 p45 subunit-related factor 2) binds to this ARE, increasing basal and inducible expression of Glo1. Activators of Nrf2 induced increased Glo1 mRNA, protein and activity. Increased expression of Glo1 decreased cellular and extracellular concentrations of MG, MG-derived protein adducts, mutagenesis and cell detachment. Hepatic, brain, heart, kidney and lung Glo1 mRNA and protein were decreased in Nrf2-/- mice, and urinary excretion of MG protein and nucleotide adducts were increased approximately 2-fold. We conclude that dicarbonyl stress is countered by up-regulation of Glo1 in the Nrf2 stress-responsive system, protecting protein and DNA from increased damage and preserving cell function.

Human DJ-1 and its homologs are novel glyoxalases

Abstract: Human DJ-1 is a genetic cause of early-onset Parkinson's disease (PD), although its biochemical function is unknown. We report here that human DJ-1 and its homologs of the mouse and Caenorhabditis elegans are novel types of glyoxalase, converting glyoxal or methylglyoxal to glycolic or lactic acid, respectively, in the absence of glutathione. Purified DJ-1 proteins exhibit typical Michaelis-Menten kinetics, which were abolished completely in the mutants of essential catalytic residues, consisting of cysteine and glutamic acid. The presence of DJ-1 protected mouse embryonic fibroblast and dopaminergically derived SH-SY5Y cells from treatments of glyoxals. Likewise, C. elegans lacking cDJR-1.1, a DJ-1 homolog expressed primarily in the intestine, protected worms from glyoxal-induced death. Sub-lethal doses of glyoxals caused significant degeneration of the dopaminergic neurons in C. elegans lacking cDJR-1.2, another DJ-1 homolog expressed primarily in the head region, including neurons. Our findings that DJ-1 serves as scavengers for reactive carbonyl species may provide a new insight into the causation of PD.

A quick, convenient and economical method for the reliable determination of methylglyoxal in millimolar concentrations: the N-acetyl-L-cysteine assay.

Abstract: The determination of methylglyoxal (MG) concentrations in vivo is gaining increasing importance as high levels of MG are linked to various health impairments including complications of diabetes. In order to standardize the measurements of MG in body fluids, it is necessary to precisely determine the concentration of MG stock solutions used as analytical standards. The “gold standard” method for the determination of MG concentration in the millimolar range is an enzyme-catalyzed endpoint assay based on the glyoxalase I catalyzed formation of S-lactoylglutathione. However, as this assay used purified glyoxalase I enzyme, it is quite expensive. Another method uses a derivation reaction with 2,4-dinitrophenylhydrazine, but this substance is explosive and needs special handling and storage. In addition, precipitation of the product methylglyoxal-bis-2,4-dinitrophenylhydrozone during the reaction limits the reliability of this method. In this study, we have evaluated a new method of MG determination based on the previously published fast reaction between MG and N-acetyl-l-cysteine at room temperature which yields an easily detectable condensation product, N-α-acetyl-S-(1-hydroxy-2-oxo-prop-1-yl)cysteine. When comparing these three different assays for the measurement of MG concentrations, we find that the N-acetyl-l-cysteine assay is the most favorable, providing an economical and robust assay without the need for the use of hazardous or expensive reagents.

Flavour chemistry of methylglyoxal and glyoxal

Abstract: Methylglyoxal (MGO) and glyoxal (GO), known as reactive carbonyl species, can be generated endogenously and exogenously (human body and food system). They are attracting increased attention because of their relationship with diabetes and flavour generation. In this review, their characteristics relating to flavour chemistry are discussed. MGO and GO can be detected in food systems by GC and HPLC after derivatization. MGO and GO formed in the Maillard reaction play important roles as precursors of aroma and colour compounds, especially in Strecker degradation, a major flavour generation reaction. When combined with amino acids they undergo Schiff base formation, decarboxylation and α-aminoketone condensation leading to heterocyclic aroma compounds such as pyrazines, pyrroles and pyridines. They attack amine groups in amino acids, peptides and proteins to form advanced glycation end products (AGEs) and cause carbonyl stress followed by oxidative stress and tissue damage. Therefore, many studies about scavengers of MGO and GO are seen. The influence of these scavengers on flavour generation is also discussed.

