A Computational Exploration of the Interactions of the Green Tea Polyphenol (–)-Epigallocatechin 3-Gallate with Cardiac Muscle Troponin C

Thanks to its polyphenols and phytochemicals, green tea is believed to have a number of health benefits, including protecting from heart disease, but its mechanism of action at the molecular level is still not understood. Here we explore, by means of atomistic simulations, how the most abundant of the green tea polyphenols, (–)-Epigallocatechin 3-Gallate (EGCg), interacts with the structural C terminal domain of cardiac muscle troponin C (cCTnC), a calcium binding protein that plays an important role in heart contractions. We find that EGCg favourably binds to the hydrophobic cleft of cCTnC consistently with solution NMR experiments. It also binds to cCTnC in the presence of the anchoring region of troponin I (cTnI(34–71)) at the interface between the E and H helices. This appears to affect the strength of the interaction between cCTnC and cTnI(34–71) and also counter-acts the effects of the Gly159Asp mutation, related to dilated cardiomyopathy. Our simulations support the picture that EGCg interacting with the C terminal domain of troponin C may help in regulating the calcium signalling either through competitive binding with the anchoring domain of cTnI or by affecting the interaction between cCTnC and cTnI(34–71).     

Promotion of Neuronal Plasticity by (−)-Epigallocatechin-3-Gallate

The consumption of (−)-epigallocatechin-3-gallate (EGCG), the major polyphenolic compound found in green tea, has been associated with various neurological benefits including cognitive improvement. The physiological basis for this effect is unknown. In this study, we used synaptic transmission between the CA3 and CA1 regions (Schaffer collateral) of the mouse hippocampus to examine the effects of EGCG on neuronal plasticity. We found that the level of high frequency stimulation-evoked long-term potentiation (LTP) was significantly enhanced when hippocampal slices were pre-incubated with 10 μM EGCG for 1 h prior to the experiment. EGCG incubation also enabled hippocampal slices prepared from Ts65Dn mice, a Down syndrome mouse model deficient in LTP, to express LTP to a level comparable to the normal controls. EGCG treatment did not alter the degree of pair-pulse inhibition; therefore, the enhancement effect of EGCG is unlikely to involve the attenuation of this inhibitory mechanism.     

(-)-Epigallocatechin-3-Gallate Protects against NO-Induced Ototoxicity through the Regulation of Caspase- 1, Caspase-3, and NF-κB Activation

Excessive nitric oxide (NO) production is toxic to the cochlea and induces hearing loss. However, the mechanism through which NO induces ototoxicity has not been completely understood. The aim of this study was to gain further insight into the mechanism mediating NO-induced toxicity in auditory HEI-OC1 cells and in ex vivo analysis. We also elucidated whether and how epigallocatechin-3-gallate (EGCG), the main component of green tea polyphenols, regulates NO-induced auditory cell damage. To investigate NO-mediated ototoxicity, S-nitroso-N-acetylpenicillamine (SNAP) was used as an NO donor. SNAP was cytotoxic, generating reactive oxygen species, releasing cytochrome c, and activating caspase-3 in auditory cells. NO-induced ototoxicity also mediated the nuclear factor (NF)-κB/caspase-1 pathway. Furthermore, SNAP destroyed the orderly arrangement of the 3 outer rows of hair cells in the basal, middle, and apical turns of the organ of Corti from the cochlea of Sprague–Dawley rats at postnatal day 2. However, EGCG counteracted this ototoxicity by suppressing the activation of caspase-3/NF-κB and preventing the destruction of hair cell arrays in the organ of Corti. These findings may lead to the development of a model for pharmacological mechanism of EGCG and potential therapies against ototoxicity.     

Biosynthesis of cyclic β-(1–3),β-(1–6) glucan in Bradyrhizobium spp.

