Study of the green tea polyphenols catechin-3-gallate (CG) and epicatechin-3-gallate (ECG) as proteasome inhibitors

The green tea polyphenol catechin-3-gallate (CG) and epicatechin-3-gallate (ECG) were synthesized enantioselectively via a Sharpless hydroxylation reaction followed by a diastereoselective cyclization. Their potencies to inhibit the proteasome activity were measured. The unnatural enantiomers were found to be equally potent to the natural compounds.     

h-ANF does not play a role in the regulation of myocardial force of contraction

In the present study, h-ANF failed to reduce myocardial force of contraction alone and in the presence of isoprenaline. This lack of effect was observed in electrically driven, isolated human papillary muscle strips and rat papillary muscles. In contrast, adenosine and carbachol exerted antiadrenergic actions in rat and human myocardium. It is concluded that h-ANF obviously does not play a role in the regulation of human and rat myocardial force of contraction, whereas adenosine and carbachol are capable to produce antiadrenergic effects in the human and rat myocardium.     

Tonic component of myocardial contraction

Calcium transients and contractions of cardiac myocytes consist of phasic component, relaxing spontaneously independently of membrane voltage and of the tonic component (TC) relaxing only upon repolarization. Experimental data reviewed in this article suggest that most Ca(2+) activating TC is released from sarcoplasmic reticulum (SR) via the ryanodine receptors (RyRs). Most likely these RyRs are activated by sustained Ca(2+) influx. However, its route may differ depending on species and state of the cells. It seems that in rat RyRs responsible for TC are activated by the sustained Ca(2+) current. In guinea-pig the blockers of Ca(2+) current or reverse mode Na(+)/Ca(2+) exchange do not inhibit TC, so these routes seem unlikely. In myocytes of the failing human hearts TC is activated mostly via the reverse mode Na(+)/Ca(2+) exchange and contribution of SR is negligible. The mechanism of TC in the normal human cardiomyocytes has not been investigated. Thus, despite investigation of TC for half a century many problems concerning the mechanism of its activation and maintenance as well as its physiological meaning remain unsolved.     

Cell cycle dysregulation by green tea polyphenol epigallocatechin-3-gallate

Epidemiological, in vitro cell culture, and in vivo animal studies have shown that green tea or its constituent polyphenols, particularly its major polyphenol epigallocatechin-3-gallate (EGCG) may protect against many cancer types. In earlier studies, we showed that green tea polyphenol EGCG causes a G0/G1-phase cell cycle arrest and apoptosis of human epidermoid carcinoma (A431) cells. We also demonstrated that these effects of EGCG may be mediated through the inhibition of nuclear factor kappa B that has been associated with cell cycle regulation and cancer. In this study, employing A431 cells, we provide evidence for the involvement of cyclin kinase inhibitor (cki)-cyclin-cyclin-dependent kinase (cdk) machinery during cell cycle deregulation by EGCG. As shown by immunoblot analysis, EGCG treatment of the cells resulted in significant dose- and time-dependent (i) upregulation of the protein expression of WAF1/p21, KIP1/p27, p16 and p18, (ii) downmodulation of the protein expression of cyclin D1, cdk4 and cdk6, but not of cyclin E and cdk2, (iii) inhibition of the kinase activities associated with cyclin E, cyclin D1, cdk2, cdk4 and cdk6. Taken together, our study suggests that EGCG causes an induction of G1-phase ckis, which inhibit the cyclin-cdk complexes operative in G0/G1 phase of the cell cycle thereby causing a G0/G1-phase arrest of the cell cycle, which is an irreversible process ultimately resulting in an apoptotic cell death. We suggest that the naturally occurring agents such as green tea polyphenols which may inhibit cell cycle progression could be developed as potent anticancer agents for the management of cancer.     