Exogenous nitric oxide alleviates high temperature induced oxidative stress in wheat (Triticum aestivum L.) seedlings by modulating the antioxidant defense and glyoxalase system

Abstract: We investigated the protective role of exogenous nitric oxide (NO) in alleviating high temperature induced damages of wheat (Triticum aestivum L. cv. Pradip) seedlings. Heat treatment (38 degrees C) alone or in combination with 0.5 mM SNP (a NO donor) was applied with nutrient solution on 8-d-old hydroponically grown seedlings for a period of 24 h and 48 h. Heat stress significantly decreased the Chl content and increased the lipid peroxidation (MDA) and H2O2 levels in time depending manners. Ascorbate (AsA) content markedly decreased upon heat treatment but glutathione (GSH) and glutathione disulfide (GSSG) content increased. Heat treatment resulted in an increase of the activities of antioxidant enzymes - ascorbate peroxidase (APX), glutathione reductase (GR), glutathione peroxidase (GPX) and glutathione S-transferase (GST). The activities of glyoxalase enzymes also increased upon heat stress. Exogenous NO supplementation in the seedlings had little influence on the nonenzymatic and enzymatic components compared to the control. However, supplementation of heat-treated seedlings with SNP significantly reduced the high temperature induced lipid peroxidation, H2O2 content and increased the content of Chl, AsA and GSH as well as the GSH/GSSG ratio. Heat treated seedlings which were supplemented with SNP also upregulated the activities of APX, MDHAR, DHAR, GR, GST, CAT and Gly I. This study concludes that an exogenous supply of NO protects wheat seedlings from high temperature induced oxidative stress by upregulating antioxidant defense and methylglyoxal (MG) detoxification system.

Mechanisms leading to oligomers and SOA through aqueous photooxidation: insights from OH radical oxidation of acetic acid and methylglyoxal

Abstract: . Previous experiments have demonstrated that the aqueous OH radical oxidation of methylglyoxal produces low volatility products including pyruvate, oxalate and oligomers. These products are found predominantly in the particle phase in the atmosphere, suggesting that methylglyoxal is a precursor of secondary organic aerosol (SOA). Acetic acid plays a central role in the aqueous oxidation of methylglyoxal and it is a ubiquitous product of gas phase photochemistry, making it a potential "aqueous" SOA precursor in its own right. However, the fate of acetic acid upon aqueous-phase oxidation is not well understood. In this research, acetic acid (20 μM–10 mM) was oxidized by OH radicals, and pyruvic acid and methylglyoxal experimental samples were analyzed using new analytical methods, in order to better understand the formation of SOA from acetic acid and methylglyoxal. Glyoxylic, glycolic, and oxalic acids formed from acetic acid and OH radicals. In contrast to the aqueous OH radical oxidation of methylglyoxal, the aqueous OH radical oxidation of acetic acid did not produce succinic acid and oligomers. This suggests that the methylgloxal-derived oligomers do not form through the acid catalyzed esterification pathway proposed previously. Using results from these experiments, radical mechanisms responsible for oligomer formation from methylglyoxal oxidation in clouds and wet aerosols are proposed. The importance of acetic acid/acetate as an SOA precursor is also discussed. We hypothesize that this and similar chemistry is central to the daytime formation of oligomers in wet aerosols.

Protection against methylglyoxal-derived AGEs by regulation of glyoxalase 1 prevents retinal neuroglial and vasodegenerative pathology

Abstract: Aims/hypothesis Methylglyoxal (MG) is an important precursor for AGEs. Normally, MG is detoxified by the glyoxalase (GLO) enzyme system (including component enzymes GLO1 and GLO2). Enhanced glycolytic metabolism in many cells during diabetes may overpower detoxification capacity and lead to AGE-related pathology. Using a transgenic rat model that overexpresses GLO1, we investigated if this enzyme can inhibit retinal AGE formation and prevent key lesions of diabetic retinopathy.

