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Showing papers on "Methylglyoxal published in 2001"


Journal ArticleDOI
TL;DR: Methylglyoxal metabolism was studied during Saccharomyces cerevisiae grown with D‐glucose as the sole carbon and energy source and metabolic fluxes of its formation and D‐lactate production were determined.

135 citations


Journal ArticleDOI
TL;DR: It is demonstrated that GO and MGO triggered two distinct signal cascades, one for PTK-dependent control of ERK and another forPTK-independent redox-linked activation of JNK/p38 MAPK and caspases in HUVECs, depending on the structure of the carbon skeleton of the chemicals.

134 citations


Journal ArticleDOI
TL;DR: It is observed that MG-induced apoptosis was associated with both rapid production of superoxide anion (O(2)(-)) followed by a marked increase in ROS and striking and temporal activation of ASK1, which suggests that activating ASK 1 at the early stage linking to production of O( 2)(-) is crucial for subsequent progression of apoptosis in MG-treated Jurkat cells.

130 citations


Journal ArticleDOI
TL;DR: A key-role of glyoxalase system to detoxify cytotoxic methylglyoxal and modulate S-D-lactoylglutathione levels in tumor cells is confirmed and a possible employment of GI inhibitors as anti-cancer drugs is suggested.
Abstract: The present work aimed to study the activities of glyoxalase system enzymes, glyoxalase I (G I) and glyoxalase II (G II), as well as the expression of their genes in human breast carcinoma. Samples of tumoral tissue and normal counterparts were drawn from several patients during surgery. They served either for preparing extracts to be used in enzyme activity evaluations or for RNA extraction and subsequent northern blot analysis. A far higher activity level of G I and G II occurs in the tumor compared with pair-matched normal tissue, as shown by both spectrophotometrical assay and electrophoretic pattern. Such increased activities of G I and G II likely result from an enhanced enzyme synthesis as a consequence of increased expression of the respective genes in the tumoral tissue, as evidenced by northern blot. The present findings confirm a key-role of glyoxalase system to detoxify cytotoxic methylglyoxal and modulate S-D-lactoylglutathione levels in tumor cells. Moreover, they suggest a possible employment of GI inhibitors as anti-cancer drugs.

119 citations


Journal ArticleDOI
TL;DR: The rice allergen, Glb33, was identified to be a novel type of plant glyoxalase I that is expressed in various plant tissues, including maturing seeds, not only in maturing Seeds of rice but also in its stem and leaf.

110 citations


Journal ArticleDOI
TL;DR: A strategy to stimulate the anaerobic pentosephosphate pathway of glycolysis by maximizing transketolase activity by thiamine supplementation is proposed, with the consequent consumption of glyceraldehyde-3-ph phosphate and increased formation of ribose-5-phosphates.
Abstract: The accumulation of triosephosphates and the increased formation of the potent glycating agent methylglyoxal in intracellular hyperglycaemia are implicated in the development of diabetic complications. A strategy to counter this is to stimulate the anaerobic pentosephosphate pathway of glycolysis by maximizing transketolase activity by thiamine supplementation, with the consequent consumption of glyceraldehyde-3-phosphate and increased formation of ribose-5-phosphate. To assess the effect of thiamine supplementation on the accumulation of triosephosphates and methylglyoxal formation in cellular hyperglycaemia, we incubated human red blood cell suspensions (50% v/v) in short-term culture with 5 mM glucose and 50 mM glucose in Krebs-Ringer phosphate buffer at 37 degrees C as models of cellular metabolism under normoglycaemic and hyperglycaemic conditions. In hyperglycaemia, there is a characteristic increase in the concentration of the triosephosphate pool of glycolytic intermediates and a consequent increase in the concentration and metabolic flux of the formation of methylglyoxal. The addition of thiamine (50-500 microM) increased the activity of transketolase, decreased the concentration of the triosephosphate pool, decreased the concentration and metabolic flux of the formation of methylglyoxal, and increased the concentration of total sedoheptulose-7-phosphate and ribose-5-phosphate. Biochemical changes implicated in the development of diabetic complications were thereby prevented. This provides a biochemical basis for high dose thiamine therapy for the prevention of diabetic complications.

