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Sulfites are commonly used as a preservative and antioxidant additives in the food industry. Sulfites are absorbed by the gastrointestinal tract and distributed essentially to all body tissues. Although sulfites have been believed to be safe food additives, some studies have shown that they exhibit adverse effects in various tissues. In this study, we examined the cytotoxic effect of sodium sulfite (Na2SO3) against rat gastric mucosal cells (RGM1) and further investigated its underlying molecular mechanism. We demonstrated that exposure to Na2SO3 exerts significant cytotoxicity in RGM1 cells through induction of oxidative stress. Exposure of RGM1 cells to Na2SO3 caused a significant formation of protein carbonyls and 8-hydroxy-2'-deoxyguanosine, major oxidative stress markers, with a concomitant accumulation of carbonylated protein-related aggregates. Furthermore, we found that incubation of lysozyme with Na2SO3 evokes protein carbonylation and aggregation via the metal ion-catalyzed free radical formation derived from Na2SO3. Our results suggest that Na2SO3 might lead to gastric tissue injury via induction of oxidative stress by the formation of Na2SO3-related free radicals.

Proteins are oxidatively modified by a large number of reactive species including reactive oxygen species, lipid peroxidation-derived aldehydes, and reducing sugars. Among divergent oxidative modifications, the introduction of carbonyl groups such as aldehyde, ketone, and lactam into the amino acid side chains of proteins is a major hallmark for oxidative damage to proteins, and is termed "protein carbonylation". Detection and quantification of protein carbonyls are commonly performed to determine the level of oxidative stress in the context of cellular damage, aging, and several age-related disorders. This review focuses on the molecular mechanisms and biological implications of protein carbonylation, and also presents current analytical approaches for determining and characterizing carbonylated proteins.

Polyphenols, especially catechol-type polyphenols, exhibit lysyl oxidase–like activity and mediate oxidative deamination of lysine residues in proteins. Previous studies have shown that polyphenol-mediated oxidative deamination of lysine residues can be associated with altered electrical properties of proteins and increased crossreactivity with natural immunoglobulin M antibodies. This interaction suggested that oxidized proteins could act as innate antigens and elicit an innate immune response. However, the structural basis for oxidatively deaminated lysine residues remains unclear. In the present study, to establish the chemistry of lysine oxidation, we characterized oxidation products obtained via incubation of the lysine analog N-biotinyl-5-aminopentylamine with eggshell membranes containing lysyl oxidase and identified a unique six-membered ring 2-piperidinol derivative equilibrated with a ring-open product (aldehyde) as the major product. By monitoring these aldehyde–2-piperidinol products, we evaluated the lysyl oxidase–like activity of polyphenols. We also observed that this reaction was mediated by some polyphenols, especially o-diphenolic-type polyphenols, in the presence of copper ions. Interestingly, the natural immunoglobulin M monoclonal antibody recognized these aldehyde–2-piperidinol products as an innate epitope. These findings establish the existence of a dynamic equilibrium of oxidized lysine and provide important insights into the chemopreventive function of dietary polyphenols for chronic diseases.

Pyrroloquinoline quinone (PQQ), a redox-active o-quinone found in various foods and mammalian tissues, has received an increasing amount of attention because of a number of health benefits that can be attributed to its ability to enhance mitochondrial biogenesis. However, its underlying molecular mechanism remains incompletely understood. We have now established that the exposure of mouse NIH/3T3 fibroblasts to a physiologically relevant concentration of PQQ significantly stimulates mitochondrial biogenesis. The exposure of NIH/3T3 cells to 10–100 nM PQQ for 48 h resulted in increased levels of Mitotracker staining, mitochondrial DNA content, and mitochondrially encoded cytochrome c oxidase subunit 1 (MTCO1) protein. Moreover, we observed that PQQ treatment induces deacetylation of the peroxisome proliferator-activated receptor-γ-coactivator 1α (PGC-1α) and facilitates its nuclear translocation and target gene expression but does not affect its protein levels, implying increased activity of the NAD+-depen...

A simple, practical, and rapid access to quinoxalin-2-ones 1, 1,2,3,4-tetrahydroquinoxalines 2, quinoxalines 3, and quinoxalin-2(1H)-ones 4 has been achieved, based on the copper-catalyzed quinoxalinone formation of 2-haloanilines and amino acids followed by their reduction and oxidation. The olfactory properties and lipid accumulation inhibitory activity in cultured hepatocytes of the quinoxaline derivatives were also evaluated.

Scope Nonalcoholic fatty liver disease (NAFLD) has emerged as the most common chronic liver disease worldwide, defined by hepatic over-accumulation of lipids without significant ethanol consumption. Pharmacological or bioactive food ingredients that suppress hepatic lipid accumulation through AMP-activated protein kinase (AMPK) signaling, which plays a critical role in the regulation of lipid metabolism, are searched. Methods and results It is found that tomatidine, the aglycone of α-tomatine abundant in green tomatoes, significantly inhibits palmitate-provoked lipid accumulation and stimulates phosphorylation of AMPK and acetyl-CoA carboxylase 1 (ACC1) in human HepG2 hepatocytes. The results also indicate that tomatidine can enhance triglyceride turnover and decline in lipogenesis by upregulating adipose triglyceride lipase (ATGL) and downregulating fatty acid synthase (FAS) via the AMPK signaling-dependent regulation of transcription factors, element-binding protein-1c (SREBP-1c) and forkhead box protein O1 (FoxO1). Furthermore, mechanistic studies demonstrate that tomatidine-stimulated AMPK phosphorylation is due to CaMKKβ activation in response to an increase in intracellular Ca2+ concentration. Finally, it is discovered that tomatidine functions as an agonist for vitamin D receptor to elicit AMPK-dependent suppression of lipid accumulation. Conclusion The in vitro study suggests the potential efficacy of tomatidine as a preventive and therapeutic treatment in obesity-related fatty liver diseases.