Advanced glycation endproducts: from precursors to RAGE: round and round we go

Abstract: The formation of advanced glycation endproducts (AGEs) occurs in diverse settings such as diabetes, aging, renal failure, inflammation and hypoxia. The chief cellular receptor for AGEs, RAGE, transduces the effects of AGEs via signal transduction, at least in part via processes requiring the RAGE cytoplasmic domain binding partner, diaphanous-1 or mDia1. Data suggest that RAGE perpetuates the inflammatory signals initiated by AGEs via multiple mechanisms. AGE-RAGE interaction stimulates generation of reactive oxygen species and inflammation--mechanisms which enhance AGE formation. Further, recent data in type 1 diabetic kidney reveal that deletion of RAGE prevents methylglyoxal accumulation, at least in part via RAGE-dependent regulation of glyoxalase-1, a major enzyme involved in methylglyoxal detoxification. Taken together, these considerations place RAGE in the center of biochemical and molecular stresses that characterize the complications of diabetes and chronic disease. Stopping RAGE-dependent signaling may hold the key to interrupting cycles of cellular perturbation and tissue damage in these disorders.

Glyoxalase 1 increases anxiety by reducing GABAA receptor agonist methylglyoxal

Abstract: 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.

Resveratrol Upregulates Nrf2 Expression To Attenuate Methylglyoxal-Induced Insulin Resistance in Hep G2 Cells

Abstract: Oxidative stress can result in insulin resistance, a primary cause of type-2 diabetes. Methylglyoxal (MG), a highly reactive dicarbonyl metabolite generated during glucose metabolism, has also been confirmed to cause pancreatic injury and induce inflammation, thereby resulting in insulin resistance. Recently, resveratrol has been reported to exert antioxidant properties, protecting cells from the generation of reactive oxygen species (ROS). The aim of this study was to evaluate resveratrol activation of nuclear factor erythroid 2-related factor 2 (Nrf2) to attenuate MG-induced insulin resistance in Hep G2 cells. Therefore, the molecular signaling events affecting resveratrol-mediated heme oxygenase-1 (HO-1) and glyoxalase expression levels were further investigated in this study. Our findings indicated that resveratrol activated the extracellular signal-regulated kinase (ERK) pathway but not the p38 or c-Jun N-terminal kinase (JNK) pathways, subsequently leading to Nrf2 nuclear translocation and elevation...

Lipid glycation and protein glycation in diabetes and atherosclerosis

Abstract: Recent instrumental analyses using a hybrid quadrupole/linear ion trap spectrometer in LC-MS/MS have demonstrated that the Maillard reaction progresses not only on proteins but also on amino residues of membrane lipids such as phosphatidylethanolamine (PE), thus forming Amadori-PE (deoxy-d-fructosyl PE) as the principal products. The plasma Amadori-PE level is 0.08 mol% of the total PE in healthy subjects and 0.15–0.29 mol% in diabetic patients. Pyridoxal 5′-phosphate and pyridoxal are the most effective lipid glycation inhibitors, and the PE-pyridoxal 5′-phosphate adduct is detectable in human red blood cells. These findings are beneficial for developing a potential clinical marker for glycemic control as well as potential compounds to prevent the pathogenesis of diabetic complications and atherosclerosis. Glucose and other aldehydes, such as glyoxal, methylglyoxal, and glycolaldehyde, react with the amino residues of proteins to form Amadori products and Heynes rearrangement products. Because several advanced glycation end-product (AGE) inhibitors such as pyridoxamine and benfotiamine inhibit the development of retinopathy and neuropathy in streptozotocin (STZ)-induced diabetic rats, AGEs may play a role in the development of diabetic complications. In the present review, we describe the recent progress and future applications of the Maillard reaction research regarding lipid and protein modifications in diabetes and atherosclerosis.

