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In this study, we found that hydrogen peroxide was generated during the separate incubation of four catechins, (-)-epicatechin, (-)-epigallocatechin, and their galloylated forms, in the absence or presence of mushroom tyrosinase (EC 1.14.18.1). In particular, autooxidation of (-)-epigallocatechin and enzymatic oxidation of (-)-epicatechin gallate contributed to the increase of hydrogen peroxide. We confirmed the hydrogen peroxide-induced inactivation of tyrosinase and found that hydrogen peroxide was also generated during the tyrosinase-catalyzed synthesis of theaflavins by selectively combining two types of catechins, diphenol- and pyrogallol-types. Elimination of hydrogen peroxide by co-incubation with bovine catalase (EC 1.11.1.6) increased the products in synthetic reactions for TF1, TF2A, TF2B, and TF3 by 15%, 16%, 45%, and 18%, respectively. Based on the prospective mechanism of the tyrosinase-catalyzed synthesis of theaflavins, the yield of TF2B increased by elevating the initial ratio of (-)-epigallocatechin/(-)-epicatechin gallate in the reaction media. Furthermore, under such conditions, there were clear ameliorative effects of catalase on the tyrosinase-catalyzed synthesis of TF2B, reaching 3.1-fold increase of the product compared to the reaction without catalase. These results support a novel strategy to use both tyrosinase and catalase for the efficient synthesis of theaflavins from catechins.

Background Polyphenols in tea are considered beneficial to human health. However, many such claims of their bioactivity still require in vitro and in vivo evidence. Results Using Drosophila melanogaster as a model multicellular organism, we assess the fat accumulation-suppressing effects of theaflavin (TF), a tea polyphenol; epitheaflagallin (ETG), which has an unknown function; and epigallocatechin gallate (EGCg), a prominent component of green tea. Dietary TF reduced the malondialdehyde accumulation related to a high-fat diet in adult flies. Other physiological and genetic responses induced by the high-fat diet, such as lipid accumulation in the fat body and expression of lipid metabolism-related genes, were ameliorated by the addition of TF, ETG, and EGCg, in some cases approaching respective levels without high-fat diet exposure. Continuous ingestion of the three polyphenols resulted in a shortened lifespan. Conclusion We provide evidence in Drosophila that tea polyphenols have a fat accumulation-suppressing effect that has received recent attention. We also suggest that tea polyphenols can provide different desirable biological activities depending on their composition and the presence or absence of other chemical components.

This study aimed to develop a novel method with tea extracts and its components, to reduce the risk of foodborne illnesses caused by the bacterial toxin staphylococcal enterotoxin A (SEA). The potential effect of tea extracts, theaflavins, and epitheaflagallin on staphylococcal growth was studied. A broth microdilution method was used to determine the minimum inhibitory concentration of these samples against an SEA-producing strain, Staphylococcus aureus C-29. The following assays were performed to evaluate various effects on concentrations of no effect on staphylococcal growth. The interactions of theaflavin-rich green tea extracts (TGE), theaflavins, and epitheaflagallin to cultured S. aureus C-29 were determined using Western blot analysis. As a result, all samples suppressed the binding affinity of the anti-SEA antibody to SEA. Since these samples could react directly with SEA, we examined whether they could bind to SEA. Our results demonstrated that binding of the anti-SEA antibody to 4 theaflavins-treated SEA was inhibited in a dose-dependent manner. On the other hand, the production of SEA was significantly decreased by treatment with TGE and epitheaflagallin. Based on the finding that TGE and epitheaflagallin inhibit the production of SEA, we further examined the relative expression levels of sea toxin-encoding genes after treatment with TGE and epitheaflagallin with real-time RT-PCR. TGE and epitheaflagallin significantly supressed the gene transcription of SEA in S. aureus C-29. We then tested whether the samples block the biological activity of SEA in murine spleen cells. TGE, theaflavins, and epitheaflagallin became inactivated the biological activity of SEA. These results suggest that edible and safe compounds in tea can be used to inactivate both pathogens and toxins.

In this study, we investigated the memory task performance and the central nervous activity after smelling two kinds of pan-fired Japanese green tea to examine the differences of their physical and psychological effects. Twenty eight subjects (14 male and 14 female) participated in this study. We used Koushun and Kouju for test samples, which were made by different manufacturing processes. After smelling each odor sample, a memory task and an arithmetic task were used to test mental stress. Electroencephalogram (EEG) was recorded before and after smelling the test samples, and EEG activity was estimated for 4 frequency bands (alpha 1, alpha 2, beta 1 and beta 2). The profiles of mood states (POMS) and the visual analog scale (VAS) after mental stress task were completed for subjective assessments. The results showed that the odor of Kouju may induce a positive emotion such as a more familiar and a less hate feeling. It may also affect the EEG band power of beta 1 at right frontal region and improve memory task performance as well.

