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Strenuous exercise induces delayed-onset muscle damage including oxidative damage of cellular components. Oxidative stress to muscle cells impairs glucose uptake via disturbance of insulin signaling pathway. We investigated glucose uptake and insulin signaling in relation to oxidative protein modification in muscle after acute strenuous exercise. ICR mice were divided into sedentary and exercise groups. Mice in the exercise group performed downhill running exercise at 30 m/min for 30 min. At 24 hr after exercise, metabolic performance and insulin-signaling proteins in muscle tissues were examined. In whole body indirect calorimetry, carbohydrate utilization was decreased in the exercised mice along with reduction of the respiratory exchange ratio compared to the rested control mice. Insulin-stimulated uptake of 2-deoxy-[<³H]glucose in damaged muscle was decreased after acute exercise. Tyrosine phosphorylation o insulin receptor substrate (IRS)-I and phosphatidyl-3-kinase/Akt signaling were impaired by exercise, leading to inhibition of the membrane translocation of glucose transporter 4. We also found that acute exercise caused 4-hydroxy-nonenal modification of IRS-1 along with elevation of oxidative stress in muscle tissue. Impairment of insulin-induced glucose uptake into damaged muscle after strenuous exercise would be related to disturbance of insulin signal transduction by oxidative modification of IRS-1. [ABSTRACT FROM AUTHOR]

Background: Secondary carnitine deficiency in patients with anorexia nervosa has been rarely reported. This study aimed to investigate the occurrence of carnitine deficiency in severely malnourished patients with eating disorders during refeeding and assess its potential adverse effects on treatment outcomes., Method: In a cohort study of 56 female inpatients with eating disorders at a single hospital from March 2010 to December 2020, we measured plasma free carnitine (FC) levels and compared to those of a healthy control group (n = 35). The patients were categorized into three groups based on FC levels: FC deficiency (FC< 20 µmol/L), FC pre-deficiency (20 µmol/L ≤ FC< 36 µmol/L), and FC normal (36 µmol/L ≤ FC)., Results: Upon admission, the patients had a median age of 26 years (interquartile range [IQR]: 21-35) and a median body mass index (BMI) of 13.8 kg/m 2 (IQR: 12.8-14.8). Carnitine deficiency or pre-deficiency was identified in 57% of the patients. Hypocarnitinemia was associated with a decline in hemoglobin levels during refeeding (odds ratio [OR]: 0.445; 95% confidence interval [CI]: 0.214-0.926, p = 0.03), BMI at admission (OR: 0.478; 95% CI: 0.217-0.874, p = 0.014), and moderate or greater hepatic impairment at admission (OR: 6.385; 95% CI: 1.170-40.833, p = 0.032)., Conclusions: Hypocarnitinemia, particularly in cases of severe undernutrition (BMI< 13 kg/m 2 at admission) was observed in severely malnourished patients with eating disorders during refeeding, a critical metabolic transition phase. Moderate or severe hepatic impairment at admission was considered a potential indicator of hypocarnitinemia. Although hypocarnitinemia was not associated with any apparent adverse events other than anemia during refeeding, the possibility that carnitine deficiency may be a risk factor for more serious complications during sudden increases in energy requirements associated with changes in physical status cannot be denied. Further research on the clinical significance of hypocarnitinemia in severely malnourished patients with eating disorders is warranted., (© 2024. The Author(s).)

Aim: Eating disorders (EDs) are complex, multifactorial psychiatric conditions. Previous studies identified pathogenic copy number variations associated with NDDs (NDD-CNVs) in ED patients. However, no statistical evidence for an association between NDD-CNVs and EDs has been demonstrated. Therefore, we examined whether NDD-CNVs confer risk for EDs., Methods: Using array comparative genomic hybridization (aCGH), we conducted a high-resolution CNV analysis of 71 severe female ED patients and 1045 female controls. According to the American College of Medical Genetics guidelines, we identified NDD-CNVs or pathogenic/likely pathogenic CNVs in NDD-linked loci. Gene set analysis was performed to examine the involvement of synaptic dysfunction in EDs. Clinical data were retrospectively examined for ED patients with NDD-CNVs., Results: Of the samples analyzed with aCGH, 70 severe ED patients (98.6%) and 1036 controls (99.1%) passed our quality control filtering. We obtained 189 and 2539 rare CNVs from patients and controls, respectively. NDD-CNVs were identified in 10.0% (7/70) of patients and 2.3% (24/1036) of controls. Statistical analysis revealed a significant association between NDD-CNVs and EDs (odds ratio = 4.69, P = 0.0023). NDD-CNVs in ED patients included 45,X and deletions at KATNAL2, DIP2A, PTPRT, RBFOX1, CNTN4, MACROD2, and FAM92B. Four of these genes were related to synaptic function. In gene set analysis, we observed a nominally significant enrichment of rare exonic CNVs in synaptic signaling in ED patients (odds ratio = 2.55, P = 0.0254)., Conclusion: Our study provides the first preliminary evidence that NDD-CNVs may confer risk for severe EDs. The pathophysiology may involve synaptic dysfunction., (© 2022 The Authors. Psychiatry and Clinical Neurosciences published by John Wiley & Sons Australia, Ltd on behalf of Japanese Society of Psychiatry and Neurology.)