Inner membranes of Bradyrhizobium japonicum strain USDA 110 produced in vitro soluble and insoluble -(1–3),-(1–6) glucans. The reaction proceeded through a 90 kDa inner membrane intermediate protein; used UDP-glucose as sugar donor and required Mg²⁺. Gel chromatography of soluble glucans resolved a cyclic -(1–3) glucan with a degree of polymerization of eleven from a family of -(1–3),-(1–6) glucans with variable degree of polymerization higher than eleven. Bradyrhizobium strains BR4406 and BR8404 isolated from tree legume nodules in Southeast Brazil produce -(1–3),-(1–6) glucans very similar to that of B. japonicum. A 100 kDa protein was identified in these strains as intermediates in the synthesis of these glucans. Inner membranes of B. japonicum USDA110, B. japonicum I17, and Bradyrhizobium strains BR4406 and BR8404 incubated with UDP-glucose were unable to synthesize -(1–2) glucan and lacked the 235 kDa intermediate protein known to be involved in the synthesis of -(1–2) glucan in Agrobacterium tumefaciens, Rhizobium meliloti and Rhizobium loti.Abbreviations EPS= exopolysaccharides - CPS= capsular polysaccharides - LPS= lipopolysaccharides - AMA= Yeast extract-mannitol medium - TY= tryptone-yeast extract - PMSF= phenyl methyl sulfonil fluoride

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Synthesis of 2-(p-trifluoroacetamidophenyl)ethylO-α-l-fucopyranosyl-(1–3)-O-(2-acetamido-2-deoxy-β-d-glucopyranosyl) (1–3)-O-β-d-galactopyranosyl-(1–4)-β-d-glucopyranoside,a tetrasaccharide fragment of a tumour-associated glycosphingolipid

The tetrasaccharide 2-(p-trifluoroacetamidophenyl)ethylO-α-l-fucopyranosyl-(1–3)-O-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-(1–3)-O-β-d-galactopyranosyl-(1–4)-β-d-glucopyranoside was synthesized from thioglycoside intermediates. The key step was a methyl triflate promoted glycosidation of a lactose-derived 3′,4′-diol with a disaccharide thioglycoside to give a β(1–3)-linked tetrasaccharide derivative in 67% yield.     

Synthesis of (–)-Muricatacin

The synthesis of (–)-muricatacin starting from 1-bromododecane and 2-pentyn-l-ol is described. 2-Pentadecyn-1-ol (4), which was prepared from 1-bromododecane (2) and 2-pentyn-1-ol (3), was converted to epoxy alcohol 6 through a two-step reaction sequence, 6 being successively submitted to tosylation, iodination, chain extension with tert-butyl lithioacetate, and acid-catalyzed cyclization to give (–)-muricatacin (1a). Recrystallization afforded optically pure 1a.     

Computational DNA binding studies of (–)-epigallocatechin-3-gallate

The catechin family of molecules that are present in the leaves of green tea has been under investigation since the antioxidant and anti-inflammatory properties of tea were discovered. Among multiple proposed therapeutic targets of these molecules, the direct interaction with nucleic acids has been proposed and experimentally observed but without clear knowledge about the potential binding modes between these ligands and DNA. One of these catechin structures, (–)-epigallocatechin gallate (EGCG), has three aromatic rings that could interact with double-stranded DNA via terminal base-pair stacking, intercalation, or through groove binding. Using enhanced sampling techniques and molecular dynamics simulations, we have found a stable complex between the EGCG ligand and DNA through intercalation of the trihydroxybenzoate aromatic ring and an ApC step. Moreover, we have calculated the absorption spectra of four possible binding modes and compared these to absorption profiles reported in the literature, and explored the possible DNA sequence preference for the EGCG ligand to bind. Our results suggest that an intercalative mode of interaction through the major groove is possible between the EGCG ligands and DNA with apparently very little DNA sequence selectivity.     

Therapeutic Implications of The Unusually Long Half-Life of (E)-5–(2–bromovinyl)uracil (Bvura) In Vivo

Abstract

The long half-life of BVUra in the plasma permits, on the one hand, the de novo formation of the antiviral drug (E)-5–(2-bromovinyl)-2′-deoxyuridine (BVdUrd) by a pentosyl transfer, and, on the other hand, the inhibition of the degradation of pyrimidine bases such as 5-fluorouracil (FUra).     

(1–3)-β-Glucan synthesis ofNeurospora crassa

(1–3)--d-Glucan synthase activity ofNeurospora crassa was localized to the plasma membrane by autoradiography of colloidal gold-labeled plasma membranes. The active site of glucan synthase for substrate hydrolysis was determined to be cytoplasmic facing. However, glucan synthase activity present in intact protoplasts was partially sensitive to Novozym 234 and to glutaraldehyde treatments, suggestive that enzyme activity is transmembrane. Enzyme activity also directed the formation of microfibrils in vitro. Taken together, these and previous results support the following scheme for glucan synthesis: 1. The sequential addition of glucose residues from UDP-glucose to glucan chains occurs on the cytoplasmically facing portion of glucan synthase. 2. As each glucan chain is synthesized, it is extruded to the extracytoplasmic side of the plasma membrane. 3. As each chain is extruded, it forms interchain hydrogen bonds with adjacent chains, resulting in glucan microfibril assembly.     