The present study was performed to investigate the immune-modulating activities of extracts from green soybean (Glycine max) in a 2,4-toluene diisocyanate (TDI)-inducing guinea pig rhinitis model and a human trial study for allergic rhinitis. Hot water extracts of green soybean were chosen for animal experimentation on the basis of their ability to regulate the production of B cell-activating factor of the TNF family and a proliferation-inducing ligand in mouse spleen cells. Green soybean extracts significantly decreased the levels of ovalubumin (OVA)-specific IgE in mice and significantly suppressed the TDI-induced nasal mucosa secretion. An open-label human pilot study was performed on 16 subjects, using Japanese cedar pollinosis. The symptom scores for Japanese cedar pollinosis were better in the long-term green soybean extracts intake group than in the withdrawal short-term intake group. Green soybean extracts had great potential as an orally active immune modulator for the treatment of various allergic diseases.

Many biological activities of green tea have been attributed to a major constituent, (minus;)-epigallocatechin gallate (EGCG). We previously reported that EGCG and an EGCG-free fraction derived from green tea modulated the gene expression of gluconeogenic enzymes, glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, in the mouse liver. EGCG is also known to affect the gene expression of enzymes related to lipid metabolism. However, it remains to be examined whether or not a constituent other than EGCG contributes to the change in gene expression of these enzymes. In this study, we prepared an EGCG-free water-soluble fraction (GT-W), and examined its effects on the hepatic gene expression of lipogenic enzymes in mice. The results of quantitative real-time PCR assays indicated that the dietary administration of GT-W for 4 weeks reduced the hepatic gene expression of lipogenic enzymes: fatty acid synthase, hydroxymethylglutaryl coenzyme A reductase, and acetyl-coenzyme A carboxylase alpha. Also, the gene expression of sterol regulatory element-binding transcription factor (Srebf)1 and/or Srebf2 was reduced, suggesting that the reduction of Srebfs contributed to the down-regulation of the lipogenic enzymes, since these transcription factors bind the promoter region to enhance their expression. The plasma levels of triglycerides and cholesterol were reduced with statistical significance in the group given a diet containing GT-W. These results suggest that in addition to EGCG, green tea contains some component(s) which may help to prevent arteriosclerosis and obesity.

The reactive oxygen species generated by an aqueous extract of the particulate phase of cigarette smoke were evaluated by an electron spin resonance (ESR) analysis, using spin-trapping agents, and by comparing with model reaction systems. The ESR signals of DMPO-OH were detected from the extract by using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). These signals were eliminated by adding superoxide dismutase, but hardly by catalase. These responses of the ESR signals to the scavengers were similar to those of a hypoxanthine-xanthine oxidase system. The results indicate that the signals of DMPO-OH from the extract were derived from a reaction product of DMPO with superoxide anion radicals and clarify the mechanism by which the extract generated superoxide anion radicals.

We recently reported that propolis suppresses tumor-induced angiogenesis through tube formation inhibition and apoptosis induction in endothelial cells. However, molecular mechanisms underlying such angiogenesis suppression by propolis have not been fully elucidated. The aim of this study was to investigate the effects of ethanol extract of Brazilian propolis (EEBP) on two major survival signals, extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt, and to elucidate whether changes in these signals were actually involved in antiangiogenic effects of the propolis. Detection by western blotting revealed that EEBP suppressed phosphorylation of ERK1/2, but not that of Akt. Pharmacological inhibition by U0126 demonstrated that ERK1/2 inactivation alone was enough to inhibit tube formation and induce apoptosis. It was also shown that EEBP and U0126 similarly induced activation of caspase-3 and cleavage of poly ADP-ribose polymerase (PARP) and lamin A/C, all of which are molecular markers of apoptosis. These results indicate that inhibition of survival signal ERK1/2, and subsequent induction of apoptosis, is a critical mechanism of angiogenesis suppression by EEBP.

Immunoglobulin E (IgE) plays an important role in allergic diseases. In this study, we found that am ethanol extract of leaves of the camphor tree Cinnamomum camphora (C. camphora )r educed the amount of IgE secreted by human myeloma U266 cells. When the methanol extract was fractionated by extraction with organic solvents, the ethyl acetate fraction showed the highest activity. The fraction was further separated into several subfractions by preparative TLC. We identified the component of one of the active subfractions as dimethylmatairesinol. Authentic dimethylmatairesinol exhibited similar activity. Thus, the extract of C. camphora and its components including dimethylmatairesinol have potential as an anti-allergic agent.

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.