Multiple studies on the dynamics of inflammatory cytokines in patients with anorexia nervosa (AN) have been published, although results are not consistent among reports. Thus the pathophysiologic roles of these cytokines are not clear. We performed an exploratory analysis that included (1) comparisons of plasma interleukin-18 (IL-18) concentrations between patients with AN ( n = 21) and healthy controls ( n = 39), and (2) correlations between body mass index (BMI) and IL-18 concentrations in both groups, exploring the relationship between malnourishment and IL-18. Plasma IL-18 levels were significantly decreased in patients with AN compared with controls. Plasma IL-18 levels correlated to BMI in controls, but not in patients with AN. These results suggest that a decline in plasma IL-18 levels in patients with AN is not only due to malnourishment, but other pathophysiologic changes as well. IL-18 has a role in the brain's reaction to sadness and chronic stress. Therefore, decreased levels of IL-18 may commonly occur in patients with chronic AN.

In schizophrenia (SCZ) and autism spectrum disorder (ASD), the dysregulation of glutamate transmission through N-methyl-D-aspartate receptors (NMDARs) has been implicated as a potential etiological mechanism. Previous studies have accumulated evidence supporting NMDAR-encoding genes' role in etiology of SCZ and ASD. We performed a screening study for exonic regions of GRIN1, GRIN2A, GRIN2C, GRIN2D, GRIN3A, and GRIN3B, which encode NMDAR subunits, in 562 participates (370 SCZ and 192 ASD). Forty rare variants were identified including 38 missense, 1 frameshift mutation in GRIN2C and 1 splice site mutation in GRIN2D. We conducted in silico analysis for all variants and detected seven missense variants with deleterious prediction. De novo analysis was conducted if pedigree samples were available. The splice site mutation in GRIN2D is predicted to result in intron retention by minigene assay. Furthermore, the frameshift mutation in GRIN2C and splice site mutation in GRIN2D were genotyped in an independent sample set comprising 1877 SCZ cases, 382 ASD cases, and 2040 controls. Both of them were revealed to be singleton. Our study gives evidence in support of the view that ultra-rare variants with loss of function (frameshift, nonsense or splice site) in NMDARs genes may contribute to possible risk of SCZ.

N-methyl-d-aspartate receptors (NMDARs) play a critical role in excitatory synaptic transmission and plasticity in the central nervous systems. Recent genetics studies in schizophrenia (SCZ) show that SCZ is susceptible to NMDARs and the NMDAR signaling complex. In autism spectrum disorder (ASD), several studies report dysregulation of NMDARs as a risk factor for ASD. To further examine the association between NMDARs and SCZ/ASD development, we conducted a mutation screening study of GRIN2B which encodes NR2B subunit of NMDARs, to identify rare mutations that potentially cause diseases, in SCZ and ASD patients (n = 574 and 152, respectively). This was followed by an association study in a large sample set of SCZ, ASD, and normal healthy controls (n = 4145, 381, and 4432, respectively). We identified five rare missense mutations through the mutation screening of GRIN2B. Although no statistically significant association between any single mutation and SCZ or ASD was found, one of its variant, K1292R, is found only in the patient group. To further examine the association between mutations in GRIN2B and SCZ/ASD development, a larger sample size and functional experiments are needed.

