茶树表没食子儿茶素-3-O-(3-O-甲基)没食子酸酯研究进展

表没食子儿茶素-3-O-(3-O-甲基)没食子酸酯(EGCG3"Me)是茶叶中最常检测到的甲基化表没食子儿茶素没食子酸酯(EGCG"Me),具有较表没食子儿茶素没食子酸酯(EGCG)更好的保健功效。本文对EGCG3"Me的理化性质、制备方法、保健功效、茶树EGCG3"Me含量影响因素、EGCG3"Me体内合成路径等国内外研究现状进行了综述,展望EGCG3"Me的体内代谢途径及其深加工产品研发将成为研究热点。     

Relationship between the anti-hemolysin activity and the structure of catechins and theaflavins

We examined the corresponding isomers of catechins and theaflavins for anti-hemolysin activities against Staphylococcus aureus alpha-toxin and Vibrio cholerae O1 hemolysin. Catechins and theaflavins showed anti-hemolysin activities in a dose-dependent manner. Among the catechins tested, (-)catechin gallate, (-)epicatechin gallate and (-)epigallocatechin gallate having galloyl groups in their molecules showed more potent anti-hemolysin activities against both toxins. On the other hand, free catechins, i. e. (-)catechin, (-)gallocatechin, (-) epicatechin and (-)epigallocatechin had low anti-hemolysin activities against alpha-toxin. Although (-)catechin or (-)gallocatechin had no effect on cholera hemolysin, (-) epicatechin and (-)epigallocatechin were slightly inhibitory. Among dextrocatechins, (+) epicatechin and (+)epigallocatechin proved to be more effective than (+)catechin and (+) gallocatechin. The anti-hemolysin activities of theaflavins against alpha-toxin and cholera hemolysin were dependent on the number of the galloyl group in their structure. These results suggest that the tertiary structure of the catechin or theaflavin and the active site of hemolysin, that affects the interaction between them, plays an important role in the anti-hemolysin activity.     

Affinity of polyphenols for lipid bilayers

Interaction of tea catechins with lipid bilayers has been investigated with liposome systems. Epicatechin gallate had the highest affinity for lipid bilayers, followed by epigallocatechin gallate, epicatechin, and epigallocatechin. Epicatechin gallate and epigallocatechin gallate in the surface of lipid bilayer perturbed the membrane structure.     

Interaction of Tea Catechins with Lipid Bilayers Investigated with Liposome Systems

Interaction of tea catechins with lipid bilayers was investigated with liposome systems, which enabled us to separate liposomes from the external medium by centrifugation. We found that epicatechin gallate had the highest affinity for lipid bilayers, followed by epigallocatechin gallate, epicatechin, and epigallocatechin. Epicatechin gallate and epigallocatechin gallate in the surface of lipid bilayer perturbed the membrane structure.     

Controlled potential enzymology of methyl transfer reactions involved in acetyl-CoA synthesis by CO dehydrogenase and the corrinoid/iron-sulfur protein from Clostridium thermoaceticum

Many anaerobic bacteria fix CO2 via the Wood pathway of acetyl-CoA synthesis. Carbon monoxide dehydrogenase (CODH), also called acetyl-CoA synthase, accepts the methyl group from the methylated corrinoid/iron-sulfur protein (C/Fe-SP), binds a carbonyl group from CO, CO2, or the carboxyl of pyruvate, and binds coenzyme A. Then CODH catalyzes the synthesis of acetyl-CoA from these enzyme-bound groups. Here, we have characterized the methyl transfer steps involved in acetyl-CoA synthesis. We have studied the reactions leading to methylation of CODH by methyl iodide and shown an absolute requirement of the C/Fe-SP in this reaction. In addition, we have discovered and partly characterized two previously unknown exchange reactions catalyzed by CODH: between the methylated C/Fe-SP and methylated CODH and between methylated CODH and the methyl moiety of acetyl-CoA. We have performed these two exchange reactions, methylation of the C/Fe-SP, and methylation of CODH at controlled potentials. The rates of all these reactions except the exchange between methylated C/Fe-SP and methylated CODH are accelerated (from 1 to 2 orders of magnitude) when run at low potentials. Our results provide strong evidence for a nucleophilic redox-active metal center on CODH as the initial acceptor of the methyl group from the methylated C/Fe-SP. This metal center also is proposed to be involved in the cleavage of acetyl-CoA in the reverse reaction.     

Interaction of tea catechins with lipid bilayers investigated with liposome systems

Interaction of tea catechins with lipid bilayers was investigated with liposome systems, which enabled us to separate liposomes from the external medium by centrifugation. We found that epicatechin gallate had the highest affinity for lipid bilayers, followed by epigallocatechin gallate, epicatechin, and epigallocatechin. Epicatechin gallate and epigallocatechin gallate in the surface of lipid bilayer perturbed the membrane structure.     