Several physical and psychological stresses frequently become triggers for gastrointestinal disorders such as ulcer. In this study, we tried to identify serum proteins as potential biomarkers for the evaluation of stress-induced gastric ulcer. By proteomic analysis using rats with gastric ulcer induced by water immersion and restraint (WIR) stress as an animal model, we found quantitative changes in several serum proteins, including creatine kinase muscle M chain (CK-M) and apolipoprotein A-IV (ApoA4) in the stressed rats. On western blotting and enzyme-linked immunosorbent assay (ELISA), we confirmed that serum CK-M was remarkably increased by WIR stress. However, ApoA4 appeared to be decreased by fasting, but not WIR stress, which is usually applied prior to WIR stress. The findings suggest that these two serum proteins might be useful as biomarkers, CK-M for stress-induced gastric ulcer and ApoA4 for starvation.

(-)-Epigallocatechin gallate (EGCg) is the major component of green tea and is known to show strong biological activity, although it can be easily oxidized under physiological conditions. In this study, we indicate that EGCg is stable in human serum and that human serum albumin (HSA) stabilizes EGCg under aerobic condition. Although EGCg is usually decomposed within 1 h in aqueous solution at neutral pH, EGCg in serum and phosphate buffer (pH 7.4) containing HSA was stable over 1 h, even at neutral and slightly alkaline pH. Under these conditions, EGCg binds to HSA non-covalently. The sulfhydryl group acts as an antioxidant for EGCg oxidation. Incubation of EGCg with HSA is accompanied by the oxidation of a free sulfhydryl group in HSA. These results suggest that the antioxidant property and the binding capacity of HSA contribute to the stabilization of EGCg in human serum.

Propolis is a resinous substance collected by honeybees from various plant sources. The antioxidant activities of propolis of various geographic origins, i.e., Argentina, Australia, Brazil, Bulgaria, Chile, China (Hebei, Hubei, and Zhejiang), Hungary, New Zealand, South Africa, Thailand, Ukraine, Uruguay, United States, and Uzbekistan were compared. Ethanol extracts of propolis (EEP) were prepared and evaluated for antioxidant activities of EEP samples by the β-carotene bleaching and 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging assay systems. Major constituents were identified in EEP by HPLC analysis with photo-diode array (PDA) and mass spectrometric (MS) detection, and quantitatively analyzed. EEP from Argentina, Australia, China, Hungary and New Zealand had relatively strong antioxidant activities, and were also correlated with the total polyphenol and flavonoid contents. Propolis with strong antioxidant activity contained antioxidative compounds such as kaempferol and phenethyl caffeate.

Gamma-terpinene is a monoterpene and a major component of essential oils made from citrus fruits and shows strong antioxidant activity in various assay systems. Plant gamma-terpinene synthase is a member of the monoterpene cyclase family, which produces a specific monoterpene through cyclization of geranyl diphosphate (GPP), but the monoterpene cyclases have not been fully characterized. It is necessary to prepare large amounts of gamma-terpinene synthase from Citrus unshiu (Satsuma mandarin) for the characterization, on this purpose we expressed the protein in Escherichia coli (E. coli) cells. As most monoterpene synthases have plastid-targeting signals, a gene lacking these signals was prepared and functionally expressed in E. coli cells harboring extra copies of the argU gene. The purified enzyme was incubated with GPP and the main product was confirmed to be gamma-terpinene by GC/MS.

We measured ubiquinone (CoQ0) and hydrogen peroxide (H2O2) formed in the process of oxidation of ubiquinol (CoQ0H2). We found that copper-zinc superoxide dismutase and manganese superoxide dismutase inhibited both the CoQ0 formation and the H2O2 formation only in the presence of chelators such as DTPA (diethylenetriaminepentaacetic acid). The amount of H2O2 was almost equal to that of CoQ0, indicating that the H2O2 formation was coupled with the CoQ0 formation. The lack of inhibitory effects of the corresponding heat-inactivated superoxide dismutase (SOD) confirmed that the inhibition by the original SOD was due to its enzymatic activity. We propose that CoQ0H2 oxidation occurs as a chain reaction with superoxide as the chain carrier and that SOD inhibits this reaction by lowering the superoxide concentration.

We established a simple method to quantify hydrogen peroxide (H2O2) generated during the aerobic heating process of (+)-catechin solution. Using this method, we found that non-enzymatic H2O2 formation from catechin depended on pH, temperature, incubation time, and the presence of O2 in the solution. The formation of oxidized products of (+)-catechin, which was estimated by measuring the absorbance of the solution at 430 nm, also depended on these factors. The H2O2 formation was inhibited by various kinds of superoxide dismutase (SOD) with almost the same dose dependency. Although the oxidation of (+)-catechin was enhanced by superoxide (O2−), neither catalase nor H2O2 had any effects on the oxidation. These results suggest that O2−, rather than H2O2, participated in the autoxidation of (+)-catechin, which was coupled with the H2O2 formation.