PSD-95 associated PSD proteins play a critical role in regulating the density and activity of glutamate receptors. Numerous previous studies have shown an association between the genes that encode these proteins and schizophrenia (SZ) and autism spectrum disorders (ASD), which share a substantial portion of genetic risks. We sequenced the protein-encoding regions of DLG1, DLG2, DLG4, DLGAP1, DLGAP2, and SynGAP in 562 cases (370 SZ and 192 ASD patients) on the Ion PGM platform. We detected 26 rare (minor allele frequency <1%), non-synonymous mutations, and conducted silico functional analysis and pedigree analysis when possible. Three variants, G344R in DLG1, G241S in DLG4, and R604C in DLGAP2, were selected for association analysis in an independent sample set of 1315 SZ patients, 382 ASD patients, and 1793 healthy controls. Neither DLG4-G241S nor DLGAP2-R604C was detected in any samples in case or control sets, whereas one additional SZ patient was found that carried DLG1-G344R. Our results suggest that rare missense mutations in the candidate PSD genes may increase susceptibility to SZ and/or ASD. These findings may strengthen the theory that rare, non-synonymous variants confer substantial genetic risks for these disorders.

Both schizophrenia (SCZ) and autism spectrum disorders (ASD) are neuropsychiatric disorders with overlapping genetic etiology. Protocadherin 15 (PCDH15), which encodes a member of the cadherin super family that contributes to neural development and function, has been cited as a risk gene for neuropsychiatric disorders. Recently, rare variants of large effect have been paid attention to understand the etiopathology of these complex disorders. Thus, we evaluated the impacts of rare, single-nucleotide variants (SNVs) in PCDH15 on SCZ or ASD. First, we conducted coding exon-targeted resequencing of PCDH15 with next-generation sequencing technology in 562 Japanese patients (370 SCZ and 192 ASD) and detected 16 heterozygous SNVs. We then performed association analyses on 2,096 cases (1,714 SCZ and 382 ASD) and 1,917 controls with six novel variants of these 16 SNVs. Of these six variants, four (p.R219K, p.T281A, p.D642N, c.3010-1G>C) were ultra-rare variants (minor allele frequency < 0.0005) that may increase disease susceptibility. Finally, no statistically significant association between any of these rare, heterozygous PCDH15 point variants and SCZ or ASD was found. Our results suggest that a larger sample size of resequencing subjects is necessary to detect associations between rare PCDH15 variants and neuropsychiatric disorders.

B-cell CLL/lymphoma 9 (BCL9) is located within the schizophrenia (SCZ) suspected locus chr1q21.1. A recent study reported that a single nucleotide polyphormism (SNP) within BCL9 (rs583583) is associated with negative symptoms of Schizophrenia, as measured by the Positive and Negative Syndrome Scale (PANSS), in the Caucasian population. We therefore investigated genetic association of rs583583, and its effect on negative symptoms in the Japanese patients. For association analysis, we used a Japanese sample set comprising 1089 SCZ and 950 controls (CON). Analysis of the effect of rs586586 on negative symptoms as examined by PANSS was investigated using 280 SCZ. Furthermore, for analysis of cognitive performance, we investigated 90 SCZ and 51 CON using the Continuous Performance Test (CPT-IP) and the Wisconsin Card Sorting Test (WCST) Keio version. We did not detect association between rs583583 and SCZ. Furthermore, rs583583 was not associated with PANSS negative scores or with CPT-IT or WCST cognitive tests. Considering the results of our previous study, combined with the results of the current study of rs583583, we argue that BCL9 most likely does not harbor a common genetic variant that can increase the risk for SCZ in the Japanese population.