Polyphenols have received a significant amount of attention in disease prevention because of their unique chemical and biological properties. However, the underlying molecular mechanism for their beneficial effects remains unclear. We have now identified a polyphenol as a source of innate epitopes detected in natural IgM and established a unique gain-of-function mechanism in the formation of innate epitopes by polyphenol via the polymerization of proteins. Upon incubation with bovine serum albumin (BSA) under physiological conditions, several polyphenols converted the protein into the innate epitopes recognized by the IgM Abs. Interestingly, piceatannol, a naturally occurring hydroxylated analogue of a red wine polyphenol, resveratrol, mediated the modification of BSA, whose polymerized form was specifically recognized by the IgMs. The piceatannol-mediated polymerization of the protein was associated with the formation of a lysine-derived cross-link, dehydrolysinonorleucine. In addition, an oxidatively deaminated product, α-aminoadipic semialdehyde, was detected as a potential precursor for the cross-link in the piceatannol-treated BSA, suggesting that the polymerization of the protein might be mediated by the oxidation of a lysine residue by piceatannol followed by a Schiff base reaction with the ε-amino group of an unoxidized lysine residue. The results of this study established a novel mechanism for the formation of innate epitopes by small dietary molecules and support the notion that many of the beneficial effects of polyphenols could be attributed, at least in part, to their lysyl oxidase-like activity. They also suggest that resveratrol may have beneficial effects on human health because of its conversion to piceatannol.

Introduction of carbonyl groups into amino acid residues is a hallmark for oxidative damage to proteins by reactive oxygen species (ROS). Protein carbonylation can have deleterious effects on cell function and viability, since it is generally unrepairable by cells and can lead to protein dysfunction and to the production of potentially harmful protein aggregates. Meanwhile, pyridoxamine (PM) is known to scavenge various toxic carbonyl species derived from either glucose or lipid degradation through nucleophilic addition. PM is also demonstrated to catalyze non-enzymatic transamination reactions between amino and α-keto acids. Here, we found that PM scavenges protein carbonyls in oxidized BSA with concomitant generation of pyridoxal and recovers oxidized lysozyme activity. Moreover, we demonstrated that the treatment of H2O2-exposed HepG2 hepatocytes with PM significantly reduced levels of cellular carbonylated proteins and aggregated proteins, and also improved cell survival rate. Our results suggest that PM may have potential efficacy in ameliorating ROS-mediated cellular dysfunction.

Short-chain fatty acids (SCFAs) such as acetate, propionate, and butyrate are generated by gut microbial fermentation of dietary fiber. SCFAs may exert multiple beneficial effects on human lipid and glucose metabolism. However, their actions and underlying mechanisms are not fully elucidated. In this study, we examined the direct effects of propionate on hepatic glucose and lipid metabolism using human HepG2 hepatocytes. Here, we demonstrate that propionate at a physiologically-relevant concentration effectively suppresses palmitate-enhanced glucose production in HepG2 cells but does not affect intracellular neutral lipid levels. Our results indicated that propionate can decline in gluconeogenesis by down-regulation of glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK) through activation of AMP-activated protein kinase (AMPK), which is a major regulator of the hepatic glucose metabolism. Mechanistic studies also revealed that propionate-stimulated AMPK phosphorylation can be ascribed to Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ) activation in response to an increase in intracellular Ca2+ concentration. Moreover, siRNA-mediated knockdown of the propionate receptor GPR43 prevented propionate-inducible activation of AMPK and abrogates the gluconeogenesis-inhibitory action. Thus, our data indicate that the binding of propionate to hepatic GPR43 elicits CaMKKβ-dependent activation of AMPK through intracellular Ca2+ increase, leading to suppression of gluconeogenesis. The present study suggests the potential efficacy of propionate in preventive and therapeutic management of diabetes.

Pyrroloquinoline quinone (PQQ), a redox-active o-quinone, is an important nutrient involved in numerous physiological and biochemical processes in mammals. Despite such beneficial functions, the underlying molecular mechanisms remain to be established. In the present study, using PQQ-immobilized Sepharose beads as a probe, we examined the presence of protein(s) that are capable of binding PQQ in mouse NIH/3T3 fibroblasts and identified five cellular proteins, including l-lactate dehydrogenase (LDH) A chain, as potential mammalian PQQ-binding proteins. In vitro studies using a purified rabbit muscle LDH show that PQQ inhibits the formation of lactate from pyruvate in the presence of NADH (forward reaction), whereas it enhances the conversion of lactate to pyruvate in the presence of NAD+ (reverse reaction). The molecular mechanism underlying PQQ-mediated regulation of LDH activity is attributed to the oxidation of NADH to NAD+ by PQQ. Indeed, the PQQ-bound LDH oxidizes NADH, generating NAD+ and significantly catalyzes the conversion of lactate to pyruvate. Furthermore, PQQ attenuates cellular lactate release and increases intracellular ATP levels in the NIH/3T3 fibroblasts. Our results suggest that PQQ, modulating LDH activity to facilitate pyruvate formation through its redox-cycling activity, may be involved in the enhanced energy production via mitochondrial TCA cycle and oxidative phosphorylation.

Nonalcoholic fatty liver disease (NAFLD) is currently the most common chronic liver disease worldwide, characterized by excessive hepatic lipid accumulation without significant ethanol consumption. We have performed a screening for medicinal foods that inhibit hepatocytic lipid accumulation through activation of AMP-activated protein kinase (AMPK), which is a critical regulator of the hepatic lipid metabolism. We found that clove (Syzygium aromaticum), which is commonly used as a spice, markedly inhibits palmitate-inducible lipid accumulation in human HepG2 hepatocytes. Analyses of the clove extracts found that β-caryophyllene, an orally-active cannabinoid, is the principal suppressor of the lipid accumulation, and stimulates the phosphorylation of AMPK and acetyl-CoA carboxylase 1 (ACC1). Our data also showed that β-caryophyllene prevents the translocation of sterol regulatory element-binding protein-1c (SREBP-1c) into the nucleus and forkhead box protein O1 (FoxO1) into the cytoplasm through AMPK signaling, and consequently, induces a significant downregulation of fatty acid synthase (FAS) and upregulation of adipose triglyceride lipase (ATGL), respectively. Moreover, we demonstrated that the β-caryophyllene-induced activation of AMPK could be mediated by the cannabinoid type 2 receptor-dependent Ca(2+) signaling pathway. Our results suggest that β-caryophyllene has the potential efficacy in preventing and ameliorating NAFLD and its associated metabolic disorders. This article is protected by copyright. All rights reserved.