106 citations


Journal ArticleDOI
TL;DR: The relative importance of aldose reductase and glyoxalase-I in the metabolic disposal of methylglyoxal is highly dependent upon the concentration of glutathione, owing to the non-catalytic pre-enzymatic reaction between methylgly oxal and glutathion.

103 citations


Journal ArticleDOI
TL;DR: The results suggest that methylglyoxal crosslinks a guanine residue of the substrate DNA and lysine and cysteine residues near the binding site of the DNA polymerase during DNA synthesis and that DNA replication is severely inhibited by the methyl glyoxal-induced DNA-DNA polymerase crosslink.
Abstract: Methylglyoxal, a known endogenous and environmental mutagen, is a reactive alpha-ketoaldehyde that can modify both DNA and proteins. To investigate the possibility that methylglyoxal induces a crosslink between DNA and DNA polymerase, we treated a 'primed template' DNA and the exonuclease-deficient Klenow fragment (KF(exo-)) of DNA polymerase I with methylglyoxal in vitro. When the reaction mixtures were analyzed by SDS-PAGE, we found that methylglyoxal induced a DNA-KF(exo-) crosslink. The specific binding complex of KF(exo-) and 'primed template' DNA was necessary for formation of the DNA-KF(exo-) crosslink. Methylglyoxal reacted with guanine residues in the single-stranded portion of the template DNA. When 2'-deoxyguanosine was incubated with Nalpha-acetyllysine or N-acetylcysteine in the presence of methylglyoxal, a crosslinked product was formed. No other amino acid derivatives tested could generate a crosslinked product. These results suggest that methylglyoxal crosslinks a guanine residue of the substrate DNA and lysine and cysteine residues near the binding site of the DNA polymerase during DNA synthesis and that DNA replication is severely inhibited by the methylglyoxal-induced DNA-DNA polymerase crosslink.

101 citations


Journal ArticleDOI
TL;DR: A novel, non-carboxymethyllysine (CML) anti-AGE antibody that recognizes serum proteins and peptides modified by 3-DG in vivo is developed that provides immunochemical evidence for the existence of six distinct AGEs in vivo among the AGE-modified proteins and proteins in the serum of diabetic patients on hemodialysis.
Abstract: The advanced stage of the Maillard reaction that leads to the formation of advanced glycation end-products (AGEs) plays an important role in the pathogenesis of angiopathy in diabetic patients and in the aging process. Recently, it has been proposed that the intermediates contributing to AGE formation include dicarbonyl intermediates such as glyoxal, methylglyoxal, and 3-deoxyglucosone (3-DG). In the present study, we developed a novel, non-carboxymethyllysine (CML) anti-AGE antibody that recognizes serum proteins and peptides modified by 3-DG in vivo. AGE-modified serum albumins were prepared by incubation of rabbit serum albumin with 3-DG or D-glucose. After immunization of rabbits, anti-AGE antisera were subjected to affinity chromatography on a Sepharose 4B column coupled with CML-BSA, or AGE-BSA created by incubation with 3-DG (AGE-6) or D-glucose (AGE-1). The AGE-Ab-6 and AGE-Ab-1 thus obtained was used to investigate AGEs in serum from diabetic patients on hemodialysis. Characterization of the novel AGE-Ab-6 obtained by immunoaffinity chromatography was performed with a competitive ELISA and immunoblot analysis. This antibody specifically cross-reacted with proteins modified by 3-DG. AGE-6 was detected in diabetic serum as three peaks with apparent molecular weights of 200, 1.15, and 0.85 kD, while AGE-1 was detected as four peaks with apparent molecular weights of 200, 65, 1.15, and 0.85 kD. This study provides new data on the pathways of AGE formation from 3-DG and methods for the immunochemical detection of AGEs. We also provide immunochemical evidence for the existence of six distinct AGEs in vivo among the AGE-modified proteins and peptides in the serum of diabetic patients on hemodialysis.