Role of Glyoxalase 1 (Glo1) and methylglyoxal (MG) in behavior: recent advances and mechanistic insights

Abstract: Glyoxalase 1 (GLO1) is a ubiquitous cellular enzyme that participates in the detoxification of methylglyoxal (MG), a cytotoxic byproduct of glycolysis that induces protein modification (advanced glycation end-products, AGEs), oxidative stress, and apoptosis. The concentration of MG is elevated under high-glucose conditions, such as diabetes. As such, GLO1 and MG have been implicated in the pathogenesis of diabetic complications. Recently, findings have linked GLO1 to numerous behavioral phenotypes, including psychiatric diseases (anxiety, depression, schizophrenia, and autism) and pain. This review highlights GLO1's association with behavioral phenotypes, describes recent discoveries that have elucidated the underlying mechanisms, and identifies opportunities for future research.

Angiogenesis impairment in diabetes: role of methylglyoxal-induced receptor for advanced glycation endproducts, autophagy and vascular endothelial growth factor receptor 2.

Abstract: Diabetes impairs physiological angiogenesis by molecular mechanisms that are not fully understood. Methylglyoxal (MGO), a metabolite of glycolysis, is increased in patients with diabetes. This study defined the role of MGO in angiogenesis impairment and tested the mechanism in diabetic animals. Endothelial cells and mouse aortas were subjected to Western blot analysis of vascular endothelial growth factor receptor 2 (VEGFR2) protein levels and angiogenesis evaluation by endothelial cell tube formation/migration and aortic ring assays. Incubation with MGO reduced VEGFR2 protein, but not mRNA, levels in a time and dose dependent manner. Genetic knockdown of the receptor for advanced glycation endproducts (RAGE) attenuated the reduction of VEGFR2. Overexpression of Glyoxalase 1, the enzyme that detoxifies MGO, reduced the MGO-protein adducts and prevented VEGFR2 reduction. The VEGFR2 reduction was associated with impaired angiogenesis. Suppression of autophagy either by inhibitors or siRNA, but not of the proteasome and caspase, normalized both the VEGFR2 protein levels and angiogenesis. Conversely, induction of autophagy either by rapamycin or overexpression of LC3 and Beclin-1 reduced VEGFR2 and angiogenesis. MGO increased endothelial LC3B and Beclin-1, markers of autophagy, which were accompanied by an increase of both autophagic flux (LC3 punctae) and co-immunoprecipitation of VEGFR2 with LC3. Pharmacological or genetic suppression of peroxynitrite (ONOO−) generation not only blocked the autophagy but also reversed the reduction of VEGFR2 and angiogenesis. Like MGO-treated aortas from normglycemic C57BL/6J mice, aortas from diabetic db/db and Akita mice presented reductions of angiogenesis or VEGFR2. Administration of either autophagy inhibitor ex vivo or superoxide scavenger in vivo abolished the reductions. Taken together, MGO reduces endothelial angiogenesis through RAGE-mediated, ONOO–dependent and autophagy-induced VEGFR2 degradation, which may represent a new mechanism for diabetic angiogenesis impairment.

Phlorotannins from brown algae (Fucus vesiculosus) inhibited the formation of advanced glycation endproducts by scavenging reactive carbonyls.

Abstract: Accumulation of advanced glycation end products (AGEs) in vivo is associated with aging, diabetes, Alzheimer's disease, renal failure, etc. The objective of this study was to investigate the inhibitory effects of brown algae Fucus vesiculosus phlorotannins on the formation of AGEs. F. vesiculosus phlorotannins were extracted using 70% acetone. The resultant extract was fractionated into dichloromethane, ethyl acetate, butanol, and water fractions. The ethyl acetate fraction was further fractionated into four subfractions (Ethyl-F1 to -F4) using a Sephadex LH-20 column. F. vesiculosus acetone extract or fractions significantly inhibited the formation of AGEs mediated by glucose and methylglyoxal in a concentration-dependent manner. The concentrations of F. vesiculosus extracts required to inhibit 50% of albumin glycation (EC(50)) in the bovine serum albumin (BSA)-methylglyoxal assay were lower than those of aminoguanidine (a drug candidate for diabetic complication), except for F. vesiculosus acetone extract and dichloromethane fraction. In the BSA-glucose assay, F. vesiculosus extracts inhibited BSA glycation more than or as effectively as aminoguanidine, except for Ethyl-F3 and -F4. The ethyl acetate fraction and its four subfractions scavenged more than 50% of methylglyoxal in two hours. The hypothesis whether F. vesiculosus phlorotannins scavenged reactive carbonyls by forming adducts was tested. Phloroglucinol, the constituent unit of phlorotannins, reacted with glyoxal and methylglyoxal. Five phloroglucinol-carbonyl adducts were detected and tentatively identified using HPLC-ESI-MS(n).