N(ε)-acetylation occurs on select lysine residues in α-crystallin of the human lens and alters its chaperone function. In this study, we investigated the effect of N(ε)-acetylation on advanced glycation end product (AGE) formation and consequences of the combined N(ε)-acetylation and AGE formation on the function of α-crystallin. Immunoprecipitation experiments revealed that N(ε)-acetylation of lysine residues and AGE formation co-occurs in both αA- and αB-crystallin of the human lens. Prior acetylation of αA- and αB-crystallin with acetic anhydride (Ac(2)O) before glycation with methylglyoxal (MGO) resulted in significant inhibition of the synthesis of two AGEs, hydroimidazolone (HI) and argpyrimidine. Similarly, synthesis of ascorbate-derived AGEs, pentosidine and N(ε)-carboxymethyl lysine (CML), was inhibited in both proteins by prior acetylation. In all cases, inhibition of AGE synthesis was positively related to the degree of acetylation. While prior acetylation further increased the chaperone activity of MGO-glycated αA-crystallin, it inhibited the loss of chaperone activity by ascorbate-glycation in both proteins. BioPORTER-mediated transfer of αA- and αB-crystallin into CHO cells resulted in significant protection against hyperthermia-induced apoptosis. This effect was enhanced in acetylated and MGO-modified αA- and αB-crystallin. Caspase-3 activity was reduced in α-crystallin transferred cells. Glycation of acetylated proteins with either MGO or ascorbate produced no significant change in the anti-apoptotic function. Collectively, these data demonstrate that lysine acetylation and AGE formation can occur concurrently in α-crystallin of human lens, and that lysine acetylation improves anti-apoptotic function of α-crystallin and prevents ascorbate-mediated loss of chaperone function., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background and Aims: Recent advancements in capsule endoscopy and double-balloon endoscopy have revealed that non-steroidal anti-inflammatory drugs (NSAIDs), such as indomethacin, can induce small intestinal mucosal damage. However, the precise pathogenesis and therapeutic strategy have not been fully revealed. The aim of the present study was to determine the upregulated proteins in the small intestine exposed to indomethacin., Methods: Indomethacin (10 mg/kg) was administered subcutaneously to male Wistar rats to induce small intestinal damage and the severity of the intestinal injury was evaluated by measuring the area of visible ulcerative lesions. The intestinal mucosal tissue samples were collected and then analyzed by two-dimensional gel electrophoresis, with matrix-assisted laser desorption/ionization time-of-flight spectrometer peptide mass fingerprinting being used to determine the differentially expressed proteins between normal and injured intestinal mucosa., Results: Among several protein spots showing differential expression, one, hemopexin (HPX), was identified as upregulated in indomethacin-induced injured intestinal mucosa using the MASCOT search engine., Conclusion: HPX was identified as upregulated protein in the small intestine exposed to indomethacin. HPX may be responsible for the development of the intestinal inflammation induced by NSAIDs., (© 2012 Journal of Gastroenterology and Hepatology Foundation and Blackwell Publishing Asia Pty Ltd.)

N(ε)-(Hexanoyl)lysine, formed by the reaction of lysine with n-6 lipid hydroperoxide, is a lipid peroxidation marker during the initial stage of oxidative stress. The aim of the present study is to indentify N(ε)-(hexanoyl)lysine-modified proteins in neoplastic transformed gastric mucosal cells by N-methyl-N'-nitro-N-nitrosoguanidine, and to compare the levels of these proteins between gastric mucosal cells and normal gastric cells. Much greater fluorescence of 2-[6-(4'-hydroxy)phenoxyl-3H-xanthen-3-on-9-yl]benzoic acid, an index of the intracellular levels of reactive oxygen species, was observed for gastric mucosal cells compared to normal gastric cells. N(ε)-(Hexanoyl)lysine-modified proteins were detected by SDS-PAGE or two-dimensional electrophoresis and Western blotting using anti-N(ε)-(hexanoyl)lysine polyclonal antibody, and a protein band of between 30-40 kDa was clearly increased in gastric mucosal cells compared to normal gastric cells. Two N(ε)-(hexanoyl)lysine-modified protein spots in gastric mucosal cells were identified as the tropomyosin 1 protein by mass spectrometry using a MASCOT search. The existence of N(ε)-(hexanoyl)lysine modification in tropomyosin 1 was confirmed by Western blotting of SDS-PAGE-separated or two-dimensional electrophoresis-separated proteins as well as by the immunoprecipitation with anti-tropomyosin 1 antibody. These data indicate that N(ε)-(hexanoyl)lysine modification of tropomyosin 1 may be related to neoplastic transformation by N-methyl-N'-nitro-N-nitrosoguanidine in gastric epithelial cells.

Methylglyoxal is a reactive dicarbonyl compound produced from cellular glycolytic intermediates that reacts nonenzymatically with proteins to form products such as argpyrimidine at arginine residues. Abnormal accumulation of methylglyoxal and methylglyoxal-derived advanced glycation end products (AGEs) occurs under hyperglycemic conditions and has been implicated in endothelium dysfunction, arterial stiffening, and microvascular complications in diabetes. However, the role of methylglyoxal in the healing process of diabetic gastric ulcers has not been fully investigated. Recently, methylglyoxal modification of peroxiredoxin-VI was found to be associated with delayed healing of diabetic gastric ulcers. Thus, inhibition of methylglyoxal modification might have therapeutic potential for the treatment of such ulcers. In this review, we present what is currently known regarding the role of methylglyoxal in the healing of diabetic gastric ulcers.