97 citations


Journal ArticleDOI
TL;DR: In vitro and in vivo assays of yeast aldose reductase activity indicate that the metabolism of methylglyoxal is stimulated under stress conditions; and they support a methyl Glyoxal degradative pathway, in which this compound is metabolised by the action of aldOSE reduct enzyme.
Abstract: The enzyme aldose reductase plays an important role in the osmo-protection mechanism of diverse organisms. Here, we show that yeast aldose reductase is encoded by the GRE3 gene. Expression of GRE3 is carbon-source independent and up-regulated by different stress conditions, such as NaCl, H2O2, 39 °C and carbon starvation. Measurements of enzyme activity and intracellular sorbitol in wild-type cells also indicate that yeast aldose reductase is stress-regulated. Overexpression of GRE3 increases methylglyoxal tolerance in Saccharomyces cerevisiae. Furthermore, high expression of GRE3 complements the deficiency of the glyoxalase system of a glo1Δ mutant strain. Consistent with this, in vitro and in vivo assays of yeast aldose reductase activity indicate that methylglyoxal is an endogenous substrate of aldose reductase. Furthermore, addition of NaCl or H2O2 to exponential-phase cells triggers an initial transient increase in the intracellular level of methylglyoxal, which is dependent on the Gre3p and Glo1p function. These observations indicate that the metabolism of methylglyoxal is stimulated under stress conditions; and they support a methylglyoxal degradative pathway, in which this compound is metabolised by the action of aldose reductase.

96 citations


Journal ArticleDOI
TL;DR: Results from this study confirm that MG-mediated arginine modifications occur in vivo and provide a method for assessing protein-arginine modification by MG in aging and diabetes.

Journal ArticleDOI
TL;DR: Through genetic engineering, appropriate combinations of enzymes have been brought together in Escherichia coli and yeast to generate 1,2-propanediol from glucose, and the optimization of these strains may yield microbial processes for the production of this widely used chemical.
Abstract: This short review covers metabolic pathways, genetics and metabolic engineering of 1,2-propanediol formation in microbes. 1,2-Propanediol production by bacteria and yeasts has been known for many years and two general pathways are recognized. One involves the metabolism of deoxyhexoses, where lactaldehyde is formed during the glycolytic reactions and is then reduced to 1,2-propanediol. The second pathway derives from the formation of methylglyoxal from dihydroxyacetonephosphate and its subsequent reduction to 1,2-propanediol. The enzymes involved in the reduction of methylglyoxal can generate isomers of lactaldehyde or acetol, which can be further reduced by specific reductases, giving chiral 1,2-propanediol as the product. The stereospecificity of the enzymes catalyzing the two reduction steps is important in deriving a complete pathway. Through genetic engineering, appropriate combinations of enzymes have been brought together in Escherichia coli and yeast to generate 1,2-propanediol from glucose. The optimization of these strains may yield microbial processes for the production of this widely used chemical.

Journal ArticleDOI
TL;DR: Anaerobic glycerol fermentation by Escherichia coli strains expressing genes from the Klebsiella pneumoniae dha regulon showed that cell growth and 1,3-propanediol production are significantly inhibited when 5 g/L or higher of Glycerol is initially present.

Journal ArticleDOI
TL;DR: In this paper, the cytotoxic effects of methylglyoxal on insulin-secreting cells, which are particularly sensitive to glucose toxicity, were investigated, and it was found that methyl glyoxal caused a concentration-dependent increase in the number of apoptotic RINm5F cells.