Microbial production of (R)-3-halolactic acid from (±)-3-halo-1,2-propanediol

Microbial oxidation of (±)-3-halo-1,2-propanediol was studied and it was found that several microorganisms accumulated (R)-3-halolactic acid. Geotrichum loubieri CBS 252.61 produced the most and gave optically pure (R)-3-chlorolactic acid and (R)-3-bromolactic acid from the corresponding diols.     

Small-molecule modulators of 14-3-3 protein–protein interactions

14-3-3 Proteins are eukaryotic adapter proteins that regulate a plethora of physiological processes by binding to several hundred partner proteins. They play a role in biological activities as diverse as signal transduction, cell cycle regulation, apoptosis, host-pathogen interactions and metabolic control. As such, 14-3-3s are implicated in disease areas like cancer, neurodegeneration, diabetes, pulmonary disease, and obesity. Targeted modulation of 14-3-3 protein–protein interactions (PPIs) by small molecules is therefore an attractive concept for disease intervention. In recent years a number of examples of inhibitors and stabilizers of 14-3-3 PPIs have been reported promising a vivid future in chemical biology and drug development for this remarkable class of proteins.     

Absorption of DNA in the region of vacuum-uv (3–25 eV)

By means of a device that might be considered a modern version of "Ulbricht's sphere" the absorption spectrum and the photoelectric emission of calf thymus DNA was measured in the region of 3 to 25 eV (400 to 50 nm). A tentative explanation of the general shape of the absorption spectrum and of its 6 maxima is given. The results permit a much better insight into some biologic effects of vacuum-uv to be gained than hitherto possible.     

Structure–activity relationship (SAR) studies of 3-(2-amino-ethyl)-5-(4-ethoxy-benzylidene)-thiazolidine-2,4-dione: Development of potential substrate-specific ERK1/2 inhibitors

A series of analogs of 3-(2-amino-ethyl)-5-(4-ethoxy-benzylidene)-thiazolidine-2,4-dione, a putative substrate-specific ERK1/2 inhibitor, were synthesized and biologically characterized in human leukemia U937 cells to define its pharmacophore. It was discovered that shift of ethoxy substitution from the 4- to the 2-position on the phenyl ring significantly improved functional activities of inhibiting cell proliferation and inducing apoptosis. This may provide access to a new lead for developing ERK1/2 substrate-specific inhibitors.     

Structure–activity relationships of the 1-amino-3-(1H-indol-1-yl)-3-phenylpropan-2-ol series of monoamine reuptake inhibitors

The SAR of a series of 1-amino-3-(1H-indol-1-yl)-3-phenylpropan-2-ols as monoamine reuptake inhibitors, with a goal to improve both potency toward inhibiting the norepinephrine transporter and selectivity over the serotonin transporter, is reported. The effect of specific substitution on both the 3-phenyl group and the indole moiety were explored. This study led to the discovery of compound 20 which inhibited the norepinephrine transporter with an IC₅₀ value of 4 nM while exhibiting 86-fold selectivity over the serotonin transporter.     

Large-scale Preparation of (R)-(–)-1,3-Butanediol from the Racemate by Candida parapsilosis

Large-scale preparation of (R)-(–)-1,3-butanediol (R-BDO), an important chiral synthon, from the racemate by Candida parapsilosis IFO 1396 was investigated. We found that ethanol accumulated during culture enhanced the secondary alcohol oxido-reduction activity of cells. Large-scale preparation of R-BDO was done using a fermentor. 3092 g of R-BDO was obtained from the racemate by the use of this strain with 94.0% enantiomeric excess.     

Tumour-specific cytotoxicity and structure–activity relationships of novel 1-[3-(2-methoxyethylthio)propionyl]-3,5-bis(benzylidene)-4-piperidones

A series of 1-acyl-3,5-bis(benzylidene)-4-piperidones 3–7 were designed and synthesized as novel cytotoxic agents. These compounds displayed potent cytotoxic properties towards human Molt4/C8, CEM, HSC-2, HSC-3 and HSC-4 neoplasms and also to murine L1210 cells. The majority of the compounds have sub-micromolar or very low micromolar IC₅₀ and CC₅₀ values and are significantly more potent than the reference alkylating drug melphalan. Evaluation of these compounds against non-malignant HGF and HPLF cells revealed the tumour-specific toxicity. In particular, 3e emerged as a promising lead cytotoxic agent which caused apoptosis and PARP1 cleavage in HSC-2 cells.     