The molecular mechanisms underlying the posttranslational modification of proteins in gastrointestinal cancer are still unknown. Here, we investigated the role of methylglyoxal modifications in gastrointestinal tumors. Methylglyoxal is a reactive dicarbonyl compound produced from cellular glycolytic intermediates that reacts non-enzymatically with proteins. By using a monoclonal antibody to methylglyoxal-modified proteins, we found that murine heat-shock protein 25 and human heat-shock protein 27 were the major adducted proteins in rat gastric carcinoma mucosal cell line and human colon cancer cell line, respectively. Furthermore, we found that heat-shock protein 27 was modified by methylglyoxal in ascending colon and rectum of patients with cancer. However, methylglyoxal-modified heat-shock protein 25/heat-shock protein 27 was not detected in non cancerous cell lines or in normal subject. Matrix-associated laser desorption/ionization mass spectrometry/mass spectrometry analysis of peptide fragments identified Arg-75, Arg-79, Arg-89, Arg-94, Arg-127, Arg-136, Arg-140, Arg-188, and Lys-123 as methylglyoxal modification sites in heat-shock protein 27 and in phosphorylated heat-shock protein 27. The transfer of methylglyoxal-modified heat-shock protein 27 into rat intestinal epithelial cell line RIE was even more effective in preventing apoptotic cell death than that of native control heat-shock protein 27. Furthermore, methylglyoxal modification of heat-shock protein 27 protected the cells against both the hydrogen peroxide- and cytochrome c-mediated caspase activation, and the hydrogen peroxide-induced production of intracellular reactive oxygen species. The levels of lactate converted from methylglyoxal were increased in carcinoma mucosal cell lines. Our results suggest that posttranslational modification of heat-shock protein 27 by methylglyoxal may have important implications for epithelial cell injury in gastrointestinal cancer., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Previous studies have shown that activated neutrophils and their myeloperoxidase (MPO)-derived products play a crucial role in the pathogenesis of non-steroidal anti-inflammatory drug (NSAID)-related small intestinal injury. The aim of the present study is to identify dihalogenated proteins in the small intestine on indomethacin administration. Intestinal damage was induced by subcutaneous administration of indomethacin (10 mg/kg) in male Wistar rats, and the severity of the injury was evaluated by measuring the area of visible ulcerative lesions. Tissue-associated MPO activity was measured in the intestinal mucosa as an index of neutrophil infiltration. The dihalogenated proteins were separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) using novel monoclonal antibodies against dibromotyrosine (DiBrY), and they were identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) peptide mass fingerprinting and a Mascot database search. Single administration of indomethacin elicited increased ulcerative area and MPO activity in the small intestine. 2D-PAGE showed an increased level of DiBrY-modified proteins in the indomethacin-induced injured intestinal mucosa and 6 modified proteins were found. Enolase-1 and albumin were found to be DiBrY modified. These proteins may be responsible for the development of neutrophil-associated intestinal injury induced by indomethacin.

Methylglyoxal is a reactive dicarbonyl compound produced from cellular glycolytic intermediates that reacts non-enzymatically with proteins to form products such as argpyrimidine at arginine residue. The aim of the present study was to investigate the role of methylglyoxal in the delayed healing of gastric ulcer in diabetes, and to identify the methylglyoxal-modified proteins as a target molecule of this modification. Using male C57BL/6 mice, diabetes was induced by a single i.p. injection of streptozotocin and gastric ulcers were produced by the focal application of 40% of acetic acid to the serosal surface of the stomach. In order to evaluate the effect of OPB-9195, an inhibitor of methylglyoxal modification, on gastric ulcer healing, mice were given orally OPB-9195 (30 mg/kg) twice daily for 14 days, one week before and after the injection of streptozotocin. The area of gastric ulcer on day 7 was significantly increased in diabetic mice compared to non-diabetic mice, indicating delayed ulcer healing. This increase in ulcer area in diabetic mice was significantly reversed by the treatment with OPB-9195 without affecting blood glucose levels. Proteomics analysis showed the methylglyoxal-modification of peroxiredoxin 6 proteins in the diabetic gastric mucosa around gastric ulcer, and this modification was markedly inhibited by the treatment with OPB-9195. In conclusion, the present study suggests a link of increased methylglyoxal modification of proteins including peroxiredoxin 6 to the delayed gastric ulcer healing in diabetes, and also shows the therapeutic potential of the inhibitor of methylglyoxal modification for the treatment of diabetic gastric ulcers.