Journal ArticleDOI
TL;DR: It is shown that the Zn ligand Glu172 can abstract the substrate C1 proton from the S enantiomer of the substrate, without being displaced from the ZN ion, and the calculated activation barrier is in excellent agreement with experimental rates.
Abstract: Hybrid density functional theory is used to study the catalytic mechanism of human glyoxalase I (GlxI). This zinc enzyme catalyzes the conversion of the hemithioacetal of toxic methylglyoxal and glutathione to nontoxic (S)-d-lactoylglutathione. GlxI can process both diastereomeric forms of the substrate, yielding the same form of the product. As a starting point for the calculations, we use a recent crystal structure of the enzyme in complex with a transition-state analogue, where it was found that the inhibitor is bound directly to the zinc by its hydroxycarbamoyl functions. It is shown that the Zn ligand Glu172 can abstract the substrate C1 proton from the S enantiomer of the substrate, without being displaced from the Zn ion. The calculated activation barrier is in excellent agreement with experimental rates. Analogously, the Zn ligand Glu99 can abstract the proton from the R form of the substrate. To account for the stereochemical findings, it is argued that the S and R reactions cannot be fully symme...

Journal ArticleDOI
TL;DR: It is proposed that this role is detoxification of MG, the first report of targeted gene disruption in the C. difficilechromosome, and inactivation of gldA in both C. beijerinckiiand Clostridium difficiles gave rise to pinpoint colonies that could not be subcultured, indicating that glycerol dehydration performs an essential function in both organisms.
Abstract: In contrast to gram-negative bacteria, little is known about the mechanisms by which gram-positive bacteria degrade the toxic metabolic intermediate methylglyoxal (MG). Clostridium beijerinckii BR54, a Tn1545 insertion mutant of the NCIMB 8052 strain, formed cultures that contained significantly more (free) MG than wild-type cultures. Moreover, BR54 was more sensitive to growth inhibition by added MG than the wild type, suggesting that it has a reduced ability to degrade MG. The single copy of Tn1545 in this strain lies just downstream from gldA, encoding glycerol dehydrogenase. As a result of antisense RNA production, cell extracts of BR54 possess significantly less glycerol dehydrogenase activity than wild-type cell extracts (H. Liyanage, M. Young, and E. R. Kashket, J. Mol. Microbiol. Biotechnol. 2:87-93, 2000). Inactivation of gldA in both C. beijerinckii and Clostridium difficile gave rise to pinpoint colonies that could not be subcultured, indicating that glycerol dehydrogenase performs an essential function in both organisms. We propose that this role is detoxification of MG. To our knowledge, this is the first report of targeted gene disruption in the C. difficile chromosome.

Journal ArticleDOI
TL;DR: GO and MGO trigger a novel pathway for chemical reaction-mediated downregulation of ERK, and evidence was provided that GO/MGO upregulated MKP-1 activity that in turn dephosphorylated possibly co-aggregated phospho-ERK efficiently for inactivation.

Journal ArticleDOI
TL;DR: The crystal structure reveals that Asp-71 and phosphate are positioned to function as the base and leaving group, respectively, in a concerted suprafacial 1,4-elimination of phosphate from the enediolic intermediate in the second step of the MGS reaction, and the relative merits of both mechanisms are discussed.
Abstract: Methylglyoxal synthase (MGS) and triosephosphate isomerase (TIM) share neither sequence nor structural similarities, yet the reactions catalyzed by both enzymes are similar, in that both initially convert dihydroxyacetone phosphate to a cis-enediolic intermediate. This enediolic intermediate is formed from the abstraction of the pro-S C3 proton of DHAP by Asp-71 of MGS or the pro-R C3 proton of DHAP by Glu-165 of TIM. MGS then catalyzes the elimination of phosphate from this enediolic intermediate to form the enol of methylglyoxal, while TIM catalyzes proton donation to C2 to form d-glyceraldehyde phosphate. A competitive inhibitor of TIM, phosphoglycolohydroxamic acid (PGH) is found to be a tight binding competitive inhibitor of MGS with a Ki of 39 nM. PGH's high affinity for MGS may be due in part to a short, strong hydrogen bond (SSHB) from the NOH of PGH to the carboxylate of Asp-71. Evidence for this SSHB is found in X-ray, 1H NMR, and fractionation factor data. The X-ray structure of the MGS homohex...