Accumulated evidence shows that some phytochemicals provide beneficial effects for human health. Recently, a number of mechanistic studies have revealed that direct interactions between phytochemicals and functional proteins play significant roles in exhibiting their bioactivities. However, their binding selectivities to biological molecules are considered to be lower due to their small and simple structures. In this study, we found that zerumbone, a bioactive sesquiterpene, binds to numerous proteins with little selectivity. Similar to heat-denatured proteins, zerumbone-modified proteins were recognized by heat shock protein 90, a constitutive molecular chaperone, leading to heat shock factor 1-dependent heat shock protein induction in hepa1c1c7 mouse hepatoma cells. Furthermore, oral administration of this phytochemical up-regulated heat shock protein expressions in the livers of Sprague-Dawley rats. Interestingly, pretreatment with zerumbone conferred a thermoresistant phenotype to hepa1c1c7 cells as well as to the nematode Caenorhabditis elegans. It is also important to note that several phytochemicals with higher hydrophobicity or electrophilicity, including phenethyl isothiocyanate and curcumin, markedly induced heat shock proteins, whereas most of the tested nutrients did not. These results suggest that non-specific protein modifications by xenobiotic phytochemicals cause mild proteostress, thereby inducing heat shock response and leading to potentiation of protein quality control systems. We considered these bioactivities to be xenohormesis, an adaptation mechanism against xenobiotic chemical stresses. Heat shock response by phytochemicals may be a fundamental mechanism underlying their various bioactivities.

Active oxygen species, e. g., hydrogen peroxide (H2O2), superoxide anion radical (O2-), hydroxyl radical (·OH) and lipid hydroperoxides, have been implicated as mediators of oxidative cell injuries in a variety of clinical conditions. Some metabolites of carcinogenic compounds, cigarette smoke and glucosone, an intermediate of the Maillard reaction, were found to produce H2O2 in their autoxidation processes, and to induce both cytotoxicity and DNA single-strand breaks in mammalian cells. Inhibition of these cell injuries by catalase indicated that H2O2 was the critical species. In contrast to the above prooxidant effects, antioxidant effects of food ingredients on H2O2-induced cytotoxicity were also investigated. Polyphenols, e. g., caffeic acid ester and flavonoids, were effective for suppression of H2O2-induced cytotoxicity on mammalian cells. A study of the structure-activity relationship revealed that the affinity of polyphenols for cell membranes and the presence of an ortho-dihydroxy moiety in their structure were essential for their protective action.

The 70% methanol soluble fraction from a licorice acetone extract was found to inhibit cell proliferation in human monoblastic leukemia U937 cells by inducing apoptosis. Separation by the methods including preparative HPLC provided us with an active compound, which was identified as licocoumarone. Several lines of evidence indicated that licocoumarone induced apoptosis in U937 cells. Thus, licocoumarone is suggested to be potentially useful as a natural anti-cancer agent.

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.

The major catechins of green tea extract are (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECg), and (-)-epigallocatechin gallate (EGCg). Recent research has indicated that catechins form complexes with human serum albumin (HSA) in blood, and differences in their binding affinity toward HSA are believed to modulate their bioavailability. In this study, we kinetically investigated the interaction between the catechins and HSA immobilized on a quartz-crystal microbalance (QCM). The association constants obtained from the frequency changes of QCM revealed interactions of ECg and EGCg with HSA that are 100 times stronger than those of EC and EGC. Furthermore, comparisons of these catechins by native-gel electrophoresis/blotting with redox-cycling staining revealed that, in a phosphate buffer, ECg and EGCg have a higher binding affinity toward HSA than EC and EGC. These observations indicate that catechins with a galloyl moiety have higher binding affinities toward HSA than catechins lacking a galloyl moiety.