The Maillard reaction plays an important role in eye lens aging and cataract formation. Methylglyoxal (MGO) is a metabolic dicarbonyl compound present in the lens. It reacts with arginine residues in lens proteins to form advanced glycation end products (AGEs), such as hydroimidazolones and argpyrimidine. alpha-Crystallin, comprising alphaA- and alphaB-crystallin, is a major protein of the lens and it functions as a chaperone protein. We have found that upon reaction with MGO, human alphaA-crystallin becomes a more effective chaperone. Modification of specific arginine residues to AGEs appears to be the reason. Mutation of these arginine residues to alanine mirrors the effect of MGO, suggesting neutralization of the positive charge on arginine residues as a cause for improved chaperone function. Reaction with MGO also blocks the loss of the chaperone function of alphaA-crystallin caused by nonenzymatic glycation by ascorbate and ribose. These findings suggest that low levels of MGO might help the lens remain transparent during aging.

Diabetic patients are prone to severe bacterial infections. The functional alterations of neutrophils by hyperglycemia are thought to be partially responsible for such infections. In this study, we investigated the functional changes of neutrophil-like differentiated cell lines (dHL-60, dTHP-1, and dNB-4) by treatment with 5.5 mM, 11 mM, or 35 mM of glucose. In dHL-60 cells, the incubation with high glucose (35 mM) resulted in the enhancement of cell aggregation, the suppression of cellular fragility, the induction of reactive-oxygen species (ROS) production by phorbol myristate acetate (PMA) stimulation, and the impairment of phagocytosis. In dTHP-1 cells, the treatment with higher glucose generated the suppression of cellular fragility and extremely impaired phagocytosis (by 35 mM), and induced ROS production due to PMA stimulation (by 11 mM). Furthermore, the higher glucose exposure to dNB-4 cells enlarged intracellular vacuoles (by 35 mM) and induced ROS production due to PMA stimulation (by 11 mM). Since the ROS generation of those cells was enhanced only after PMA stimulation under the higher glucose conditions, glucose may have a priming effect rather than a triggering effect. These extraordinary sensitivities caused by the higher glucose treatments may reflect the dysfunction or overactivation of neutrophils.

Heat shock protein 27 (Hsp27) is a stress-inducible protein in cells that functions as a molecular chaperone and also as an anti-apoptotic protein. Methylglyoxal (MGO) is a reactive dicarbonyl compound produced from cellular glycolytic intermediates that reacts non-enzymatically with proteins to form products such as argpyrimidine. We found considerable amount of Hsp27 in phosphorylated form (pHsp27) in human cataractous lenses. pHsp27 was the major argpyrimidine-modified protein in brunescent cataractous lenses. Modification by MGO enhanced the chaperone function of both pHsp27 and native Hsp27, but the effect on Hsp27 was at least three-times greater than on pHsp27. Phosphorylation of Hsp27 abolished its chaperone function. Transfer of Hsp27 using a cationic lipid inhibited staurosporine (SP)-induced apoptotic cell death by 53% in a human lens epithelial cell line (HLE B-3). MGO-modified Hsp27 had an even greater effect (62% inhibition). SP-induced reactive oxygen species in HLE-B3 cells was significantly lower in cells transferred with MGO-modified Hsp27 when compared to native Hsp27. In vitro incubation experiments showed that MGO-modified Hsp27 reduced the activity of caspase-9, and MGO-modified pHsp27 reduced activities of both caspase-9 and caspase-3. Based on these results, we propose that Hsp27 becomes a better anti-apoptotic protein after modification by MGO, which may be due to multiple mechanisms that include enhancement of chaperone function, reduction in oxidative stress, and inhibition of activity of caspases. Our results suggest that MGO modification and phosphorylation of Hsp27 may have important consequences for lens transparency and cataract development.

We reported previously that chemical modification of human alphaA-crystallin by a metabolic dicarbonyl compound, methylglyoxal (MGO), enhances its chaperone-like function, a phenomenon which we attributed to formation of argpyrimidine at arginine residues (R) 21, 49, and 103. This structural change removes the positive charge on the arginine residues. To explore this mechanism further, we replaced these three R residues with a neutral alanine (A) residue one at a time or in combination and examined the impact on the structure and chaperone function. Measurement of intrinsic tryptophan fluorescence and near-UV CD spectra revealed alteration of the microenvironment of aromatic amino acid residues in mutant proteins. When compared to wild-type (wt) alphaA-crystallin, the chaperone function of R21A and R103A mutants increased 20% and 18% as measured by the insulin aggregation assay and increased it as much as 39% and 28% when measured by the citrate synthase (CS) aggregation assay. While the R49A mutant lost most of its chaperone function, R21A/R103A and R21A/R49A/R103A mutants had slightly better function (6-14% and 10-14%) than the wt protein in these assays. R21A and R103A mutants had higher surface hydrophobicity than wt alphaA-crystallin, but the R49A mutant had lower hydrophobicity. R21A and R103A mutants, but not the R49A mutant, were more efficient than wt protein in refolding guanidine hydrochloride-treated malate dehydrogenase to its native state. Our findings indicate that the positive charges on R21, R49, and R103 are important determinants of the chaperone function of alphaA-crystallin and suggest that chemical modification of arginine residues may play a role in protein aggregation during lens aging and cataract formation.