Insulin resistance is a pathological hallmark of type 2 diabetes mellitus and is characterized by defects in insulin signaling. Protein tyrosine phosphatase 1B (PTP1B) negatively regulates insulin signaling by tyrosine dephosphorylation of insulin receptor, and increased activity and expression of PTP1B is implicated in the pathogenesis of insulin resistance. Therefore, inhibition of PTP1B is anticipated to improve insulin resistance in type 2 diabetic subjects. Pyrroloquinoline quinone (PQQ), a redox cofactor for bacterial dehydrogenases, inhibits PTP1B to oxidatively modify the catalytic cysteine through its redox cycling activity. Here, we report that PQQ induces the ligand-independent activation of insulin signaling by inhibiting cellular PTP1B and enhances glucose uptake through the translocation of glucose transporter 4 in mouse C2C12 myotubes. Furthermore, we demonstrated that oral administration of PQQ improved impaired glucose tolerance in type 2 diabetic KK-A(y) mice. Our results strongly suggest that PQQ can be useful in anti-diabetic treatment for type 2 diabetic subjects.

Pyrroloquinoline quinone (PQQ), a redox cofactor for bacterial dehydrogenases, has been implicated to be an important nutrient in mammals functioning as a potent growth factor. However, the underlying molecular mechanisms have not been elucidated. The present study revealed that PQQ induces the activation (tyrosine autophosphorylation) of epidermal growth factor receptor (EGFR) and its downstream signaling in a ligand-independent manner, leading to increased cellular proliferation in an epithelial cell line A431. PQQ inhibited protein tyrosine phosphatase 1B (PTP1B), which negatively regulates the EGFR signaling by tyrosine dephosphorylation, to oxidatively modify the catalytic cysteine through its redox cycling activity to generate H(2)O(2). PQQ-inducible intracellular ROS production and EGFR activation were significantly suppressed by the pre-treatment with antioxidants. The intracellular redox state regulates the EGFR signaling through the redox-sensitive catalytic cysteine of PTP1B and modulates cell proliferation. Our data suggest that PQQ may stimulate epithelial cell proliferation by activating EGFR by oxidation and subsequent inactivation of PTP1B via its redox cycling. Our results provide novel insight into the mechanisms by which PQQ may function as a growth factor to contribute to mammalian growth.

Acrolein, a ubiquitous pollutant in the environment, is endogenously formed through oxidation reactions and is believed to be involved in cytopathological effects observed during oxidative stress. Acrolein exerts these effects because of its facile reactivity with biological materials, particularly proteins. In the present study, we quantitatively analyzed the acrolein-specific adducts generated during lipid peroxidation-modification of proteins and identified the acrolein adduct most abundantly generated in the in vitro oxidized low-density lipoproteins (LDL). Taking advantage of the fact that the acrolein-lysine adducts, N(ε)-(3-formyl-3,4-dehydropiperidino)lysine (FDP-lysine) and N(ε)-(3-methylpyridinium)lysine (MP-lysine), have stable core structures resistant to the acid hydrolysis condition of proteins, we examined the formation of these adducts in proteins using high performance liquid chromatography with online electrospray ionization tandem mass spectrometry. However, only MP-lysine was detected as a minor product in the iron/ascorbate-mediated oxidation of polyunsaturated fatty acids in the presence of proteins and in the oxidized low-density lipoproteins (LDL). However, using a reductive amination-based pyridylamination method, we analyzed the acrolein-specific adducts with a carbonyl functionality and found that acrolein modification of the protein produced a number of carbonylated amino acids, including an acrolein-histidine adduct. On the basis of the chemical and spectroscopic evidence, this adduct was identified as N(τ)-(3-propanal)histidine. More notably, N(τ)-(3-propanal)histidine appeared to be one of the major adducts generated in the oxidized LDL. These data suggest that acrolein generated during lipid peroxidation may primarily react with histidine residues of proteins to form N(τ)-(3-propanal)histidine.

(−)-Epigallocatechin-3-gallate (EGCG), the most abundant and biologically active polyphenol in green tea, induces apoptosis and suppresses proliferation of cancer cells by modulating multiple signal transduction pathways. However, the fundamental mechanisms responsible for these cancer-preventive effects have not been clearly elucidated. Recently, we found that EGCG can covalently bind to cysteine residues in proteins through autoxidation and subsequently modulate protein function. In this study, we demonstrate the direct binding of EGCG to cellular proteins in AZ521 human gastric cancer cells by redox-cycle staining. We comprehensively explored the binding targets of EGCG from EGCG-treated AZ521 cells by proteomics techniques combined with the boronate-affinity pull-down method. The DEAD-box RNA helicase p68, which is overexpressed in a variety of tumor cells and plays an important role in cancer development and progression, was identified as a novel EGCG-binding target. Exposure of AZ521 cells to EGCG lowered the p68 level dose dependently. The present findings show that EGCG inhibits AZ521 cell proliferation by preventing β-catenin oncogenic signaling through proteasomal degradation of p68 and provide a new perspective on the molecular mechanism of EGCG action.

Catechins are polyphenolic antioxidants found in green tea leaves. Recent studies have reported that various polyphenolic compounds, including catechins, cause protein carbonyl formation in proteins via their pro-oxidant actions. In this study, we evaluate the formation of protein carbonyl in human serum albumin (HSA) by tea catechins and investigate the relationship between catechin chemical structure and its pro-oxidant property. To assess the formation of protein carbonyl in HSA, HSA was incubated with four individual catechins under physiological conditions to generate biotin-LC-hydrazide labeled protein carbonyls. Comparison of catechins using Western blotting revealed that the formation of protein carbonyl in HSA was higher for pyrogallol-type catechins than the corresponding catechol-type catechins. In addition, the formation of protein carbonyl was also found to be higher for the catechins having a galloyl group than the corresponding catechins lacking a galloyl group. The importance of the pyrogallol structural motif in the B-ring and the galloyl group was confirmed using methylated catechins and phenolic acids. These results indicate that the most important structural element contributing to the formation of protein carbonyl in HSA by tea catechins is the pyrogallol structural motif in the B-ring, followed by the galloyl group. The oxidation stability and binding affinity of tea catechins with proteins are responsible for the formation of protein carbonyl, and consequently the difference in these properties of each catechin may contribute to the magnitude of their biological activities.