Antioxidative activity of pea bean (Phaseolus vulgaris L.) extract was evaluated by using a linoleic acid system, and the methanol extract exhibited strong antioxidative activity as measured by the thiocyanate method. The crude methanol extract was partitioned between then-butanol phase (BP) and the water phase (WP). Then, the antioxidative activity of the BP and the WP was determined by using a linoleic acid system. The WP showed strong antioxidative activity, while BP showed only weak activity as measured by the thiocyanate method. Next, the synergistic antioxidative action of WP with α-tocopherol was examined by using linoleic acid and liposome systems. The WP had a synergistic effect with α-tocopherol in both the food model and liposome systems. For purification and isolation of the antioxidative substances of the pea bean, preparative high-performance liquid chromatography was carried out with an octadecylsilyl column. Five fractions were collected, and antioxidative activity was determined in a linoleic acid system. Although fraction 1 had strong activity by the thiocyanate method, the purification of this active fraction was difficult; therefore, the partly characterized active fraction was investigated. The contents of total phenolics and sugars were 0.31±0.01 mg/g of fraction 1 and 406.1±0.1 mg/g, respectively. The ninhydrin chromogenic reaction was positive, and the ultraviolet absorption spectral λ max value in distilled water was 264.0 nm, indicating that the water-soluble antioxidative components from pea bean may be a new type of antioxidant. Isolation and identification are currently being investigated.

BACKGROUND: Lectins are proteins that bind specifically to the carbohydrate moiety of glyco-conjugates. Japanese mistletoe lectin given intragastrically affected cytokine gene expression in the mouse intestine. This study examines the actions of Lens culinaris agglutinin (LCA) on the gene expression of gluconeogenic enzymes in the intestine. RESULTS: The results of quantitative real-time reverse transcription–polymerase chain reaction indicated that LCA caused an up-regulation of the gene expression of glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK). This change was correlated with an increase in the expression of two transcription factors, HNF1α and HNF4α. Experiments using human colonic cancer Caco-2 cells demonstrated that LCA up-regulated the gene expression of G6Pase and PEPCK whereas insulin had the opposite effect. In addition, the observed up-regulation of HNF4α gene expression in the duodenum raises the possibility that the lectin promotes the colorectal cancer. CONCLUSION: Lentil beans should be cooked well to avoid unfavourable effects of LCA. Copyright © 2011 Society of Chemical Industry

We found that glucosone (D-arabino-hexosulose), a typical enediol product formed in the Maillard reaction or in γ-radiolysis of sugars, was cytotoxic on Chinese hamster lung V79 cells in the presence of cupric ion (Cu 2+ ). Several other metal ions showed no effects. The cytotoxicity was assessed by colony formation assay and measurement of cell growth rate. In both assays, catalase inhibited the cytotoric effects. To confirm the role of H 2 O 2 on the cytotoxicity, we detected H 2 O 2 in the reaction mixture of glucosone and Cu 2+

Glucosone (D-arabino-hexos-2-ulose), a typical enediol product formed both in the Maillard reaction and gamma-radiolysis of sugars, decreased survival of Chinese hamster lung V79 cells, which were incubated under MEM for 4 h. Inhibition of the decrease in cell survival by catalase and SOD suggests the role of active oxygen species, namely H2O2 and O2-, in the biological effects of glucosone. H2O2 was formed in the medium during oxidative degradation of glucosone. Inhibition of the formation of H2O2 by SOD indicates that the formation of H2O2 and the consequent decrease of the cell survival was enhanced by O2-. These results suggest that the mechanisms of the effects of glucosone on the mammalian cells in the absence of Cu2+ are different from those in the presence of Cu2+.

Green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) has various beneficial properties including chemopreventive, anticarcinogenic, and antioxidant actions. The interaction with proteins known as EGCG-binding targets may be related to the anticancer effects. However, the binding mechanisms for this activity remain poorly understood. Using mass spectrometry and chemical detection methods, we found that EGCG forms covalent adducts with cysteinyl thiol residues in proteins through autoxidation. To investigate the functional modulation caused by binding of EGCG, we examined the interaction between EGCG and a thiol enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Concentration-dependent covalent binding of EGCG to GAPDH was found to be coupled to the irreversible inhibition of GAPDH activity. Mutation experiments revealed that EGCG is primarily bound to the cysteinyl thiol group of the active center, indicating that the irreversible inhibition of GAPDH is due to the covalent attachment of EGCG to the active-center cysteine. Moreover, using EGCG-treated cancer cells, we identified GAPDH as a target of EGCG covalent binding through specific interactions between catechols and aminophenyl boronate agarose resin. Based on these findings, we propose that the covalent modification of proteins by EGCG may be a novel pathway related to the biological activity of EGCG.

Malondialdehyde (MDA), a lipid peroxidation product, reacts with lysine residues in proteins. Human serum albumin (HSA) is a major target of MDA-modification of serum proteins. To identify, the modification sites of HSA by MDA in vitro, MDA-treated HSA was digested with a protease and the resulting peptides were subjected to liquid chromatography-tandem mass spectrometry. We identified six peptides, which contained a N-propenal adduct at Lys136, Lys174, Lys240, Lys281, Lys525, and Lys541, and revealed that Lys525 is the most reactive residue for MDA modification. Analysis of electrostatic surface potential of a 3-D model structure of HSA indicates that Lys525 is located at the center of positively charged grooves. The results of this study indicate that the modification of proteins by lipid-derived aldehydes may be influenced by the electrostatic potential of the protein surface.