Glycation or the Maillard reaction in proteins forms advanced glycation end products (AGEs) that contribute to age- and diabetes-associated changes in tissues. Dideoxyosones, which are formed by the long-range carbonyl shift of the Amadori product, are newly discovered intermediates in the process of AGE formation in proteins. They react with o-phenylenediamine (OPD) to produce quinoxalines. We developed a monoclonal antibody against 2-methylquinoxaline-6-carboxylate coupled to keyhole limpet hemocyanin. The antibody reacted strongly with ribose and fructose (+OPD)-modified RNase A and weakly with glucose and ascorbate (+OPD)-modified RNase A. Reaction with substituted quinoxalines indicated that this antibody favored the 2-methyl group on the quinoxaline ring. We used high performance liquid chromatography to isolate and purify three antibody-reactive products from a reaction mixture of N alpha-hippuryl-L-lysine+ribose+OPD. The two most reactive products were identified as diastereoisomers of N1-benzoylglycyl-N6-(2-hydroxy-3-quinoxalin-2-ylpropyl)lysine and the other less reactive product as N1-benzoylglycyl-N6-[2-hydroxy-2-(3-methylquinoxalin-2-yl)ethyl]lysine. Our study confirms that dideoxyosone intermediates form during glycation and offers a new tool for the study of this important pathway in diabetes and aging.

Methylglyoxal (MGO) is an alpha-dicarbonyl compound produced from triose phosphate intermediates of glycolysis. It reacts rapidly with proteins to produce advanced glycation products. We have studied the effect of MGO modification of fibronectin on retinal capillary cell viability. Our studies show that pericytes grown on MGO-modified fibronectin (FN) undergo enhanced apoptosis through the p38MAPK-mediated oxidative pathway and that alphaB-crystallin, a stress protein present in pericytes, can protect them from MGO-mediated apoptosis. Our studies with vascular endothelial cells show that hyperglycemia-induced apoptosis is inhibited by overexpression of alphaB-crystallin. These observations suggest a novel role of alphaB-crystallin in hyperglycemia-mediated damage to vascular cells in diabetes.

The molecular chaperone function of alpha-crystallin in the lens prevents the aggregation and insolubilization of lens proteins that occur during the process of aging. We found that chemical modification of alpha-crystallin by a physiological alpha-dicarbonyl compound, methylglyoxal (MG), enhances its chaperone function. Protein-modifying sugars and ascorbate have no such effect and actually reduce chaperone function. Chaperone assay after immunoprecipitation or with immunoaffinity-purified argpyrimidine-alpha-crystallin indicates that 50-60% of the increased chaperone function is due to argpyrimidine-modified protein. Incubation of alpha-crystallin with DL-glyceraldehyde and arginine-modifying agents also enhances chaperone function, and we believe that the increased chaperone activity depends on the extent of arginine modification. Far- and near-UV circular dichroism spectra indicate modest changes in secondary and tertiary structure of MG-modified alpha-crystallin. LC MS/MS analysis of MG-modified alpha-crystallin following chymotryptic digestion revealed that R21, R49, and R103 in alphaA-crystallin were converted to argpyrimidine. 1,1'-Bis(4-anilino)naphthalene-5,5'-disulfonic acid binding, an indicator of hydrophobicity of proteins, increased in alpha-crystallin modified by low concentrations of MG (2-100 microM). MG similarly enhances chaperone function of another small heat shock protein, Hsp27. Our results show that posttranslational modification by a metabolic product can enhance the chaperone function of alpha-crystallin and Hsp27 and suggest that such modification may be a protective mechanism against environmental and metabolic stresses. Augmentation of the chaperone function of alpha-crystallin might have evolved to protect the lens from deleterious protein modifications associated with aging.