4-Hydroxy-2-nonenal (HNE), a major racemic product of lipid peroxidation, preferentially reacts with cysteine residues to form a stable HNE-cysteine Michael addition adduct possessing three chiral centers. Here, to gain more insight into sulfhydryl modification by HNE, we characterized the stereochemical configuration of the HNE-cysteine adducts and investigated their stereoselective formation in redox-regulated proteins. To characterize the HNE-cysteine adducts by NMR, the authentic (R)-HNE- and (S)-HNE-cysteine adducts were prepared by incubating N-acetylcysteine with each HNE enantiomer, both of which provided two peaks in reversed-phase high performance liquid chromatography (HPLC). The NMR analysis revealed that each peak was a mixture of anomeric isomers. In addition, mutarotation at the anomeric center was also observed in the analysis of the nuclear Overhauser effect. To analyze these adducts in proteins, we adapted a pyridylamination-based approach, using 2-aminopyridine in the presence of sodium cyanoborohydride, which enabled analyzing the individual (R)-HNE- and (S)-HNE-cysteine adducts by reversed-phase HPLC following acid hydrolysis. Using the pyridylamination method along with mass spectrometry, we characterized the stereoselective formation of the HNE-cysteine adducts in human thioredoxin and found that HNE preferentially modifies Cys(73) and, to the lesser extent, the active site Cys(32). More interestingly, the (R)-HNE- and (S)-HNE-cysteine adducts were almost equally formed at Cys(73), whereas Cys(32) exhibited a remarkable preference for the adduct formation with (R)-HNE. Finally, the utility of the method for the determination of the HNE-cysteine adducts was confirmed by an in vitro study using HeLa cells. The present results not only offer structural insight into sulfhydryl modification by lipid peroxidation products but also provide a platform for the chemical analysis of protein S-associated aldehydes in vitro and in vivo.

The oxidation of dietary polyphenols with a catechol structure leads to the formation of an o-quinone structure, which rapidly reacts with sulfhydryls such as glutathione and protein cysteine residues. This modification may be important for understanding the redox regulation of cell functions by polyphenols. In this study, to investigate the catechol modification of protein sulfhydryls, we used 3,4-dihydroxyphenyl acetic acid (DPA) as a model catechol compound and developed a new probe to directly detect protein modification by catechol type polyphenols using a biotinylated DPA (Bio-DPA). The oxidation-dependent electrophilic reactivity of DPA with peptide sulfhydryls was confirmed by both mass spectrometry and nuclear magnetic resonance spectroscopy. When RL34 cells were treated with Bio-DPA, the significant incorporation of Bio-DPA into a 40 kDa protein was observed by Western blot analysis. The band was identified by mass spectrometry as the cytoskeletal protein, beta-actin. This identification was confirmed by the pull-down assay with anti-beta-actin antibody. To examine the reactivity of the catechol type polyphenols, such as flavonoids, to endogenous beta-actin, RL34 cells were coexposed to Bio-DPA and the flavonoids quercetin, (-)-epicatechin, and (-)-epicatechin gallate. Upon exposure of the cells to Bio-DPA in the presence of the flavonoids, we observed a significant decrease in the DPA-modified beta-actin. These results indicate that beta-actin is one of the major targets of protein modification by catechol type polyphenols and that Bio-DPA is an useful probe for understanding the redox regulation by dietary polyphenols. Furthermore, Keap1, a scaffold protein to the actin cytoskeleton controlling cytoprotective enzyme genes, was also identified as another plausible target of the catechol type polyphenols by oxidative modification of the intracellular sulfhydryls. These results provide an alternative approach to understand that catechol type polyphenol is a potential modifier of redox-dependent cellular events through sulfhydryl modification.

The oxidative modification of proteins is believed to play a critical role in the etiology and/or progression of several diseases. alpha-Aminoadipic semialdehyde (AAS) and gamma-glutamic semialdehyde (GGS) residues represent major oxidized amino acids generated in oxidized proteins. This paper describes a novel procedure for the specific and sensitive determination of AAS and GGS after their reductive amination with sodium cyanoborohydride and p-aminobenzoic acid, a fluorescence reagent, to their corresponding derivatives, followed by a high-performance liquid chromatography (HPLC) analysis. This fluorescent labeling of protein-associated aldehyde moieties is a simple and accurate technique that may be widely used to reveal increased levels of oxidatively modified proteins with reactive oxygen species during aging and disease.

Hypoallergenic wheat flour was produced by polished-graded method using a rice polishing machine. Eight fractions (C1–C8) of polished-graded wheat flours were obtained step wise from the outer layer of whole wheat grains by 10% of the total weight, and the distribution of allergenic protein in each fraction was determined. The salt-soluble (albumin/globulin), salt-insoluble (glutenin) and alcohol-soluble (gliadin) proteins obtained from the polished-graded wheat flours were tested for the allergen assay with immunodetection using the sera of wheat allergenic patients. Immunoblotting results confirmed that the innermost fraction (C8) contained a smaller amount of allergenic proteins. Albumin/globulin groups in all fractions (C1–C8) showed different IgE-reactivity patterns, the 60–75 kDa proteins appeared in all of fraction flours. It was higher in C3–C5 fractions. Fractions C3 and C4 contained higher amount of specific wheat allergenic protein including 60–75, 35, 22, and < 20 kDa. IgE-antibody also bound ...

Synthesised E- and Z-ajoene were used to determine their amounts in food oils containing various fresh garlics. The best yield of E-ajoene (172.0 μg/g of garlic) and Z-ajoene (476.0 μg/g of garlic) was obtained from freshly prepared Japanese garlic with rice oil which was heated at 80 °C. Determination of E- and Z-ajoene from soybean oil containing 15% Japanese garlic samples prepared at 80 °C for 0.5 h gave the amount of E-ajoene (170.0 μg/g of garlic) and Z-ajoene (127.0 μg/g of garlic). After 9-month storage, 54.0% E- and 11.0% Z-ajoene remained in Japanese garlic with rice oil. Ajoene (0.1 mM) in ethyl acetate was incubated under UV-light (253.7 nm) for 3 days, 81.7% E- and 56.9% Z-ajoene remained. 4.3% and 0.5% E- and Z-ajoene remained when ajoene (0.1 mM in ethyl acetate) was incubated at 100 °C.

