A functional arginine residue in rabbit-muscle aldolase.
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Rabbit muscle aldolase is irreversibly modified by the arginine-selective alpha-dicarbonyl, phenylglyoxal, loss of activity correlating with the unique modifications of one arginine residue per subunit, as determined by amino acid analysis, and (7-14C)phenylglyoxal incorporation. The affinity of the modified enzyme for dihydroxyacetone phosphate is significantly reduced while substantial protection against inactivation is afforded by fructose 1,6-disphosphate, dihydroxyacetone phosphate or phosphate ion. The nature of the substrate C-1 phosphate binding site in this enzyme is discussed in the light of these and other results. (+info)
Induction of 1,2-dicarbonyl compounds, intermediates in the formation of advanced glycation end-products, during heat-sterilization of glucose-based peritoneal dialysis fluids.
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OBJECTIVE: To study the presence of 1,2-dicarbonyl compounds in peritoneal dialysis (PD) fluids, their concentration in effluents with increasing dwell time, and their role in the formation of advanced glycation end-products (AGEs). MEASUREMENTS: Dicarbonyl compounds in heat- and filter-sterilized PD fluids were quantified by reverse-phase high performance liquid chromatography (HPLC) after derivatization to dimethoxyquinoxaline derivatives. Kinetics of the in vitro formation of AGEs upon incubation of 1,2-dicarbonyl compounds or PD fluids with albumin, with or without aminoguanidine, were measured by AGE fluorescence (excitation/emission wavelengths of 350 nm/430 nm). PATIENTS: AGEs and dicarbonyl compounds were measured in effluents collected from standardized 4-hour dwells from 8 continuous cycling peritoneal dialysis patients. RESULTS: In PD fluids, 3-deoxyglucosone (3-DG) has been identified as the major dicarbonyl compound formed during the process of heat sterilization. The process also formed glyoxal (GO) and methylglyoxal (MGO), with the amount of 3-DG being approximately 25-60 times higher than GO and MGO. When incubated with albumin, the identified 1,2-dicarbonyl compounds rapidly formed AGEs. The formation of AGEs was more pronounced in conventional heat-sterilized PD fluids compared with filter-sterilized PD fluids, and was completely inhibited by aminoguanidine. In effluents, the concentration of MGO, GO, and 3-DG decreased with increasing dwell time, with a concomitant increase in AGE fluorescence. CONCLUSIONS: The dicarbonyl compounds 3-DG, MGO, and GO are potent promoters of AGE formation. The presence of these and possibly other dicarbonyl compounds formed during heat sterilization of glucose-based PD fluids is, to a large extent, responsible for the in vitroAGE formation by these fluids, as evidenced by the speed of AGE formation in PD fluids and the complete inhibition by aminoguanidine. Because 3-DG, MGO, and GO are rapidly cleared from PD fluids during dialysis, these compounds may contribute to the in vivo AGE formation in PD patients. (+info)
A new method of DNA denaturation mapping.
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Described is a new method for DNA denaturation mapping utilizing glyoxal (ethanedial) to stabilize the denatured regions. The extent of glyoxal reaction can be easily and sensitively measured using an assay based on the intercalation of ethidium into duplex DNA. Thus denturation maps can be produced in a controlled way under a wide variety of conditions. (+info)
Formation of glyoxal, methylglyoxal and 3-deoxyglucosone in the glycation of proteins by glucose.
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The glycation of proteins by glucose has been linked to the development of diabetic complications and other diseases. Early glycation is thought to involve the reaction of glucose with N-terminal and lysyl side chain amino groups to form Schiff's base and fructosamine adducts. The formation of the alpha-oxoaldehydes, glyoxal, methylglyoxal and 3-deoxyglucosone, in early glycation was investigated. Glucose (50 mM) degraded slowly at pH 7.4 and 37 degrees C to form glyoxal, methylglyoxal and 3-deoxyglucosone throughout a 3-week incubation period. Addition of t-BOC-lysine and human serum albumin increased the rate of formation of alpha-oxoaldehydes - except glyoxal and methylglyoxal concentrations were low with albumin, as expected from the high reactivity of glyoxal and methylglyoxal with arginine residues. The degradation of fructosyl-lysine also formed glyoxal, methylglyoxal and 3-deoxyglucosone. alpha-Oxoaldehyde formation was dependent on the concentration of phosphate buffer and availability of trace metal ions. This suggests that alpha-oxoaldehydes were formed in early glycation from the degradation of glucose and Schiff's base adduct. Since alpha-oxoaldehydes are important precursors of advanced glycation adducts, these adducts may be formed from early and advanced glycation processes. Short periods of hyperglycaemia, as occur in impaired glucose tolerance, may be sufficient to increase the concentrations of alpha-oxoaldehydes in vivo. (+info)
Immunological evidence that non-carboxymethyllysine advanced glycation end-products are produced from short chain sugars and dicarbonyl compounds in vivo.