Potent antioxidative hydroxyflavanones were produced with Aspergillus saitoi from hesperidin or naringin, which are flavanone glycosides in citrus fruit with weak antioxidative activity. The hydroxyflavanone produced from hesperidin was identified as 8-hydroxyhesperetin (8-HHE), a novel substance, and those from naringin were identified as carthamidin (6-hydroxynaringenin) and isocarthamidin (8-hydroxynaringenin) by FAB-MS, 1H-NMR and 13C-NMR analyses. The antioxidative activity of these hydroxyflavanones was examined by using the free radical-scavenging system of 1,1-diphenyl-2-picrylhydrazyl (DPPH) and the methyl linoleate oxidation system. The hydroxyflavanones (8-HHE, carthamidin, and isocarthamidin) exhibited stronger activity than the flavanone glycosides (hesperidin or naringin) and their aglycones (hesperetin or naringenin). The activity of 8-HHE and isocarthamidin was comparable to that of alpha-tocopherol, and that of carthamidin was weaker than that of isocarthamidin. The hydroxyflavanones, which were hydroxylated on A ring of flavanone by Aspergillus saitoi, were obtained as potent antioxidants.

6-Hydroxydopamine is a neurotoxin specific to dopamine neurons, and this neurotoxin at 20 muM was confirmed to induce mainly apoptosis in human dopaminergic neuroblastoma SH-SY5Y cells. During the apoptotic process, translocation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) into nuclei was observed, which is now proposed as an apoptogenic signal in various types of apoptosis. However, it remains to be clarified, how GAPDH is translocated into nuclei. In this paper, GAPDH translocation was followed by fluoro- and electron-microscopic observation using antibody against GAPDH. Before the nuclear translocation, a condensed mass of GAPDH protein was detected in the Golgi apparatus, which was identified by 6-([N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]hexanoyl)-sphingosine, a selective marker of Golgi apparatus. These results suggest that the Golgi apparatus may be involved in the compartmentalization of GAPDH from cytosol to nuclei.

In the present study, we find that cyclopentenone prostaglandins (PGs) of the J(2) series, naturally occurring derivatives of PGD(2), are potential inducers of intracellular oxidative stress that mediates cell degeneration. Based on an extensive screening of diverse chemical agents on induction of intracellular production of reactive oxygen species (ROS), we found that the cyclopentenone PGs, such as PGA(2), PGJ(2), Delta(12)-PGJ(2), and 15-deoxy-Delta(12,14)-PGJ(2), showed the most potent pro-oxidant effect on SH-SY5Y human neuroblastoma cells. As the intracellular events that mediate the PG cytotoxicity, we observed (i) the cellular redox alteration represented by depletion of antioxidant defenses, such as glutathione and glutathione peroxidase; (ii) a transient decrease in the mitochondrial membrane potential (Deltapsi); (iii) the production of protein-bound lipid peroxidation products, such as acrolein and 4-hydroxy-2-nonenal; and (iv) the accumulation of ubiquitinated proteins. These events correlated well with the reduction in cell viability. In addition, the thiol compound, N-acetylcysteine, could significantly inhibit the PG-induced ROS production, thereby preventing cytotoxicity, suggesting that the redox alteration is closely related to the pro-oxidant effect of cyclopentenone PGs. More strikingly, the lipid peroxidation end products, acrolein and 4-hydroxy-2-nonenal, detected in the PG-treated cells potently induced the ROS production, which was accompanied by the accumulation of ubiquitinated proteins and cell death, suggesting that the membrane lipid peroxidation products may represent one of the causative factors that potentiate the cytotoxic effect of cyclopentenone PGs by accelerating intracellular oxidative stress. These data suggest that the intracellular oxidative stress, represented by ROS production/lipid peroxidation and redox alteration, may underlie the well documented biological effects, such as antiproliferative and antitumor activities, of cyclopentenone PGs.

It has recently been shown that the lipid peroxidation product 4-hydroxy-2-nonenal (HNE) forms a fluorescent hydroxyiminodihydropyrrole derivative with the epsilon-amino group of lysine residue. In this study, we raised a monoclonal antibody (mAb2C12) directed to the fluorophore-protein conjugate and found that the antibody was specific to the chromophore structure of the compound. Immunohistochemical analysis of atherosclerotic lesions from the human aorta showed that the fluorophore was indeed present in the lesions, in which intense positivity was primarily associated with macrophage-derived foam cells and thickening of the neointima of the arterial walls. Antigenic materials were also detected in the oxidatively modified low-density lipoprotein (LDL) with Cu(2+) and in the oxidatively modified bovine serum albumin with an iron/linoleic acid autoxidation system, indicating that the HNE, which originated from the peroxidation of polyunsaturated fatty acids, could be a potential source of the fluorescent chromophore in oxidized LDL.