Studies on the Mechanism of the Oxidation of Tea Leaf Catechins

In order to understand the mechanism of the enzymic oxidation of epigallocatechin, ethyl gallate was taken up as a model substrate on account of its close structural similarity to the w-trihydroxyphenyl group, which indeed is supposed to be attacked by oxidase, when epigallocatechin is oxidized enzymically. By the action of tea or apple oxidase at pH 5.5, it gave a characteristic polyphenolic substance, which was obtained in prisms, C₁₈H₁₈O₁₀·2H₂O, and proved to be diethyl 4,4′,5,5′,6,6′-hexahydroxydiphenate, as the product was converted into ellagic acid by hydrolysis of ester linkage and then lactone formation.     

Enzymatic production of epigallocatechin by using an epigallocatechin gallate hydrolase induced from Aspergillus oryzae

Catechins are a group of polyphenolic compounds that are antioxidants having beneficial biological activities. There are four main catechins in green tea, and each has its own biological features. In order to fully exploit prominent biological activities of specific catechins and to develop new medicine from catechins, it is necessary to obtain pure catechin preparations by isolation from natural sources, by chemical synthesis, or by biotransformation reactions with high yield and specificity. In this study epigallocatechin gallate (EGCG) can be hydrolyzed to epigallocatechin (EGC) by a hydrolase from Aspergillus oryzae after induction by addition of EGCG to the cultures. However, cultures without EGCG induction did not show any EGCG hydrolysis activity. The yield of EGC could reach at least 70%. Thin layer chromatography and high performance liquid chromatography were applied to separate and quantify EGCG and EGC.     

Preparation and specificity of antibodies directed toward the ribose methylated nucleotide, 2'-O-methylguanosine 5'-monophosphate

Antibodies have been produced that recognize the ribosome methylated nucleotide, 2'-O-methylguanosine 5'-monophosphate, pGm. Specificity was tested in a radioimmunoassay. Other ribose methylated nucleotides, deoxyguanosine, and guanosine 5'-monophosphate exhibited negligible cross-reactivity. Elements of antibody recognition, in descending order of importance, were the parent base, the methylated ribose moiety, and the phosphate group.     

结直肠癌患者血浆甲基化Sept9基因的表达及临床意义

目的:探讨甲基化Sept9基因在结直肠癌患者血浆中的表达及临床意义。方法:选择2015年1月~2017年3月经陕西省人民医院病理证实的结直肠癌患者87例(结直肠癌组)、结直肠息肉患者79例(结直肠息肉组)、健康体检者93例(健康对照组)作为研究对象,采用实时荧光定量聚合酶链式反应(PCR)技术检测其外周血血浆Sept9基因甲基化情况,比较三组甲基化Sept9基因阳性表达率,分析甲基化Sept9基因阳性表达与结直肠癌病理特征的关系。结果:血浆甲基化Sept9基因在结直肠癌组、结直肠息肉组、健康对照组的阳性表达率分别为71.26%(62/87)、5.06%(4/79)、3.23%(3/93),差异有统计学意义(P0.05);血浆甲基化Sept9基因阳性表达与结直肠癌患者的性别、年龄、肿瘤部位、病理分型、血管侵犯、神经侵犯无关(P0.05),与肿瘤最大径、浸润深度、分化程度、淋巴结转移、TNM分期有关(P0.05);结直肠癌患者血浆甲基化Sept9基因阳性表达率为71.26%(62/87),高于血清CEA的54.02%(47/87)、CA199的35.63%(31/87)、CA724的33.33%(29/87)、CA125的21.84%(19/87),差异有统计学意义(P0.05)。结论:结直肠癌患者外周血血浆甲基化Sept9基因呈高表达状态,早期检测甲基化Sept9基因表达水平在结直肠癌的诊断及病情评估中有重要意义。     

Stability of N-acyl groups in methylated α2 → 8-linked oligosialosyl chains toward methanolysis. Analysis by chemical ionization-mass spectrometry

The stability of N-acetyl group of methylated trisaccharide of N-acetylneuraminic acid toward methanolysis under conditions used in methylation analysis was investigated. The analysis of the products obtained after a reaction sequence, methylation-methanolysis-deuterioacetylation, by chemical ionization-mass spectrometry has led to unequivocal conclusion that N-acetyl group of internal 8-O-substituted residue of the methylated oligosialosyl compound is de-N-acetylated under conditions sufficient to cleave glycosidic linkages, whereas the fully methylated nonreducing terminal residue of neuraminic acid is completely resistant to de-N-acetylation. The reaction mechanism to explain these observations is presented.     