Purification of theN-acetylglucosaminide α(1–3/4) fucosyltransferase of human milk

TheN-acetylglucosaminide (1–3/4)fucosyltransferase has been purified 1.8×10⁶-fold from human milk by ion-exchange chromatography, affinity chromatography of GDP-agarose and HPLC. The (1–3/4)fucosyltransferase behaves in gel filtration-HPLC as a molecule of M_r 98 000, and differs from the (1–3)fucosyltransferase which behaves like a molecule of about M_r 47 000. The enzyme is a glycoprotein, and the purified preparation appears in SDS polyacrylamide gel electrophoresis as a band of M_r 44 000. The results present the first purification of human milk (1–3/4)fucosyltransferase to apparent homogeneity, and suggest that the (1–3/4)- and (1–3)fucosyltransferases of human milk differ in their native molecular sizes, the former being a dimer of two subunits.     

Comparison of two β-glucosidases for the enzymatic synthesis of β-(1-6)-β-(1-3)-gluco-oligosaccharides

A domain of epiglucan was synthesized by beta-glucosidases. Two beta-glucosidases, an extracellular beta-glucosidase derived from Sclerotinia sclerotiorum grown on xylose, and a commercial lyophilized preparation of beta-glucosidase from Aspergillus niger, were used to synthesize gluco-oligosaccharides from cellobiose and, specially, beta-(1-6) branched beta-(1-3) gluco-oligosaccharides, corresponding to the structure of epiglucan. Gentiobiose, cellotriose, cellotetraose, beta-Glc-(1-3)-beta-Glc-(1-4)-Glc, beta-Glc-(1-6)-beta-Glc-(1-4)-Glc and beta-Glc-(1-6)-beta-Glc-(1-3)-Glc were synthesized from cellobiose by both enzymes. The latter compound was preferentially synthesized by the beta-glycosidase from Sclerotinia sclerotiorum. Under the best conditions, only 7 g l(-1) of beta-Glc-(1-6)-beta-Glc-(1-3)-Glc was synthesized by the beta-glycosidase from Aspergillus niger compared to 20 g l(-1) synthesized with beta-glycosidase from Sclerotinia sclerotiorum.     

Dose-dependent Incorporation of Tea Catechins, (–)-Epigallocatechin-3-gallate and (–)-Epigallocatechin,into Human Plasma

Tea catechins, (–)-epigallocatechin-3-gallate (EGCg) and (–)-epigallocatechin (EGC), have been reported to suppress oxidation of plasma low density lipoprotein (LDL) in vitro. If dietary catechins can be efficiently incorporated into human blood plasma, anti-atherosclerotic effects in preventing oxidative modification of LDL would be expected. In this study, a newly developed chemiluminescence detection-high pressure liquid chromatography (CL-HPLC) method for measuring plasma catechins was used and the incorporation of EGCg and EGC into human plasma was investigated. Healthy subjects orally ingested 3, 5, or 7 capsules of green tea extract (corresponding to 225, 375, and 525 mg EGCg and 7.5, 12.5, and 17.5 mg EGC, respectively). The plasma EGCg and EGC concentrations before the administration were all below the detection limit (< 2 pmol/ml), but 90 min after, significantly and dose-dependently increased to 657, 4300, and 4410 pmol EGCg/ml, and 35, 144, and 255 pmol EGC/ml, in the subjects who received 3, 5, and 7 capsules, respectively. Both EGCg and EGC levels detected in plasma corresponded to 0.2–2.0% of the ingested amount. Catechin intake had no effect on the basal level of endogenous antioxidants (α-tocopherol, β-carotene, and lycopene) or of lipids in plasma. These results suggested that drinking green tea daily would contribute to maintain plasma catechin levels sufficient to exert antioxidant activity against oxidative modification of lipoproteins in blood circulation systems.     

Synthesis of 2-(3-Deoxy-β-D-erythropentofuranosyl)-thiazole-4-carboxamide (3′-Deoxytiazofurin)

Abstract

2-(3-Deoxy-β-D-erythropentofuranosyl)-thiazole-4-carboxamide was synthesized in four steps from its β-D-ribofuranosyl nucleoside precursor.