Nox regulation of smooth muscle contraction

The catalytic subunit gp91phox (Nox2) of the NADPH oxidase of mammalian phagocytes is activated by microbes and immune mediators to produce large amounts of reactive oxygen species (ROS) which participate in microbial killing. Homologs of gp91phox, the Nox and Duox enzymes, were recently described in a range of organisms, including plants, vertebrates, and invertebrates such as Drosophila melanogaster. While their enzymology and cell biology are being extensively studied in many laboratories, little is known about in vivo functions of Noxes. Here, we establish and use an inducible system for RNAi to discover functions of dNox, an ortholog of human Nox5 in Drosophila. We report here that depletion of dNox in musculature causes retention of mature eggs within ovaries, leading to female sterility. In dNox-depleted ovaries and ovaries treated with a Nox inhibitor, muscular contractions induced by the neuropeptide proctolin are markedly inhibited. This functional defect results from a requirement for dNox-for the proctolin-induced calcium flux in Drosophila ovaries. Thus, these studies demonstrate a novel biological role for Nox-generated ROS in mediating agonist-induced calcium flux and smooth muscle contraction.     

Calcium regulation of muscle contraction.

Calcium triggers contraction by reaction with regulatory proteins that in the absence of calcium prevent interaction of actin and myosin. Two different regulatory systems are found in different muscles. In actin-linked regulation troponin and tropomyosin regulate actin by blocking sites on actin required for complex formation with myosin; in myosin-linked regulation sites on myosin are blocked in the absence of calcium. The major features of actin control are as follows: there is a requirement for tropomyosin and for a troponin complex having three different subunits with different functions; the actin displays a cooperative behavior; and a movement of tropomyosin occurs controlled by the calcium binding on troponin. Myosin regulation is controlled by a regulatory subunit that can be dissociated in scallop myosin reversibly by removing divalent cations with EDTA. Myosin control can function with pure actin in the absence of tropomyosin. Calcium binding and regulation of molluscan myosins depend on the presence of regulatory light chains. It is proposed that the light chains function by sterically blocking myosin sites in the absence of calcium, and that the "off" state of myosin requires cooperation between the two myosin heads. Both myosin control and actin control are widely distributed in different organisms. Many invertebrates have muscles with both types of regulation. Actin control is absent in the muscles of molluscs and in several minor phyla that lack troponin. Myosin control is not found in striated vertebrate muscles and in the fast muscles of crustacean decapods, although regulatory light chains are present. While in vivo myosin control may not be excluded from vertebrate striated muscles, myosin control may be absent as a result of mutations of the myosin heavy chain.     

Targeting polyamines and biogenic amines by green tea epigallocatechin-3-gallate

Biogenic amines and polyamines are organic polycations derived from aromatic or cationic amino acids. They exert pleiotropic effects, more related to intercellular communication in the case of biogenic amines, and to intracellular signaling in the case of polyamines. The bioactive compound epigallocatechin-3-gallate (EGCG), a major component of green tea, has been shown to target key enzyme of biogenic amine and polyamine metabolic pathways. Herein, we review the specific effects of EGCG on concrete molecular targets of both biogenic amine and polyamine metabolic pathways, and discuss the relevance of these data to support the potential therapeutic interest of this compound.     

Induction of apoptosis by epigallocatechin-3-gallate in human lymphoblastoid B cells

(-)-Epigallocatechin-3-gallate (EGCG), a major constituent of green tea polyphenols, has been shown to suppress cancer cell proliferation and induce apoptosis. In this study we investigated its efficacy and the mechanism underlying its effect using human B lymphoblastoid cell line Ramos, and effect of co-treatment with EGCG and a chemotherapeutic agent on apoptotic cell death. EGCG induced dose- and time-dependent apoptotic cell death accompanied by loss of mitochondrial transmembrane potential, release of cytochrome c into the cytosol, and cleavage of pro-caspase-9 to its active form. EGCG also enhanced production of intracellular reactive oxygen species (ROS). Pretreatment with diphenylene iodonium chloride, an inhibitor of NAD(P)H oxidase and an antioxidant, partially suppressed both EGCG-induced apoptosis and production of ROS, implying that oxidative stress is involved in the apoptotic response. Furthermore, we showed that combined-treatment with EGCG and a chemotherapeutic agent, etoposide, synergistically induced apoptosis in Ramos cells.     