Journal ArticleDOI
TL;DR: It was shown that Ov-GloI is expressed at elevated levels under conditions of oxidative stress and the role of Ov-gloI as a potential vaccine candidate or serodiagnostic tool was assessed.
Abstract: Glyoxal, methylglyoxal and other physiological alpha-oxoaldehydes are formed by the lipid peroxidation, glycation and degradation of glycolytic intermediates. They are detoxified enzymically by the glyoxalase system. To investigate the physiological function of glyoxalase I in parasitic organisms, the cDNA for glyoxalase I from the filarial nematode Onchocerca volvulus (designated Ov-GloI) has been cloned and characterized. The isolated cDNA contains an open reading frame of 579 bp encoding a protein with a calculated molecular mass of 21930 Da. Owing to the high degree of sequence identity (60%) with human glyoxalase I, for which the X-ray structure is available, it has been possible to build a three-dimensional model of Ov-GloI. The modelled core of Ov-GloI is conserved compared with the human glyoxalase I; however, there are critical differences in the residues lining the hydrophobic substrate-binding pocket of Ov-GloI. A 22 kDa protein was obtained by heterologous expression in Escherichia coli. A homogeneous enzyme preparation was obtained by affinity purification and functional characterization of the recombinant enzyme included the determination of kinetic constants for methylglyoxal and phenylglyoxal as well as inhibition studies. Gel filtration demonstrated a dimeric structure. To assess the role of Ov-GloI as a potential vaccine candidate or serodiagnostic tool, the serological reactivity of the recombinant Ov-GloI was analysed with sera from microfilaria carriers and specific IgG1 antibodies were detected. The effects of oxidative insult, namely plumbagin and xanthine/xanthine oxidase, on the gene transcript level of Ov-GloI were investigated. By using a semi-quantitative PCR ELISA it was shown that Ov-GloI is expressed at elevated levels under conditions of oxidative stress.

Journal ArticleDOI
TL;DR: This novel fluorescent arginine Maillard modification represents the first amino acid modification reported in beer retaining the full backbone of the original amino acid.
Abstract: Nδ-(5-Hydroxy-4,6-dimethylpyrimidine-2-yl)-l-ornithine, or Argpyrimidine, was identified and quantified in beer by high-performance liquid chromatography (HPLC) and coupled gas chromatography−mass spectrometry (HRGC−MS). This novel fluorescent arginine Maillard modification represents the first amino acid modification reported in beer retaining the full backbone of the original amino acid. Two mechanisms of formation could be verified: the major pathway via methylglyoxal and the minor pathway via 5-deoxypentoses. Argpyrimidine concentrations, determined in 35 lager-type beer varieties, reached up to 27 nmol/L and could be positively correlated to beer color and wort content. Within this context, 5-deoxy-d-ribose was identified as a novel intermediate of the Maillard reaction of maltose by HRGC−MS and independent synthesis. Keywords: Nδ-(5-Hydroxy-4,6-dimethylpyrimidine-2-yl)-l-ornithine; Argpyrimidine; 5-deoxypentose; 5-deoxy-d-ribose; Maillard reaction; beer; amino acid modification