(-)-Epigallocatechin-3-O-gallate (EGCG), the most abundant polyphenol in green tea, mediates the oxidative modification of proteins, generating protein carbonyls. However, the underlying molecular mechanism remains unclear. Here we analyzed the EGCG-derived intermediates generated upon incubation with the human serum albumin (HSA) and established that EGCG selectively oxidized the lysine residues via its oxidative deamination activity. In addition, we characterized the EGCG-oxidized proteins and discovered that the EGCG could be an endogenous source of the electrically-transformed proteins that could be recognized by the natural antibodies. When HSA was incubated with EGCG in the phosphate-buffered saline (pH 7.4) at 37°C, the protein carbonylation was associated with the formation of EGCG-derived products, such as the protein-bound EGCG, oxidized EGCG, and aminated EGCG. The aminated EGCG was also detected in the sera from the mice treated with EGCG in vivo. EGCG selectively oxidized lysine residues at the EGCG-binding domains in HSA to generate an oxidatively deaminated product, aminoadipic semialdehyde. In addition, EGCG treatment results in the increased negative charge of the protein due to the oxidative deamination of the lysine residues. More strikingly, the formation of protein carbonyls by EGCG markedly increased its cross-reactivity with the natural IgM antibodies. These findings suggest that many of the beneficial effects of EGCG may be partly attributed to its oxidative deamination activity, generating the oxidized proteins as a target of natural antibodies.

Several lines of evidence indicate that the nonenzymatic oxidative modification of proteins and the subsequent accumulation of the modified proteins have been found in cells during aging and oxidative stress and in various pathological states, including premature diseases, muscular dystrophy, rheumatoid arthritis, and atherosclerosis. Our previous work suggested the existence of molecular mimicry between antibodies raised against hydroxy-2-nonenal (HNE)-modified protein and anti-DNA autoantibodies, a serologic hallmark of systemic lupus erythematosus (SLE). In the present study, we investigated the possible involvement of HNE-modified proteins as the endogenous source of the anti-DNA antibodies. Accumulation of the antigen recognized by the antibody against the HNE-modified protein was observed in the nucleus of almost all of the epidermal cells from patients with autoimmune diseases, including SLE. The SLE patients also showed significantly higher serum levels of the anti-HNE titer than healthy individuals. To determine if a specific anti-DNA response could be initiated by the HNE-derived epitopes, we immunized BALB/c mice with the HNE-modified protein and observed a progressive increase in the anti-DNA response. Moreover, we generated the monoclonal antibodies, showing recognition specificity toward DNA, and found that they can bind to two structurally distinct antigens (i.e. the native DNA and protein-bound 4-oxo-2-nonenal). The findings in this study provide evidence to suspect an etiologic role for lipid peroxidation in autoimmune diseases.

Wheat can cause severe IgE-mediated systematic reactions, but knowledge on relevant wheat allergens at the molecular level is scanty. The aim of the present study was to achieve a more detailed and comprehensive characterization of the wheat allergens involved in food allergy to wheat using proteomic strategies, referred to as "allergenomics". Whole flour proteins were separated by two-dimensional gel electrophoresis with isoelectric focusing and lithium dodecyl sulfate-polyacrylamide gel electrophoresis. Then, IgE-binding proteins were detected by immunoblotting with sera of patients with a food allergy to wheat. After tryptic digestion, the peptides of IgE-binding proteins were analyzed by matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry. In this study, we identified four previously reported wheat allergens or their sequentially homologous proteins [serpin, alpha-amylase inhibitor, gamma-gliadin, and low molecular weight (LMW) glutenin] by a database search. As a result of the high resolution of two-dimensional gel electrophoresis, nine subunits of LMW glutenins were identified as the most predominant IgE-binding antigens. The two-dimensional allergen map can be beneficial in many ways. It could be used, for example, for precise diagnosis of wheat-allergic patients and assessment of wheat allergens in food. Additionally, we compared allergenomics to conventional biochemical methods and evaluated the usefulness of a proteomic strategy for identifying putative allergens to wheat allergy.

Since variant Creutzfeldt-Jakob disease (vCJD) has been suspected to be attributable to the infectious agents associated with bovine spongiform encephalopathy (BSE), it is important to prevent the transmission of pathogenic forms of prion protein (PrP Sc ) through contaminated feeding materials such as meat and bone meal (MBM). Here, we demonstrate that the Maillard reaction employing a formulation of glucose in combination with sodium hydrogen carbonates effectively reduced the infectivity (approximately 5.9-log reduction) of a scrapie-infected hamster brain homogenate. In addition to a bioassay, a protein misfolding cyclic amplification (PMCA) technique, in which PrP Sc can be amplified in vitro , was used as a rapid test for assessing PrP Sc inactivation. The PMCA analysis also indicated that the PrP Sc level in the infected material significantly decreased following the Maillard reaction. Therefore, the Maillard reaction can be employed for the decontamination of large amounts of byproducts such as MBM.

Facilitative glucose uptake transport systems are ubiquitous in animal cells and are responsible for transporting glucose across cell surface membranes. Evaluation of glucose uptake is crucial in the study of numerous diseases and metabolic disorders such as myocardial ischemia, diabetes mellitus, and cancer. Detailed in this unit are laboratory methods for assessing glucose uptake into mammalian cells. The unit is divided into five sections: (1) a brief overview of glucose uptake assays in cultured cells; (2) a method for measuring glucose uptake using radiolabeled 3-O-methylglucose; (3) a method for measuring glucose uptake using radiolabeled 2-deoxyglucose (2DG); (4) a microplate method for measuring 2DG-uptake using an enzymatic, fluorometric assay; and (5) a microplate-based method using a fluorescent analog of 2DG. © 2015 by John Wiley & Sons, Inc. Keywords: glucose uptake; cell-based assay; 3-O-methylglucose; 2-deoxyglucose; radioisotope; nonradioisotope; 2-NBDG,fluorescence

Pyrroloquinoline quinone (PQQ), an aromatic tricyclic o-quinone, was identified initially as a redox cofactor for bacterial dehydrogenases. Although PQQ is not biosynthesized in mammals, trace amounts of PQQ have been found in human and rat tissues because of its wide distribution in dietary sources. Importantly, nutritional studies in rodents have revealed that PQQ deficiency exhibits diverse systemic responses, including growth impairment, immune dysfunction, and abnormal reproductive performance. Although PQQ is not currently classified as a vitamin, PQQ has been implicated as an important nutrient in mammals. In recent years, PQQ has been receiving much attention owing to its physiological importance and pharmacological effects. In this article, we review the potential health benefits of PQQ with a focus on its growth-promoting activity, anti-diabetic effect, anti-oxidative action, and neuroprotective function. Additionally, we provide an update of its basic pharmacokinetics and safety information in oral ingestion.