Two elementary models for the regulation of skeletal muscle contraction by calcium

It is shown by use of an extremely simple explicit two-state model that two basic ideas may be sufficient to understand at least qualitatively the sensitive activation of isometric muscle contraction by Ca2+. (a) Ca2+ binds much more strongly on troponin if myosin is already attached to actin. The steady state analogue of this is that the single rate constant (in the two-state model) for myosin attachment plus Pi release is much larger if Ca2+ is bound to troponin. (b) End-to-end tropomyosin interactions are responsible for positive cooperativity. Although these ideas seem to be sufficient, this of course does not mean that they are necessary. These same ingredients were used in two previous, more elaborate models for the cooperative equilibrium binding of myosin subfragment-1 on actin-tropomyosin-troponin, with and without Ca2+, and for a study of the steady state ATPase activity of the same system. Essentially as an appendix, the above-mentioned simple treatment is extended to a somewhat more realistic and complicated model of isometric contraction.     

Structural basis for the regulation of muscle contraction by troponin and tropomyosin

The molecular switching mechanism governing skeletal and cardiac muscle contraction couples the binding of Ca²⁺ on troponin to the movement of tropomyosin on actin filaments. Despite years of investigation, this mechanism remains unclear because it has not yet been possible to directly assess the structural influence of troponin on tropomyosin that causes actin filaments, and hence myosin-crossbridge cycling and contraction, to switch on and off. A C-terminal domain of troponin I is thought to be intimately involved in inducing tropomyosin movement to an inhibitory position that blocks myosin-crossbridge interaction. Release of this regulatory, latching domain from actin after Ca²⁺ binding to TnC (the Ca²⁺ sensor of troponin that relieves inhibition) presumably allows tropomyosin movement away from the inhibitory position on actin, thus initiating contraction. However, the structural interactions of the regulatory domain of TnI (the “inhibitory” subunit of troponin) with tropomyosin and actin that cause tropomyosin movement are unknown, and thus, the regulatory process is not well defined. Here, thin filaments were labeled with an engineered construct representing C-terminal TnI, and then, 3D electron microscopy was used to resolve where troponin is anchored on actin-tropomyosin. Electron microscopy reconstruction showed how TnI binding to both actin and tropomyosin at low Ca²⁺ competes with tropomyosin for a common site on actin and drives tropomyosin movement to a constrained, relaxing position to inhibit myosin-crossbridge association. Thus, the observations reported reveal the structural mechanism responsible for troponin-tropomyosin-mediated steric interference of actin-myosin interaction that regulates muscle contraction.     

Glucuronidation of the green tea catechins, (-)-epigallocatechin-3-gallate and (-)-epicatechin-3-gallate, by rat hepatic and intestinal microsomes

The flavonoids (-)-epigallocatechin-3-gallate (EGCg) and (-)-epicatechin-3-gallate (ECg) are major components of green tea and show numerous biological effects. We investigated the glucuronidation of these compounds and of quercetin by microsomes. Quercetin was almost fully glucuronidated by liver microsomes after 3 h, whereas ECg and ECGg were conjugated to a lesser extent ([Formula: See Text] and [Formula: See Text] respectively). The intestinal microsomes also glucuronidated quercetin much more efficiently than ECg and EGCg. Although the rates were lower than quercetin, intestinal microsomes exhibited higher activity on the galloyl group of ECg and EGCg compared to the flavonoid ring, whereas hepatic glucuronidation was higher on the flavonoid ring of EGCg and ECg compared to the galloyl groups. The low glucuronidation rates could partially explain why these flavanols are present in plasma as unconjugated forms.     

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。     

Glucuronidation of the Green Tea Catechins, (-)-epigallocatechin-3-gallate and (-)-epicatechin-3-gallate,by Rat Hepatic and Intestinal Microsomes

The flavonoids (-)-epigallocatechin-3-gallate (EGCg) and (-)-epicatechin-3-gallate (ECg) are major components of green tea and show numerous biological effects. We investigated the glucuronidation of these compounds and of quercetin by microsomes. Quercetin was almost fully glucuronidated by liver microsomes after 3 h, whereas ECg and ECGg were conjugated to a lesser extent ([Formula: See Text] and [Formula: See Text] respectively). The intestinal microsomes also glucuronidated quercetin much more efficiently than ECg and EGCg. Although the rates were lower than quercetin, intestinal microsomes exhibited higher activity on the galloyl group of ECg and EGCg compared to the flavonoid ring, whereas hepatic glucuronidation was higher on the flavonoid ring of EGCg and ECg compared to the galloyl groups. The low glucuronidation rates could partially explain why these flavanols are present in plasma as unconjugated forms.     