Journal ArticleDOI
TL;DR: G glycated albumin may contribute to an imbalance between intraperitoneal formation and degradation of fibrin that causes peritoneal structural alterations, with subsequent membrane failure, and the present data support the clinical relevance of the formation of glycated proteins.
Abstract: ¨ Objective: The continuous contact of glucose-containing peritoneal dialysis (PD) fluids with the peritoneum results in the intraperitoneal formation of early and advanced glycation end-products. This nonenzymatic glycation of proteins may cause morphological and functional alterations to the peritoneum, which may contribute to patient dropout from PD therapy. Because fibrinolytic system components have been demonstrated to play an important role in the balance of intraperitoneal generation and degradation of fibrin, we studied the effect of early and advanced glycated human serum albumin, methylglyoxal, and 3-deoxyglucosone on the synthesis of tissue-type plasminogen activator (tPA), as well as its specific inhibitor (PAI-1), in human peritoneal mesothelial cells (HPMC). ¨ Methods: Antigen concentrations in the supernatants of cultured HPMC were measured by ELISA. Northern blot analysis was conducted for mRNA expression. Electrophoretic mobility shift assays were applied to demonstrate the involvement of the transcription factors nuclear factor kappa B (NF-kB) and activator protein-1 (AP-1) in signal transduction. ¨ Results: Incubation of HPMC with early glycated albumin (GHSA) resulted in a time- and concentration-dependent increase in PAI-1 mRNA expression and antigen secretion. In contrast, no changes in PAI-1 synthesis occurred after stimulation with either the 1,2-dicarbonyl compounds methylglyoxal and 3-deoxyglucosone, or with late advanced glycation end-products. tPA synthesis was not affected by any of the tested components. Furthermore, HPMC exposed to GHSA induced NF-kB and AP-1 DNA binding activity, suggesting that GHSA-induced overexpression of PAI-1 is transcriptionally regulated by both transcription factors. ¨ Conclusions: We conclude that Amadori modified glycated albumin upregulates PAI-1 synthesis in HPMC, possibly mediated through the activation of the transcription factors NF-k kk kB and AP-1. The present data support the clinical relevance of the formation of glycated proteins and their involvement in pathological processes in PD patients. Thus, glycated albumin may contribute to an imbalance between intraperitoneal formation and degradation of fibrin that causes peritoneal structural alterations, with subsequent membrane failure.

Journal ArticleDOI
TL;DR: The morphofunctional effect of methylglyoxal on the common toad B. bufo embryo mitochondria is investigated to verify if its dysmorphogenetic action might be also ascribed to impairment of mitochondrial functions.

Journal ArticleDOI
TL;DR: This study shows that troglitazone is a selective inhibitor of the glyoxalase system, which could contribute to trog litazone's hepatotoxic action which has previously been reported in a small percentage of individuals.
Abstract: Aims/hypothesis: The hyperglycaemia associated with diabetes causes excessive production of cytotoxic methylglyoxal, an α-oxo-aldehyde. The glyoxalase system, composed of glyoxalase I and glyoxalase II, with glutathione (GSH) as the cofactor, plays an important role in the detoxification of α-oxo-aldehydes. We tested the hypothesis that troglitazone, an insulin-sensitizing drug previously used in the treatment of Type II (non-insulin-dependent) diabetes mellitus, up-regulates the glyoxalase system either by increasing phase 2 enzyme activities and thereby increasing cellular GSH, or, by inducing glyoxalase enzyme activities.

Journal ArticleDOI
TL;DR: CHS 828 does not appear to share any major mechanisms of action with the polyamine synthesis inhibitor MGBG, and further studies will be required to define the exact mechanism of action.

Journal ArticleDOI
TL;DR: Data suggest that MG causes oxidative stress in cells, which is due at least in part to ferric iron reduction by MG and to the modification of amino acids e.g. L-arginine by MG, which was catalyzed by iron redox cycling.

01 Jan 2001
TL;DR: It is believed that 6 chemicals may effectively quench GDPs and inhibit AGE cross-link formation, in a manner different from that of aminoguanidine and thiazolium.
Abstract: The present study evaluated the inhibiting effect of various chemicals on the advanced glycation end-product (AGEs) cross-linking caused in protein by glucose degradation products (GDPs). We evaluated a few dozen organic and inorganic chemicals--in addition to previously reported AGE inhibitors, such as thiazolium derivatives and aminoguanidine--for their inhibiting effect. Collagen IV (from human placenta) or human serum albumin (HSA) was incubated with an AGE accelerator and one of the selected chemicals in phosphate buffer solution at 37 degrees C for as long as 14 days. Fluorescence intensity (440 nm) was determined after a given incubation time. Among 36 chemicals tested, 8 new chemicals and 5 previously known AGE inhibitors significantly suppressed the increase in fluorescence intensity seen after incubation of HSA with methylglyoxal. We believe that 6 chemicals may effectively quench GDPs and inhibit AGE cross-link formation, in a manner different from that of aminoguanidine and thiazolium.