4-Hydroxy-2-nonenal (HNE), a racemic mixture of 4R- and 4S-enantiomers, is a major product of lipid peroxidation and is believed to be largely responsible for the cytopathological effects observed during oxidative stress. HNE reacts with histidine to form a stable HNE-histidine Michael addition-type adduct possessing three chiral centers in the cyclic hemiacetal structure. We have previously raised the mAbs, anti-RmAb 310 and anti-SmAb S412, that enantioselectively recognized theR-HNE-histidine andR-HNE-histidine adducts, respectively, and demonstrated the presence of both epitopesin vivo. In the present study, to further investigate the anti-HNE immune response, we analyzed the variable genes and primary structure of these Abs and found that the sequence of R310 was highly homologous to anti-DNA autoantibodies, the hallmark of systemic lupus erythematosus. An x-ray crystallographic analysis of the R310 Fab fragment showed that theR-HNE-histidine adduct binds to a hydrophobic pocket in the antigen-binding site. Despite the structural identity to the anti-DNA autoantibodies, however, R310 showed only a slight crossreactivity with the native double-stranded DNA, whereas the Ab immunoreactivity was dramatically enhanced by the treatment of the DNA with 4-oxo-2-nonenal (ONE), an analog of HNE. Moreover, the 7-(2-oxo-heptyl)-substituted 1,N2-etheno-type ONE-2′-deoxynucleoside adducts were identified as alternative epitopes of R310. Molecular mimicry between theR-HNE-histidine configurational isomers and the ONE-DNA base adducts is proposed for the dual crossreactivity.

Oxidative deamination by various polyphenolic compounds is presumed to be due to the oxidative conversion of polyphenols to the corresponding quinones through autoxidation. Here we examined the oxidative deamination by the polyphenol-rich beverages green tea, black tea, and coffee at a physiological pH and temperature. Green tea, black tea, and coffee extracts oxidatively deaminated benzylamine and the lysine residues of bovine serum albumin to benzaldehyde and alpha-aminoadipic delta-semialdehyde residues, respectively, in sodium phosphate buffer (pH 7.4) at 37 degrees C in both the presence and absence of Cu2+, indicating the occurrence of an amine (lysyl) oxidase-like reaction. We also examined the effects of pH and metal ions on the reaction. The possible biological effects of drinking polyphenol-rich beverages on human are also discussed.

Recent research has demonstrated that nonenzymatic glycation (the Maillard reaction) lead to the formation of carbonyl groups and advanced glycation end products (AGEs) in proteins. Such oxidative modifications are a major contributing factor to diabetic complications and aging. alpha-Aminoadipic semialdehyde (AAS) and gamma-glutamic semialdehyde (GGS) have been identified as the major carbonyl products in oxidized proteins both in vitro and in vivo. AAS is an oxidative deamination product of lysine residue, while GGS originates from arginine and proline residues. To evaluate oxidative damage to proteins by the Maillard reaction, we developed a method of detecting AAS and GGS by high-performance liquid chromatography (HPLC). The aldehydic residues in proteins were derivatized by reductive amination with NaCNBH3 and p-aminobenzoic acid (ABA), a fluorescence regent. After acid hydrolysis of the ABA-derivatized protein, ABA-AAS and ABA-GGS were measured by fluorometric HPLC. Thus, AAS and GGS could be detected in various proteins such as human plasma protein using the present method. Accumulation of both aldehydic residues was observed in oxidized proteins by reactive oxygen species. Furthermore, AAS and GGS were markedly formed in the incubation of BSA with ascorbic acid. The formation of both aldehydic residues was also observed in the incubation of BSA with 100 mM glucose or 1.0 mM methylglyoxal in the absence and presence of 100 microM Fe3+ for 2 weeks. These results suggest that the Maillard reaction can contribute to the formation of AAS and GGS in vivo.

The lysine residue of bovine serum albumin was deaminated to allysine (α-aminoadipic-δ-semialdehyde) during the incubation with glucose, 3-deoxyglucosone (3-DG), and methylglyoxal (MG) in the presence of Cu2+ at a physiological pH and temperature but not with glyoxal. Further, glucose, 3-DG, and MG oxidatively deaminated benzylamine to benzaldehyde in the presence of Cu2+. The formation of benzaldehyde was greatest with Cu2+, and was accelerated in the presence of oxygen. EDTA, catalase, and dimethyl sulfoxide (DMSO) significantly inhibited the oxidation. Analysis of plasma proteins revealed significantly higher levels of allysine in streptozotocin (STZ)-induced diabetic rats compared with normal controls. From these findings, we propose a novel mechanism for the oxidative modification of proteins in diabetes via the Maillard reaction.