Bioactivity-Guided Identification and Cell Signaling Technology to Delineate the Lactate Dehydrogenase A Inhibition Effects of Spatholobus suberectus on Breast Cancer

Aerobic glycolysis is an important feature of cancer cells. In recent years, lactate dehydrogenase A (LDH-A) is emerging as a novel therapeutic target for cancer treatment. Seeking LDH-A inhibitors from natural resources has been paid much attention for drug discovery. Spatholobus suberectus (SS) is a common herbal medicine used in China for treating blood-stasis related diseases such as cancer. This study aims to explore the potential medicinal application of SS for LDH-A inhibition on breast cancer and to determine its bioactive compounds. We found that SS manifested apoptosis-inducing, cell cycle arresting and anti-LDH-A activities in both estrogen-dependent human MCF-7 cells and estrogen-independent MDA-MB-231 cell. Oral herbal extracts (1 g/kg/d) administration attenuated tumor growth and LDH-A expression in both breast cancer xenografts. Bioactivity-guided fractionation finally identified epigallocatechin as a key compound in SS inhibiting LDH-A activity. Further studies revealed that LDH-A plays a critical role in mediating the apoptosis-induction effects of epigallocatechin. The inhibited LDH-A activities by epigallocatechin is attributed to disassociation of Hsp90 from HIF-1α and subsequent accelerated HIF-1α proteasome degradation. In vivo study also demonstrated that epigallocatechin could significantly inhibit breast cancer growth, HIF-1α/LDH-A expression and trigger apoptosis without bringing toxic effects. The preclinical study thus suggests that the potential medicinal application of SS for inhibiting cancer LDH-A activity and the possibility to consider epigallocatechin as a lead compound to develop LDH-A inhibitors. Future studies of SS for chemoprevention or chemosensitization against breast cancer are thus warranted.     

Impact of the green tea ingredient epigallocatechin gallate and a short pentapeptide (Ile-Ile-Ala-Glu-Lys) on the structural organization of mixed micelles and the related uptake of cholesterol

Background

High levels of blood cholesterol are conventionally linked to an increased risk of developing cardiovascular disease (Grundy, 1986). Here we examine the molecular mode of action of natural products with known cholesterol-lowering activity, such as for example the green tea ingredient epigallocatechin gallate and a short pentapeptide, Ile-Ile-Ala-Glu-Lys.

The Impact of EGCG and RG108 on SOCS1 Promoter DNA Methylation and Expression in U937 Leukemia Cells

Background:The available evidence has increasingly demonstrated that a combination of genetic and epigenetic factors, such as DNA methylation, could be considered as causing leukemia. Epigenetic changes and methylation of the suppressor of the cytokine signaling 1 promoter (SOCS1) CpG region silence SOCS1 expression in cancer. In the current study, we evaluated the impact of epigallocatechin gallate (EGCG) and RG108 on SOCS1 promoter methylation and expression in U937 cells.Methods:In the current study, U937 leukemic cells were treated with EGCG and RG108 for 12, 24, 48, and 72 h and SOCS1 promoter methylation and its expression were measured by methylation-specific PCR (MSP) and quantitative real-time PCR, respectively.Results:The outcomes indicated that the SOCS1 promoter is methylated in U937 cells, and treatment of these cells with either EGCG or RG108 reduced its methylation. Moreover, we observed that SOCS1 expression was significantly upregulated in a time-dependent manner by both EGCG and RG108 in U937 cells compared with control cells. In the RG108-treated group at 12, 24, 48, and 72 h, SOCS1 expression was upregulated by 1, 4.2, 16.6, and 32.6 -fold respectively, and in the EGCG-treated group, by 0.5, 3.2, 10.8, and 22.3 -fold, respectively. Conclusion:Treatment with either EGCG or RG108 reduced SOCS1 promoter methylation and increased SOCS1 expression in U937 cells in a time-dependent manner, which may play a role in leukemia therapy.Key Words: DNA Methylation, EGCG, Leukemia, RG108, SOCS1     

Novel eye-specific calmodulin methylation characterized by protein mapping in Drosophila melanogaster

Post-translational methylation of the epsilon-amino group of lysine residues regulates a number of protein functions. Calmodulin, a key modulator of intracellular calcium signaling, is methylated on lysine 115 in many species. Although the amino acid sequence of calmodulin is highly conserved in eukaryotes, it has been shown that lysine 115 is not methylated in Drosophila calmodulin and no other methylation site has been reported. In this study, we characterized in vivo modification states of Drosophila calmodulin using proteomic methodology involving the protein mapping of microdissected Drosophila tissues on 2-D gels. We found that Drosophila calmodulin was highly expressed in methylated forms in the compound eye, whereas its methylation was hardly detected in other tissues. We identified that lysine 94 located in an EF-hand III is the methylation site in Drosophila calmodulin. The predominance of methylated calmodulin in the compound eye may imply the involvement of calmodulin in photoreceptor-specific functions through methylation.     