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BACKGROUND: The Maillard reaction that leads to the formation of advanced glycation end-products (AGE) plays an important role in the pathogenesis of angiopathy in diabetic patients and in the aging process. Recently, it was proposed that AGE were not only created by glucose, but also by dicarbonyl compounds derived from the Maillard reaction, autoxidation of sugars and other metabolic pathways of glucose. In this study, we developed four types of non-carboxymethyllysine (CML) anti-AGE antibodies that recognized proteins modified by incubation with short chain sugars and dicarbonyl compounds. MATERIALS AND METHODS: AGE-modified serum albumins were prepared by incubation of rabbit serum albumin with glyceraldehyde, glycolaldehyde, methylglyoxal or glyoxal. After immunization of rabbits, four types of AGE-specific antisera were obtained that were specific for the AGE modification. To separate non-CML AGE antibodies (Ab) (non-CML AGE-Ab-2, -3, -4, and -5), these anti-AGE antisera were subjected to affinity chromatography on a matrix coupled with four kinds of AGE bovine serum albumin (BSA) or CML-BSA. These non-CML AGE antibodies were used to investigate the AGE content of serum obtained from diabetic patients on hemodialysis. RESULTS: Characterization of the four types of non-CML AGE antibodies obtained by immunoaffinity chromatography was performed by competitive ELISA and immunoblot analysis. Non-CML AGE-Ab-2 crossreacted with the protein modified by glyceraldehyde or glycolaldehyde. Non-CML AGE-Ab-3 and -Ab-4 specifically cross-reacted with protein modified by glycolaldehyde and methylglyoxal, respectively. NonCML AGE-Ab-5 cross-reacted with protein modified with glyoxal as well as methylglyoxal and glycolaldehyde. Three kinds of non-CML AGE (AGE-2, -4, and -5) were detected in diabetic serum as three peaks with apparent molecular weights of 200, 1.15, and 0.85 kD; whereas, AGE-3 was detected as two peaks with apparent molecular weights of 200 and 0.85 kD. CONCLUSION: We propose that various types of non-CML AGE are formed by the Maillard reaction, sugar autoxidation and sugar metabolism. These antibodies enable us to identify such compounds created by the Maillard reaction in vivo. (+info)
A comparison of mutation spectra detected by the Escherichia coli lac(+) reversion assay and the Salmonella typhimurium his(+) reversion assay.
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Each of the Escherichia coli tester strains in the WP3101P-WP3106P series contains an F' plasmid with a different base substitution mutation within the lacZ gene. Each of the six possible base substitution mutations, therefore, can be assayed with these strains by Lac(+) reversion. We used the strains to characterize the mutational profiles of 21 chemical mutagens, including alkylating agents, base analogs and oxidative compounds. We also assayed the mutagens with Salmonella typhimurium tester strains TA7002, TA7004 and TA7005, which detect A.T-->T.A, G.C-->A.T and G.C-->T.A mutations, respectively, and we compared the sensitivity and specificity of the two systems. Escherichia coli strain WP3102P was more sensitive than the S.TYPHIMURIUM: strains to G.C-->A.T transitions induced by N(4)-aminocytidine, 5-azacytidine, cumene hydroperoxide (CHP), t-butyl hydroperoxide (BHP), N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG), methyl methane sulfonate and N-ethyl-N-nitrosourea (ENU), while the reverse was true for G.C-->A.T transitions induced by 2-aminopurine and phosmet. Escherichia coli strain WP3104P, which detects G.C-->T.A transversions, was superior to the S.TYPHIMURIUM: strains in detecting transversions induced by N(4)-aminocytidine, 5-azacytidine, 5-diazouracil, CHP, BHP, ENNG, ENU, 4-nitroquinoline 1-oxide (4-NQO) and 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX). Escherichia coli WP3105P was also more sensitive than S. TYPHIMURIUM: to A.T-->T.A transversions induced by N-methyl-N- nitrosourea (MNU), CHP and 4-NQO, but it was less sensitive to those induced by ENNG, ENU and 2-aminopurine. The present results indicate that the E.COLI: Lac(+) reversion system with tester strains WP3101P-WP3106P is as sensitive as the S.TYPHIMURIUM: His(+) reversion system for the detection of specific mutations induced by a variety of direct mutagens. (+info)
Selection for high immunogenicity in drug-resistant sublines of murine lymphomas demonstrated by plaque assay.