Ellagic acid, a new antiglycating agent: its inhibition of Nϵ-(carboxymethyl)lysine.

Abstract: Non-enzymatic glycation is a complex series of reactions between reducing sugars and amino groups of proteins. Accumulation of AGEs (advanced glycation end-products) due to non-enzymatic glycation has been related to several diseases associated with aging and diabetes. The formation of AGEs is accelerated in hyperglycaemic conditions, which alters the structure and function of long-lived proteins, thereby contributing to long-term diabetic complications. The present study describes AGE inhibition and the mechanism of action of a new antiglycating agent, EA (ellagic acid), a flavonoid present in many dietary sources. Inhibition of AGE formation by EA was demonstrated with different proteins, namely eye lens TSP (total soluble protein), Hb (haemoglobin), lysozyme and BSA, using different glycating agents such as fructose, ribose and methylglyoxal by a set of complementary methods. These results suggest that the antiglycating action of EA seems to involve, apart from inhibition of a few fluorescent AGEs, predominantly inhibition of CEL [Nϵ-(carboxyethyl)lysine] through scavenging of the dicarbonyl compounds. Furthermore, MALDI-TOF-MS (matrix-assisted laser-desorption ionisation-time-of-flight MS) analysis confirms inhibition of the formation of CEL on lysozyme on in vitro glycation by EA. Prevention of glycation-mediated β-sheet formation in Hb and lysozyme by EA confirm its antiglycating ability. Inhibition of glycosylated Hb formation in human blood under ex vivo high-glucose conditions signifies the physiological antiglycating potential of EA. We have also determined the effectiveness of EA against loss of eye lens transparency through inhibition of AGEs in the lens organ culture system. These findings establish the antiglycating potential of EA and its in vivo utility in controlling AGE-mediated diabetic pathologies.

Pathological significance of mitochondrial glycation.

Abstract: Glycation, the nonenzymatic glycosylation of biomolecules, is commonly observed in diabetes and ageing. Reactive dicarbonyl species such as methylglyoxal and glyoxal are thought to be major physiological precursors of glycation. Because these dicarbonyls tend to be formed intracellularly, the levels of advanced glycation end products on cellular proteins are higher than on extracellular ones. The formation of glycation adducts within cells can have severe functional consequences such as inhibition of protein activity and promotion of DNA mutations. Although several lines of evidence suggest that there are specific mitochondrial targets of glycation, and mitochondrial dysfunction itself has been implicated in disease and ageing, it is unclear if glycation of biomolecules specifically within mitochondria induces dysfunction and contributes to disease pathology. We discuss here the possibility that mitochondrial glycation contributes to disease, focussing on diabetes, ageing, cancer, and neurodegeneration, and highlight the current limitations in our understanding of the pathological significance of mitochondrial glycation.

A novel source of methylglyoxal and glyoxal in retina: implications for age-related macular degeneration.