The mechanisms for tight-junction (TJ) stabilization by beta-lactoglobulin (beta-Lg) were studied. Treatment of Caco-2-SF cells with inhibitors for some enzymes in the intracellular signal transduction pathways and a cytoskeleton-disturbing agent (cytochalasin D) reduced the TJ-stabilizing activity of beta-Lg. So beta-Lg is suggested to modulate the cytoskeletal structure through the activation of phospholipase C and protein kinase C, resulting in the TJ stabilization.

Propolis, a resinous substance collected by honeybees from various plant sources, possesses various physiological activities such as antitumor effects. We have previously shown that propolis of Brazilian origin was composed mainly of artepillin C and that its constituents were quite different from those of propolis of European origin. In this report, we examined an antiangiogenic effects of Brazilian propolis and investigated whether artepillin C was responsible for such effects. In an in vivo angiogenesis assay using ICR mice, we found that the ethanol extract of Brazilian propolis (EEBP) significantly reduced the number of newly formed vessels. EEBP also showed antiangiogenic effects in an in vitro tube formation assay. When compared with other constituents of EEBP, only artepillin C was found to significantly inhibit the tube formation of HUVECs in a concentration-dependent manner (3.13-50microg/ml). In addition, artepillin C significantly suppressed the proliferation of HUVECs in a concentration-dependent manner (3.13-50microg/ml). Furthermore, artepillin C significantly reduced the number of newly formed vessels in an in vivo angiogenesis assay. Judging from its antiangiogenic activity in vitro and in vivo, we concluded that artepillin C at least in part is responsible for the antiangiogenic activity of EEBP in vivo. Artepillin C may prove useful in the development of agents and foods with therapeutic or preventive activity against tumor angiogenesis.

Malondialdehyde (MDA), a naturally occurring dialdehyde produced in the membrane by lipid peroxidation, is a strong alkylating agent of primary amino groups. We recently raised a monoclonal antibody (mAb1F83) directed to the lipofuscin-like MDA--lysine adduct and demonstrated the presence of immunoreactivity to the antibody in the atherosclerotic lesions, in which intense positivity was associated primarily with macrophage-derived foam cells (Yamada et al., (2001) J. Lipid Res. 42, 1187-1196). To identify the structure of the epitope in the protein recognized by mAb1F83, in the present study, we exposed chain B from bovine insulin (insulin B chain) to MDA and characterized the MDA adducts by mass spectrometry. The MDA-modified insulin B chain was digested with V8 protease, and the resulting peptides were subjected to liquid chromatography--electrospray ionization--mass spectrometry (LC--ESI--MS/MS). The MS/MS analyses confirmed the formation of N-propenal- (+54 Da) and dihydropyridine-type (DHP, +134 Da) adducts in both Lys29 and the N-terminus of insulin B chain. The ELISA analysis of HPLC fractions of peptides, including the DHP adducts using mAb1F83, showed that the immunoreactivity of the DHP--lysine adduct was more significant than the DHP--N-terminus adduct. The results of this study chemically characterized that the MDA adducts such as DHP-type adducts generated in the epsilon-amino group of lysine and N-terminal amino acid residues in the protein and the structure of the epitope recognized by mAb1F83 were DHP--lysine adducts in protein.

We assessed the inhibitory effects of curcumin and its chemically modified homologues against H2O2-induced cytotoxicity toward Chinese hamster lung fibroblasts V79 cells with a colony formation assay. Among 4 curcuminoids, dihydroxycurcumin and dihydroxytetrahydrocurcumin suppressed H2O2-induced cytotoxicity. On the contrary, neither curcumin nor tetrahydrocurcumin showed any suppressive effects. These results support our empirical rule that the o-dihydroxy (catechol) moiety is essential for the inhibitory effects.

Cytotoxicity and DNA single-strand breaks caused by H2O2 were assessed by a colony formation assay and a DNA precipitation assay, respectively, with Chinese hamster V79 cells. In both assays, caffeic acid ethyl ester showed protective effects. The structure-activity relationship showed that the o-dihydroxy structure of caffeic acid ethyl ester was essential for the protective effects.

To identify the plant origin of Brazilian propolis directly, we observed the behavior of honeybees in Minas Gerais State of Brazil. Honeybee workers bit and chewed leaves of the plant, Baccharis dracunculifolia, packed the material into their pollen basket, brought it back to their nest, and used it as propolis. We collected the leaves of B. dracunculifolia and propolis, and compared their constituents by liquid chromatography-mass spectrometry (LC/MS) analysis. There was no difference between the chemical constituents of the ethanol extracts of B. dracunculifolia and those of propolis. This indicates directly that the plant origin of Brazilian propolis is B. dracunculifolia.