01 Jan 2001
TL;DR: The results strongly suggest that this anticancer formulation with methylglyoxal as the lead ingredient is by far more superior than other present forms of treatment against cancer.
Abstract: Based on our previous in vitro studies with human cells and in vivo studies with animals we had developed an anticancer formulation with methylglyoxal as the lead ingredient. This formation has a tumoricidal effect by inhibiting specifically in cancerous cells, the electron flow and the transfer of reducing equivalent necessary for the production of adenosine-5'-triphosphate (ATP), the cellular energy currency. By keeping this remarkable property in mind, we had treated 24 patients suffering from different types of malignancy (mostly in very advanced stage of the disease) with this methylglyoxal-based formulation. The results indicate a dramatic positive effect on the patients. Out of the 24 patients, 11 are in excellent physical condition, the condition of 5 patients can be considered stable. The rest had either opted out from the treatment or died during the course of study. These results strongly suggest that this formulation is by far more superior than other present forms of treatment against cancer. It is imperative that this formulation be widely used in treating cancer patients, as well as to attempt the improvement of its efficacy.

Journal ArticleDOI
TL;DR: Results suggest that the inhibitory effects of GSNO on cell proliferation and DNA synthesis might be at least partly due to inactivation of Glo I, and glyoxalase I activity was consistently repressed during exposure to GSNO.

Journal ArticleDOI
TL;DR: This work reports here a simple approach that allows the preparation and isolation of milligram quantities of sugar-mediated AGE Lys-Lys-like crosslinks from glycation mixtures, and fractionation of these preparations over a monomeric avidin column afforded a complete separation of cane sugar modifications and the crosslinks.
Abstract: Advanced glycation end products (AGEs) and, specifically, protein-protein AGE crosslinks have long been studied for their potential role in aging, diabetic complications and Alzheimer disease. With few exceptions, the chemical nature of these structures remains unknown. We report here a simple approach that allows the preparation and isolation of milligram quantities of sugar-mediated AGE Lys-Lys-like crosslinks from glycation mixtures. The method is based on a sugar-dependent incorporation of Nα-biotinyl-l-Lys into cysteaminyldisulfide Sepharose 6B (AE-S-S-Sepharose 6B). Glycation mixtures with six different sugars showed a time- and sugar-dependent decrease in the concentration of the support-bound primary amino groups and accounted for almost 90% loss of cysteaminyl amino groups at the end of the various incubation periods. 4-Hydroxyazobenzene-2-carboxylic acid-avidin assays indicated the incorporation of Nα-biotinyl-l-Lys equal to 8% of the total support amino groups with methylglyoxal after 7d and 1% with fructose and glucose after 1 mo of incubation. Treatment of the washed, sugar-modified supports with 2-mercaptoethanol released the bulk of the bound AGE modifications and the crosslinks. Subsequent fractionation of these preparations over a monomeric avidin column afforded a complete separation of sugar-mediated AGE modifications and the crosslinks. Depending on the sugar employed, micromolar amounts of biotinylated Lys-Lys-like crosslinks were generated by this two-step procedure from 8 mL of the original AE-S-S-Sepharose 6B.

Journal ArticleDOI
TL;DR: In this article, the authors show that the addition of organylthiols to 2-ethoxypropenal follows the Markovnikov rule to give 2-ETHO-2-organylthiopropanals which spontaneously isomerize to 1-ETHOO-1-organYLthiopro-anones in storage or in the presence of an acid catalyst.