Abstract: Aging of retinal pigment epithelial (RPE) cells of the eye is marked by accumulations of bisretinoid fluorophores; two of the compounds within this lipofuscin mixture are A2E and all-trans-retinal dimer. These pigments are implicated in pathological mechanisms involved in some vision-threatening disorders including age-related macular degeneration (AMD). Studies have shown that bisretinoids are photosensitive compounds that undergo photooxidation and photodegradation when irradiated with short wavelength visible light. Utilizing ultra performance liquid chromatography (UPLC) with electrospray ionization mass spectrometry (ESI-MS) we demonstrate that photodegradation of A2E and all-trans-retinal dimer generates the dicarbonyls glyoxal (GO) and methylglyoxal (MG), that are known to modify proteins by advanced glycation endproduct (AGE) formation. By extracellular trapping with aminoguanidine, we established that these oxo-aldehydes are released from irradiated A2E-containing RPE cells. Enzyme-linked immunosorbant assays (ELISA) revealed that the substrate underlying A2E-containing RPE was AGE-modified after irradiation. This AGE deposition was suppressed by prior treatment of the cells with aminoguanidine. AGE-modification causes structural and functional impairment of proteins. In chronic diseases such as diabetes and atherosclerosis, MG and GO modify proteins by non-enzymatic glycation and oxidation reactions. AGE-modified proteins are also components of drusen, the sub-RPE deposits that confer increased risk of AMD onset. These results indicate that photodegraded RPE bisretinoid is likely to be a previously unknown source of MG and GO in the eye.

Regulation of proline metabolism in mycobacteria and its role in carbon metabolism under hypoxia

Abstract: Genes with a role in proline metabolism are strongly expressed when mycobacterial cells are exposed to nutrient starvation and hypoxia. Here we show that proline metabolism in mycobacteria is mediated by the monofunctional enzymes Δ(1) -pyrroline-5-carboxylate dehydrogenase (PruA) and proline dehydrogenase (PruB). Proline metabolism was controlled by a unique membrane-associated DNA-binding protein PruC. Under hypoxia, addition of proline led to higher biomass production than in the absence of proline despite excess carbon and nitrogen. To identify the mechanism responsible for this enhanced growth, microarray analysis of wild-type Mycobacterium smegmatis versus pruC mutant was performed. Expression of the DNA repair machinery and glyoxalases was increased in the pruC mutant. Glyoxalases are proposed to degrade methylglyoxal, a toxic metabolite produced by various bacteria due to an imbalance in intermediary metabolism, suggesting the pruC mutant was under methylglyoxal stress. Consistent with this notion, pruB and pruC mutants were hypersensitive to methylglyoxal. Δ(1) -pyrroline-5-carboxylate is reported to react with methylglyoxal to form non-toxic 2-acetyl-1-pyrroline, thus providing a link between proline metabolism and methylglyoxal detoxification. In support of this mechanism, we show that proline metabolism protects mycobacterial cells from methylglyoxal toxicity and that functional proline dehydrogenase, but not Δ(1) -pyrroline-5-carboxylate dehydrogenase, is essential for this protective effect.

Accumulation of argpyrimidine, a methylglyoxal-derived advanced glycation end product, increases apoptosis of lens epithelial cells both in vitro and in vivo

Abstract: The formation of advanced glycation end products (AGEs) has been considered to be a potential causative factor of injury to lens epithelial cells (LECs). Damage of LECs is believed to contribute to cataract formation. The purpose of this study was to investigate the cytotoxic effect of AGEs on LECs both in vitro and in vivo. We examined the accumulation of argpyrimidine, a methylglyoxal-derived AGE, and the expression of apoptosis-related molecules including nuclear factor-kappaB (NF-κB), Bax, and Bcl-2 in the human LEC line HLE-B3 and in cataractous lenses of Zucker diabetic fatty (ZDF) rats, an animal model of type 2 diabetes. In cataractous lenses from twenty-one-week-old ZDF rats, LEC apoptosis was markedly increased, and the accumulation of argpyrimidine as well as subsequent activation of NF-κB in LECs were significantly enhanced. The ratio of Bax to Bcl-2 protein levels was also increased. In addition, the accumulation of argpyrimidine triggered apoptosis in methylglyoxal-treated HLE-B3 cells. However, the presence of pyridoxamine (an AGEs inhibitor) and pyrrolidine dithiocarbamate (a NF-κB inhibitor) prevented apoptosis in HLE-B3 cells through the inhibition of argpyrimidine formation and the blockage of NF-κB nuclear translocalization, respectively. These results suggest that the cellular accumulation of argpyrimidine in LECs is NF-κB-dependent and pro-apoptotic.