The levels of alpha-aminoadipic-delta-semialdehyde residue, the oxidative deamination product of lysine residue, in plasma protein from streptozotocin-induced diabetic rats were evaluated. alpha-Aminoadipic-delta-semialdehyde was converted to a bisphenol derivative by acid hydrolysis in the presence of phenol, and determined by high performance liquid chromatography. Analysis of plasma proteins revealed three times higher levels of alpha-aminoadipic-delta-semialdehyde in diabetic subjects compared with normal controls. Furthermore, we explored the oxidative deamination via the Maillard reaction and demonstrated that the lysine residue of bovine serum albumin is oxidatively deaminated during the incubation with various carbohydrates in the presence of Cu2+ at a physiological pH and temperature. This experiment showed that 3-deoxyglucosone and methylglyoxal are the most efficient oxidants of the lysine residue. When the reaction was initiated from glucose, a significant amount of alpha-aminoadipic-delta-semialdehyde was also formed in the presence of Cu2+. The reaction was significantly inhibited by deoxygenation, catalase, and a hydroxyl radical scavenger. The mechanism we propose for the oxidative deamination is the Strecker-type reaction and the reactive oxygen species-mediated oxidation. Based on these findings, we propose a novel mechanism for the oxidative modification of proteins in diabetes, namely the oxidative deamination of the lysine residue via the Maillard reaction.

Various amines, including lysine residue of bovine serum albumin, were oxidatively deaminated to form the corresponding aldehydes by a H2O2/Cu2+ oxidation system at physiological pH and temperature. The resulting aldehydes were measured by high-performance liquid chromatography. We investigated the effects of metal ions, pH, inhibitors, and O2 on the oxidative deamination of benzylamine by H202. The formation of benzaldehyde was the greatest with Cu2+, and catalysis occurred with Co2+, VO2+, and Fe3+. The reaction was greatly accelerated as the pH value rose and was markedly inhibited by EDTA and catalase. Dimethyl sulfoxide and thiourea, which are hydroxyl radical scavengers, were also effective in inhibiting the generation of benzaldehyde, indicating that the reaction is a hydroxyl radical-mediated reaction. Superoxide dismutase greatly stimulated the reaction, probably due to the formation of hydroxyl radicals. O2 was not required in the oxidation, and instead slightly inhibited the reaction. We also examined several oxidation systems. Ascorbic acid/O2/Cu2+ and hemoglobin/H2O2 systems also converted benzylamine to benzaldehyde. The proposed mechanism of the oxidative deamination by H2O2/Cu2+ system is discussed.

Obesity-associated insulin resistance is a major pathogenesis of type 2 diabetes mellitus and is characterized by defects in insulin signaling. High concentrations of plasma free fatty acids (FFAs) are involved in the etiology of obesity-associated insulin resistance. However, the detailed mechanism by which FFAs contribute to the development of insulin resistance is not yet fully understood. We investigated the molecular basis of insulin resistance elicited by FFAs using the human hepatocyte cell line HepG2. Among major human FFAs, palmitate markedly inhibited insulin-stimulated phosphorylation of key insulin signaling molecules such as insulin receptor, insulin receptor substrate-1, and Akt, indicating that palmitate is the principal inducer of insulin resistance. We revealed that palmitate facilitates ubiquitination of the key insulin signaling molecules, and subsequently elicits their proteasomal degradation. Furthermore, we demonstrated that inhibition of ubiquitination by the ubiquitin-activating enzyme E1 inhibitor PYR41 significantly prevents palmitate-inducible insulin resistance but not by the proteasome inhibitor MG132, implying that ubiquitinated signaling molecules may be dysfunctional. In conclusion, inhibition of ubiquitination of the key insulin signaling molecules may be a potential strategy for preventing and treating obesity-associated insulin resistance.

N-Acylhomoserine lactones (AHLs) are quorum sensing signals produced by Gram-negative bacteria. We here report the affinity purification of AHL synthases using beads conjugated with an enzyme inhibitor, which was designed based on the catalytic intermediate acyl-SAM.

Polyphenols in several oxidation systems gained amine oxidase-like activity, probably due to the formation of the corresponding quinones. In the presence of Cu(II), o- and p-phenolic compounds exhibited amine oxidase-like activity, whereas only the o-phenolic compounds showed the activity in the presence of 1,1-diphenyl-2-picrylhydrazyl radical. The activity was determined by measuring the conversion of benzylamine to benzaldehyde by HPLC. Moreover, gallic acid, chlorogenic acid, and caffeic acid, which are plant polyphenols, converted the lysine residue of bovine serum albumin to alpha-amino-adipic semialdehyde residue, indicating lysyl oxidase-like activity. We also characterized the activity of pyrocatechol, hydroquinone, and pyrogallol in the presence of Cu(II). The oxidative deamination was accelerated at a higher pH, and required O2 and transition metal ions. Furthermore, EDTA markedly inhibited the reaction but not beta-aminopropionitrile, which is a specific inhibitor of lysyl oxidase. Catalase significantly inhibited the oxidation, implying the participation of hydroxyl radical in the reaction, but superoxide dismutase stimulated the oxidation, probably due to its radical formation activity. We discussed the mechanism of the oxidative deamination by polyphenols and the possible significance of the activity for biological systems.

We examined the formation of quaternary pyridinium crosslinks of elastin formed by condensation of lysine and allysine residues using the model compounds propanal (allysine) and n-butylamine (lysine) under quasi-physiological conditions. The resulting pyridinium compounds were characterized and the structure compared with the known pyridinium crosslinks. Three pyridinium compounds were identified and the structures were identical with the skeleton of the crosslinking amino acids, desmosine (DES), isodesmosine (IDE), and pentasine. We concluded that a non-enzymatic pathway is available for the spontaneous generation of pyridinium crosslinks. To elucidate the intermediates and the mechanism of the formation of DES and IDE, we synthesized model intermediates from propanal and n-butylamine, and they were allowed to react in three kinds of solvents. Then, the products were analyzed by an ion-pair reverse-phase HPLC. The results of this model system indicated that DES and IDE can be formed by condensation of dehydromerodesmosine with dehydrolysinonorleucine and by condensation of allysine with dehydrolysinonorleucine, respectively. We also describe the mechanism of DES and IDE crosslinking.