4-Hydroxy-2-nonenal (HNE), a major lipid peroxidation-derived reactive aldehyde, is a potent inhibitor of sulfhydryl enzymes, such as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). It has been suggested that HNE exerts an inhibitory effect on the enzyme due to the modification of the cysteine residue (Cys-149) at the catalytic site generating the HNE-cysteine Michael addition-type adduct [Uchida, K., and Stadtman, E. R. (1993) J. Biol. Chem. 268, 6388-6393]. In the study presented here, to elucidate the mechanism for the inactivation of GAPDH by HNE, we attempted to identify the modification sites of the enzyme by monitoring the formation of the HNE Michael adducts by mass spectrometric methods. Incubation of GAPDH (1 mg/mL) with 1 mM HNE in 50 mM sodium phosphate buffer (pH 7.4) at 37 degrees C resulted in a time-dependent loss of enzyme activity, which was associated with the covalent binding of HNE to the enzyme. To identify the site of modification of GAPDH by HNE, both the HNE-pretreated and untreated GAPDH were digested with trypsin and V8 protease, and the resulting peptides were subjected to electrospray ionization liquid chromatography-mass spectrometry (ESI-LC-MS). This technique identified five peptides, which contained the HNE adducts at His-164, Cys-244, Cys-281, His-327, and Lys-331 and revealed that both His-164 and Cys-281 were very rapidly modified at 5 min, followed by Cys-244 at 15 min and His-327 and Lys-331 at 30 min. These observations and the observation that the HNE modification of the catalytic center, Cys-149, was not observed suggest that the HNE inactivation of GAPDH is not due to the modification of the catalytic center but to the selective modification of amino acids primarily located in the surface of the GAPDH molecule.

The ethanol extract from Japanese horseradish wasabi was found to inhibit cell proliferation in human monoblastic leukemia U937 cells by inducing apoptotic cell death. Separation by methods including silica gel chromatography and preparative HPLC gave an active compound, which was identified as 6-methylsulfinylhexyl isothiocyanate (6-HITC). Several lines of evidence indicated that 6-HITC induced apoptosis in U937 cells and human stomach cancer MKN45 cells. Thus, 6-HITC is potentially useful as a natural anti-cancer agent.

The cytotoxicity of hydrogen peroxide (H2O2) toward Chinese hamster V79 cells was assessed with a colony-formation assay. Decylubiquinone, a coenzyme Q homolog, suppressed H2O2-induced cytotoxicity. Almost all the incorporated decylubiquinone was detected as decylubiquinol after incubating the cells for 4h. These results suggest that decylubiquinol reduced in the cells acted as an antioxidant against H2O2-induced oxidative cell injury.

Background The activity of creatine kinase (CK) in serum has recently been reported to be potentially associated with several types of depression. The aim of this study is to evaluate whether serum enzymes, including CK, vary even in a healthy population with depressive symptoms caused by work-related stress. We gave questionnaires and blood examinations to 93 healthy female nursing home workers and did an enzyme-linked immunosorbent assay for the quantitative detection of CK isozyme muscle-type M chain (CK-MM) in serum. Findings Depressive symptoms were determined using the Center for Epidemiologic Studies Depression (CES-D) scale and compared with the results of the blood examination and serum CK-MM levels. The CES-D results showed significant negative correlations with total CK and lactate dehydrogenase (LDH) activities and CK-MM level (r = -0.29, p = 0.0062; r = -0.29, p = 0.0065; r = -0.33, p = 0.0016, respectively). Conclusions Total CK and LDH activities and serum CK-MM level appear to be associated with the depressive symptoms of healthy nurses working in stressful environments, although the significance level was relatively low. The simultaneous detection of serum CK and LDH activities or serum CK-MM level and LDH activity may be useful as an indicator of depressive symptoms, at least for female nursing staff with work-related stress.

Three galloyl monosaccharides contained in medicinal plants were examined for apoptosis-inducing activity in human histiocytic lymphoma U937 cells. Tetragalloyl glucose (TgG) induced apoptosis as found by chromatin condensation, DNA ladder formation, and inhibition by a caspase inhibitor. Digalloyl hamamelose had moderate activity, while monogalloyl glucose was only marginally active. These findings suggest that the number and disposition of their phenolic groups are important for apoptosis induction. TgG induced apoptosis in human colon and stomach cancer cell lines as well, indicating it is potentially useful as an anti-cancer agent.