Hydration and recognition of methylated CpG steps in DNA.

The analysis of the hydration pattern around methylated CpG steps in three high resolution (1.7, 2.15 and 2.2 A) crystal structures of A-DNA decamers reveals that the methyl groups of cytosine residues are well hydrated. In comparing the native structure with two structurally distinct forms of the decamer d(CCGCCGGCGG) fully methylated at its CpG steps, this study shows also that in certain structural and sequence contexts, the methylated cytosine base can be more hydrated that the unmodified one. These water molecules seem to be stabilized in front of the methyl group through the formation C-H...O interactions. In addition, these structures provide the first observation of magnesium cations bound to the major groove of A-DNA and reveal two distinct modes of metal binding in methylated and native duplexes. These findings suggest that methylated cytosine bases could be recognized by protein or DNA polar residues through their tightly bound water molecules.     

Isolation and analysis by the reductive-cleavage method of linkage positions and ring forms in the Mycobacterium smegmatis cell-wall arabinogalactan

The Mycobacterium smegmatis arabinogalactan polysaccharide has been isolated from the cell wall by saponification and extraction to remove lipids and subsequent solubilization by treatment with lysozyme. Analysis for neutral sugars demonstrated the presence of D-arabinose and D-galactose in a ratio of 3:1, respectively. Reductive cleavage of the fully methylated polysaccharide in the presence of triethylsilane and trimethylsilyl trifluoromethanesulfonate and subsequent acetylation in situ gave six partially methylated 1,4-anhydroalditol acetates as the major products and three partially methylated 1,5-anhydroalditol acetates as minor products. Partially methylated 1,5-anhydroalditol acetates were not formed when reductive cleavage was accomplished with triethylsilane and a mixture of trimethylsilyl methanesulfonate and boron trifluoride etherate as the catalyst, demonstrating that the polysaccharide is exclusively comprised of furanosyl residues. The partially methylated anhydroalditols so produced were identified by comparison to authentic standards. Their identifies are consistent with the presence in the M. smegmatis arabinogalactan of an octasaccharide repeating unit comprised of a nonreducing terminal D-arabinofuranosyl group, a 2-O-linked D-arabinofuranosyl residue, three 5-O-linked D-arabinofuranosyl residues, a 3,5-di-O-linked D-arabinofuranosyl residue, a 5-O-linked D-galactofuranosyl residue, and a 6-O-linked D-galactofuranosyl residue.     

Carboxyl methylation and COOH-terminal processing of the brain G-protein gamma-subunit

The enzymatic methylation of the guanine nucleotide-binding proteins (G-proteins) gamma-subunit was investigated in brain membranes. Brain membranes were methylated in vitro using [3H-methyl]S-adenosylmethionine, and the G-protein beta gamma-complex was purified using an anti-beta antibody to assay for the protein during purification. The isolated G-protein beta gamma-complex was found to be carboxyl methylated on the gamma-subunit. The methyl group was localized by tryptic digestion to the carboxyl-terminal of the protein. The methylated tryptic peptides contained a modified cysteine and were very hydrophobic, suggesting additional modification by lipidation. The evidence suggests that the COOH-terminal of G-gamma is modified in a manner similar to the processing that occurs with the ras proteins.     

Effect of Pre‐ and Post–Combined Multidoses of Epigallocatechin Gallate and Coenzyme Q10 on Cisplatin‐Induced Oxidative Stress in Rat Kidney

The nephroprotective effect of coenzyme Q10 and epigallocatechin gallate was investigated in rats with acute renal injury induced by a single nephrotoxic dose of cisplatin. Two days prior to cisplatin administration, epigallocatechin gallate and coenzyme Q10 alone and in four different combinations were given for 6 days. The treatment with antioxidants significantly protected the cisplatin‐induced increase in the levels of blood urea nitrogen and serum creatinine. Both the antioxidants alone or in different combinations significantly compensated the increased malondialdehyde and reduced glutathione levels. Moreover, the decrease in the activities of superoxide dismutase, catalase, and glutathione peroxidase and the concentration of selenium, zinc, and copper ions were significantly attenuated in renal tissue. In conclusion, epigallocatechin gallate and coenzyme Q10 are equally effective against cisplatin‐induced nephrotoxicity, whereas the intervention by combining these two antioxidants was found to be highly effective at low doses in attenuating oxidative stress in rat kidney.