Scope Protein tyrosine phosphatase 1B (PTP1B) negatively regulates insulin signaling by tyrosine dephosphorylation of insulin receptor, and its increased activity and expression is implicated in the pathogenesis of insulin resistance. Hence, PTP1B inhibition is anticipated to improve insulin resistance in type 2 diabetic subjects. The aim of this study was to find a novel PTP1B inhibitor from medicinal food and to evaluate its antidiabetic effects. Methods and results We found that saffron (Crocus sativus L.), which is used both as a spice and as a traditional medicine, potently inhibits PTP1B activity. Analyses of saffron extracts demonstrated that safranal, the saffron's aroma compound, is a principal PTP1B inhibitor, and induces a ligand-independent activation of insulin signaling in cultured myotubes. Our data implied that the molecular mechanism underlying the inactivation of PTP1B could be attributed to the covalent modification of the catalytic cysteinyl thiol by safranal through a Michael addition. Furthermore, safranal significantly enhanced glucose uptake through the translocation of glucose transporter 4. We also demonstrated that 2-wk oral administration of 20 mg/kg/day safranal improved impaired glucose tolerance in type 2 diabetic KK-Ay mice. Conclusion Our results strongly suggest the usefulness of safranal in antidiabetic treatment for type 2 diabetic subjects.

The breakdown of collagenous networks with aging results in hypoactive changes in the skin. Accordingly, reviving stagnant collagen synthesis can help protect dermal homeostasis against aging. We searched for type I collagen biosynthesis-inducing substances in various foods using human dermal fibroblasts and found that cinnamon extract facilitates collagen biosynthesis. Cinnamon extract potently up-regulated both mRNA and protein expression levels of type I collagen without cytotoxicity. We identified cinnamaldehyde as a major active component promoting the expression of collagen by HPLC and NMR analysis. Since insulin-like growth factor-I (IGF-I) is the most potent stimulator of collagen biosynthesis in fibroblasts, we examined the effect of cinnamaldehyde on IGF-I signaling. Treatment with cinnamaldehyde significantly increased the phosphorylation levels of the IGF-I receptor and its downstream signaling molecules such as insulin receptor substrate-1 and Erk1/2 in an IGF-I-independent manner. These results suggested that cinnamon extract is useful in antiaging treatment of skin.

Human serum albumin (HSA) contributes to the stabilization of (-)-epigallocatechin gallate (EGCg) in serum. We characterize in the present study the mechanisms for preventing EGCg oxidation by HSA. EGCg was stable in human serum or buffers with HSA, but (-)-epigallocatechin (EGC) was unstable. We show by comparing EGCg and EGC in a neutral buffer that EGCg had a higher binding affinity than EGC. This indicates that the galloyl moiety participated in the interaction of EGCg with HSA and that this interaction was of critical importance in preventing EGCg oxidation. The binding affinity of EGCg for HSA and protein carbonyl formation in HSA were enhanced in an alkaline buffer. These results suggest the reversible covalent modification of EGCg via Schiff-base formation, and that the immobilization of EGCg to HSA, through the formation of a stable complex, prevented the polymerization and decomposition of EGCg in human serum.

We developed a procedure to measure 4-hydroxy-2-nonenal (HNE)-amino acid adducts using the fluorescent probe 2-aminopyridine (2-AP). The method is based on the fact that HNE forms Michael addition-type amino acid adducts possessing an aldehyde functionality, which upon reaction with 2-AP in the presence of NaBH₃CN can be converted to their pyridylaminated derivatives. The HNE-amino acid adducts, namely Michael addition-type HNE-cysteine, HNE-histidine, and HNE-lysine adducts, after pyridylamination were resistant to conventional acid-hydrolysis conditions for protein (6N HCl/110°C/24 h) and could be detected by HPLC with a fluorescence detector. The reductive amination-based fluorescent labeling of HNE adducts is a simple and accurate technique that may be widely used to reveal increased levels of covalently modified proteins with HNE and its related aldehydes during aging and disease.

In this study, we investigated the relationship between the stability of catechins and their electrophilic reactivity with proteins. The stability of catechins was evaluated by HPLC analysis. Catechol-type catechins were stable in a neutral buffer, but pyrogallol-type catechins, such as (-)-epigallocatechin gallate (EGCg), were unstable. The electrophilic reactivity of catechins with thiol groups in a model peptide and a protein was confirmed by both mass spectrometry and electrophoresis/blotting with redox-cycling staining. In a comparison of several catechins, pyrogallol-type catechins had higher reactivity with protein thiols than catechol-type catechins. The instability and reactivity of EGCg were enhanced in an alkaline pH buffer. The reactivity of EGCg was reduced by antioxidants due to their ability to prevent EGCg autoxidation. These results indicate that the instability against oxidation of catechins is profoundly related to their electrophilic reactivity. Consequently, the difference in these properties of tea catechins can contribute to the magnitude of their biological activities.

The collapse of collagenous networks with aging results in comprehensive changes in the functional properties of skin. α-Lipoic acid (LA) is known to possess beneficial effects against skin aging, effects often presumed to be its antioxidant potential. However, the effects of LA on fibrillogenesis in dermal fibroblasts have not been adequately assessed. In this study, we demonstrated for the first time that LA enhances the biosynthesis of new collagen in normal human dermal fibroblasts (NHDFs). By using a quantitative dye-binding method and immunochemical approaches, we showed that LA effectively increased the expression and subsequently the deposition of type I collagen in NHDFs. LA also facilitated the expression of a collagen-processing enzyme, prolyl-4-hydroxylase, pointing to the existence of a posttranslational mechanism among the LA-mediated effects on collagen synthesis. In addition, we determined that both Smad 2/3 were rapidly phosphorylated by treatment with LA within 30 min, indicating that LA enhances type I collagen synthesis through the activation of Smad signaling. Pretreatment of SB431542, a specific transforming growth factor-β (TGF-β) receptor type I (TβRI) kinase inhibitor, blocked LA-mediated Smad 2/3 phosphorylations and both type I collagen and prolyl-4-hydroxylase expression, suggesting that LA-mediated cell responses are regulated by TβRI kinase-dependent pathway. Levels of TGF-β secretion after 4 hr of treatment with LA were not remarkably elevated, indicating that LA may be able to mimic TGF-β-mediated cell response. The study results produced new insights into the molecular pharmacology of LA in NHDFs, with potential applications in the treatment of aging skin.

Pyrroloquinoline quinone (PQQ), a putative essential nutrient, is a redox modulator in cell and animal models. Here we characterized PQQ-induced protein oxidative modifications in a model peptide and protein, and we propose that the mechanism of protein modification by PQQ is redox cycling-mediated oxidation. PQQ may contribute to the regulation of intracellular protein functions through its prooxidant action.