The tight junction (TJ) of the human intestinal Caco-2-SF monolayer was shown not to have been stably formed. TJ was stabilized by incubating the cell monolayer with beta-lactoglobulin (beta-Lg) and bovine serum albumin (BSA). Thus, the Caco-2-SF cell-culture system will provide a useful model for studying the factors which stabilize TJ and for investigating the mechanism of TJ regulation.

The both enantiomers of (Z)-5-(1-decenyl)oxacyclopentan-2-one were synthesized starting from resolved acetylenic intermediates. The (R)-form of this lactone is the pheromone of Popillia japonica. The optical purities of the final products were about 90%.

The both enantiomers of (Z)-5-(1-decenyl) oxacyclopentan-2-one were synthesized starting from resolved acetylenic intermediates. The (R)-form of this lactone is the pheromone of Popillia japonica. The optical purities of the final products were about 90%.

Epidemiological evidence suggests that smoking is a major cause of human lung cancer. However, the mechanism by which cigarette smoke induces the cancer remains obscure, although in tobacco carcinogenesis, promotion and/or co-carcinogenesis may have crucial roles. The epidemiological data show that if an individual stops smoking, the risk of his contracting lung cancer increases no further. Moreover, laboratory experiments show that cigarette smoke condensate (CSC) exhibits co-carcinogenic and promoting activities in tumour production and malignant transformation. Clastogenic action is thought to be intimately involved in tumour promotion, and it is therefore interesting that visible chromosome changes such as chromosome aberrations and sister chromatid exchanges are known to be caused by cigarette smoke. However, there has been no previous direct demonstration that cigarette smoke can cause single-strand breaks (SSB) in DNA. Here we report that cigarette smoke induces considerable numbers of DNA SSB in cultured human cells, and that such strand breaks may be ascribed to active oxygen generated from cigarette smoke.

Autoxidation of active metabolites of naphthylamine and aminoazo dyes in neutral buffer generated hydrogen peroxide (H2O2) and superoxide anion (O-.2), as detected by the titanium sulfate method and nitro blue tetrazolium method, respectively. 2-Amino-1-naphthol, 1-amino-2-naphthol, 1-amino-4-naphthol, N-hydroxy-2-aminonaphthalene, N-hydroxy-1-aminonaphthalene, N-hydroxy-4-aminoazobenzene and N-hydroxy-4-methylaminoazobenzene generated H2O2 and O-.2, whereas 1-nitrosonaphthalene, 2-nitrosonaphthalene, 1-naphtylamine, 2-naphthylamine and non-carcinogenic aminonaphthols and naphthols generated no active oxygens. Catalase and superoxide dismutase were used to identify the formation of these active oxygens. For all compounds tested except nitrosonaphthalenes, good parallelism was found between the active oxygen formation and convertibility to free radicals. These results suggest a possible role of free radicals and subsequently formed active oxygens in aromatic amine carcinogenesis.

We have established, for the first time, a reliable method to quantitate hydrogen peroxide (H2O2) generated in aqueous extracts of cigarette smoke tar. The aqueous tar extract was passed through a short reverse-phase column and its H2O2 concentration determined by differential pulse polarography using an automatic reference subtraction system. The H2O2 concentration increased with aging, pH and temperature; the presence of superoxide dismutase lead to lower H2O2 concentrations. This method was applied to many kinds of research and commercial cigarettes. With a few exceptions, the amount of H2O2 formed after a fixed time from each cigarette smoke was proportional to its tar yield.

The accumulation of fluorescent age pigment or lipofuscin is a frequently observed age-associated cellular alteration in a variety of postmitotic cells of many species. These pigments are observed within granules composed, in part, of damaged protein and lipid. Modification of various biomolecules by aldehyde products of lipid peroxidation is believed to contribute to lipofuscin and ceroid formation. In the present study, we raised a monoclonal antibody (MAb 1F83) directed to the malondialdehyde-modified protein and identified a lipofuscin-like dihydropyridine fluorophore as the major epitope. This antibody was used to conclusively demonstrate that the fluorophore forms on oxidatively modified low density lipoproteins. In addition, we demonstrated that the materials immunoreactive to MAb 1F83 indeed constituted the atherosclerotic lesions, in which intense positivity was associated primarily with macrophage-derived foam cells. The results of this study suggest that the reaction between the lipid peroxidation-derived aldehyde and primary amino groups of protein might represent a process common to the formation of the lipofuscin-like fluorophore during aging and its related diseases. —Yamada, S., S. Kumazawa, T. Ishii, T. Nakayama, K. Itakura, N. Shibata, M. Kobayashi, K. Sakai, T. Osawa, and K. Uchida. Immunochemical detection of a lipofuscin-like fluorophore derived from malondialdehyde and lysine.