Regulation of nodularin-induced apoptosis by epigallocatechin-3-gallate on fish lymphocytes in vitro

Nodularin is one of the most conspicuous and widespread pollutants that elicit water ecological hazards to fish, causing serious damage on the immune system and physiological functions. Nodularin can cause oxidative stress-induced apoptosis on fish lymphocytes. The regulatory effects of epigallocatechin-3-gallate (EGCG) at 10, 100, and 1000 μg/L levels on the antioxidant defense system and apoptosis of Carassius auratus lymphocytes exposed to a high dose of nodularin (100 μg/L) were quantified in vitro. EGCG reduced nodularin-induced oxidative damage on fish immune cells. This compound significantly increased the activities of superoxide dismutase and catalase and the level of glutathione but decreased the levels of intracellular reactive oxygen species and malondialdehyde. Flow cytometry results showed that the percentages of apoptotic cells after treatment with 10, 100, and 1000 μg/L EGCG for 12 h reached 27.9%, 19.1%, and 13.7%, respectively. By contrast, the nodularin alone-induced group showed a high percentage of apoptosis (44.2%). Western blot analysis showed the increased expression of bcl-2 and the decreased expression of bax and caspase-3 in EGCG-treated fish lymphocytes. EGCG also inhibited the potential collapse of the mitochondrial membrane. Overall, EGCG can inhibit nodularin-induced apoptosis and protect the normal immunity of fish by regulating bax/bcl-2 and blocking the downstream of mitochondrial apoptosis pathway with increased intracellular antioxidant enzyme activity.     

Decavanadate as a biochemical tool in the elucidation of muscle contraction regulation

Recently reported decameric vanadate (V(10)) high affinity binding site in myosin S1, suggests that it can be used as a tool in the muscle contraction regulation. In the present article, it is shown that V(10) species induces myosin S1 cleavage, upon irradiation, at the 23 and 74 kDa sites, the latter being prevented by actin and the former blocked by the presence of ATP. Identical cleavage patterns were found for meta- and decavanadate solutions, indicating that V(10) and tetrameric vanadate (V(4)) have the same binding sites in myosin S1. Concentrations as low as 50 muM decavanadate (5 muM V(10) species) induces 30% of protein cleavage, whereas 500 muM metavanadate is needed to attain the same extent of cleavage. After irradiation, V(10) species is rapidly decomposed, upon protein addition, forming vanadyl (V(4+)) species during the process. It was also observed by NMR line broadening experiments that, V(10) competes with V(4) for the myosin S1 binding sites, having a higher affinity. In addition, V(4) interaction with myosin S1 is highly affected by the products release during ATP hydrolysis in the presence or absence of actin, whereas V(10) appears to be affected at a much lower extent. From these results it is proposed that the binding of vanadate oligomers to myosin S1 at the phosphate loop (23 kDa site) is probably the cause of the actin stimulated myosin ATPase inhibition by the prevention of ATP/ADP exchange, and that this interaction is favoured for higher vanadate anions, such as V(10).     

表没食子儿茶素没食子酸酯对细胞信号转导通路的影响

表没食子儿茶素没食子酸酯（epigallocatechin-3-gallate,EGCG）是绿茶中含量最丰富的儿茶素,具有抗氧化、抗炎、抗动脉粥样硬化、抗癌等多种作用。研究发现EGCG通过多条细胞信号转导通路发挥作用。本文简要综述EGCG对MAPK、NF-κB、PI3K/Akt、JAK/STAT、TGF-β/Smad、Wnt/β-catenin等多条信号转导通路的影响。