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The immunogenicity of lymphoma L1210 and three L1210 sublines, resistant to methylglyoxal bis(guanylhydrazone), 4,4'-diacetyldiphenylurea bis(guanylhydrazone), or guanazole (L1210/GZL), respectively, was evaluated. Syngeneic DBA/2J mice were given a single i.p. injection of serially diluted suspension of irradiated cells from L1210 or L1210 sublines. Five days later spleen cells from the immunized mice were tested for the presence of plaque-forming cells using the immunizing lymphoma cell lines as target. Sera collected from the animals were examined for cytolytic antibody activity by lysis in gel using the same target cells. For comparison, the H-2 immunogenicity of L1210 and its sublines was investigated in H-2-incompatible allogeneic mice. The following results were obtained. (a) All the sublines showed increased immunogenicity and susceptibility to lysis as compared to L1210 cells. The number of plaque-forming cells/spleen ranged from 100 for L1210 to 4450 for L1210/GZL, the most immunogenic subline, and the antibody titer ranged from 1/8 for L1210 to 1/128 for L1210/GZL. (b) All the sublines carried common tumor-associated antigens that apparently made primary contributions to the increased immunogenicity. (c) The common tumor-associated antigens were also expressed on L1210 cells, although in a lesser defree, as evidenced by the definite, albeit low, capacity of L1210 cells to absorb DBA/2J anti-L1210/GZL antibodies. (d) Spleen and thymus cells of DBA/2J mice as well as unrelated murine ascites tumor cells did not cause significant absorption of these antibodies. (e) Only a partial inverse relationship could be demonstrated between tumor-associated antigens but the lowest for H-2. The above results would seem compatible with the hypothesis that the increased immunogenicity of drug-resistant L1210 sublines is attributable to the selection of preexisting highly immunogenic cells during immunosuppression by treatments selecting for drug resistance. (+info)
Mechanism of the inhibitory effect of OPB-9195 [(+/-)-2-isopropylidenehydrazono-4-oxo-thiazolidin-5-yla cetanilide] on advanced glycation end product and advanced lipoxidation end product formation.
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The accumulation in uremic plasma of reactive carbonyl compounds (RCO) derived from both carbohydrates and lipids ("carbonyl stress") contributes to uremic toxicity by accelerating the advanced glycation and lipoxidation of proteins. It was previously demonstrated that OPB-9195 [(+/-)-2-isopropylidenehydrazono-4-oxo- thiazolidin-5-ylacetanilide] inhibited the in vitro formation of advanced glycation end products (AGE) in uremic plasma. This study was designed to elucidate the mechanism of action of OPB-9195 by further delineating the AGE and advanced lipoxidation end product (ALE) precursors targeted by this drug. The inhibitory effects of OPB-9195 on the formation of two AGE (N:epsilon-carboxymethyllysine and pentosidine) on bovine serum albumin incubated with various AGE precursors were examined. Inhibition of N:epsilon-carboxymethyllysine and pentosidine formation with OPB-9195 was more efficient than with aminoguanidine. OPB-9195 also proved effective in blocking the carbonyl amine chemical processes involved in the formation of two ALE (malondialdehyde-lysine and 4-hydroxynonenal-protein adduct). The efficiency of OPB-9195 was similar to that of aminoguanidine. When glucose-based peritoneal dialysis fluid was incubated in the presence of OPB-9195, a similar inhibition of AGE formation was observed. The direct effect of OPB-9195 on major glucose-derived RCO in peritoneal dialysis fluids was then evaluated. The effects of OPB-9195 could be accounted for by its ability to trap RCO. The concentrations of three major glucose-derived RCO (glyoxal, methylglyoxal, and 3-deoxy-glucosone) were significantly lower in the presence of OPB-9195 than in its absence. Aminoguanidine had a similar effect. In conclusion, OPB-9195 inhibits both AGE and ALE formation, probably through its ability to trap RCO. OPB-9195 might prove to be a useful tool to inhibit some of the effects of RCO-related uremic toxicity. (+info)