Published Inhibitory effect of luteolin on hepatocyte growth factor/scatter factor -induced HepG2 cell invasion involving both MAPK/ERKs and PI3K-Akt pathways Abstract: Hepatocyte growth factor (HGF), also known as scatter factor (SF), and its receptor, the c-Met tyrosine kinase, play roles in cancer invasion and metastasis in a wide variety of tumor cells. Clinical observations suggest that HGF can promote metastasis of hepatoma cells while stimulating tumor invasiveness. We use HGF as an invasive inducer of human hepatoma HepG2 cells to investigate the effect of flavonoids on anti-invasion. In our preliminary study, we investigated the effect of flavonoids including luteolin, quercetin, baicalein, genistein, taxifolin, and catechin on HGF-mediated migration and invasion of HepG2 cells. We found that luteolin presented the most potent potential on anti-migration and anti-invasion by Boyden chamber assay. Furthermore, luteolin inhibited HGF-induced cell scattering and cytoskeleton change such as filopodia and lamellipodia was determined by both phase-contrast and fluorescence microscopy studies. In addition, Western blotting and immunoprecipitation were performed to confirm luteolin suppressed the phosphorylation of c-Met, the membrane receptor of HGF, as well as ERK1/2 and Akt, but not JNK1/2, which is activated by HGF. Our investigation demonstrated that luteolin similar to PD98059, which acts as a specific inhibitor of MEK, an up stream kinase regulating ERK1/2, and wortmannin, a PI3K inhibitor, inhibited the invasiveness induced by HGF. In conclusion, the luteolin inhibited HGF-induced HepG2 cell invasion involving both MAPK/ERKs and PI3K-Akt pathways. Anal. Chem., ASAP Article 10.1021/ac070675l S0003-2700(07)00675-0 Web Release Date: November 6, 2007 High-Throughput Automated Luminescent Magnetic Particle-Based Immunoassay to Monitor Human Exposure to Pyrethroid Insecticides Ki Chang Ahn, Pete Lohstroh, # Shirley J. Gee, Nancy A. Gee, Bill Lasley, and Bruce D. Hammock* Department of Entomology and UCD Cancer Research Center, and Center for Health and Environment, University of California, Davis, California 95616 Received for review April 5, 2007. Accepted September 18, 2007. Abstract: We have developed a sensitive, automated, competitive chemiluminescent immunoassay for the detection of 3-phenoxybenzoic acid (3-PBA), a metabolite common to many pyrethroid insecticides. The system uses a competitive hapten-protein conjugate that has been labeled with an acridinium ester as the chemiluminescent probe and secondary antibody-coated paramagnetic particles for the separation. After the immunoassay reagents and samples are combined for the competitive incubation step, a fully automated system is used to load the postincubation mixture into a delivery cuvette, facilitating the subsequent magnetic separation of the immunocomplex and the measurement of chemiluminescent signal for quantification. The immunoassay format described here supports the requirement for high throughput necessary for monitoring large numbers of samples in population-based studies. The optimized immunoassay was more sensitive than the conventional enzyme immunoassay in buffer (IC50 = 0.1 and 2 g/L, respectively). The mixed-mode solid-phase extraction used for sample preparation to reduce possible urinary matrix effects allowed the accurate measurement of 3-PBA levels as low as 1 g/L. The automated chemiluminescent immunoassay described here is sensitive, simple to use, and more rapid than the previously reported standard microplate assay. Inorg. Chem., 46 (24), 10143 -10152. Mononuclear and Polynuclear Copper(I) Complexes with a New N,N',S-Donor Ligand and with Structural Analogies to the Copper Thionein Core Abstract: The N,N',S-donor ligand 4-methoxy-3,5-dimethyl-2-((3-(2-(methylthio)phenyl)-1H -pyrazol-1-yl)methyl)pyridine (L) was prepared from 2-(chloromethyl)-4-methoxy3,5-dimethylpyridine hydrochloride and 3-(2-(methylthio)phenyl)-1H-pyrazole. The Cu(I) complexes [Cu2(L)2CH3CN] [Cu(L)CH3CN](BF4)3 (1), [Cu(L)PPh3]BF4 (2), and [Cu6(L)2(C6F5S)6] (3) were prepared and characterized by X-ray crystallography (PPh3 = triphenylphosphine, C6F5S- = pentafluorothiophenolate). The unit cell of compound 1 consists of cocrystallized mononuclear and dinuclear entities in which all of the copper atoms exhibit distorted tetrahedral coordination. Compound 2 is monomeric with L bound in the 3-N,N',S mode and a PPh3 molecule that completes the coordination environment. Compound 2 presents a fluxional behavior in CDCl3 solution due to the boat inversion of the six-membered N,N' chelate ring ( H = +43.6(3) kJ mol-1, S = -16(1) J mol-1 K-1). Crystallization of 3 in acetonitrile leads to a polynuclear structure that contains a CH3CN molecule coordinated to one of the copper atoms: [Cu6(L)2(C6F5S)6CH3CN] (3a). The core of 3a partially resembles a {Cu4S6} adamantane-like moiety, the only difference being that the Cu-NCCH3 interaction leads to the opening of the cluster by disrupting a Cu-Cu interaction. Part of this assembly is found in the yeast metallothionein copper(I)-cysteinate core whose crystal structure has recently been reported. Two additional [Cu(L)]+ peripheral moieties interact with the cluster by means of bridging thiolates. ESI-mass spectrometry, conductivity measurements, and 1H/19F pulsed gradient spin echo (PGSE) NMR experiments suggest that 3a dissociates in acetonitrile solution: 3a + CH3CN [Cu4(C6F5S)6]2- + 2[Cu(L)CH3CN]+. The stability of the cluster with respect to the hypothetical mononuclear species, [Cu(L)(C6F5S)], is confirmed by DFT calculations (B3LYP), which illustrate the exergonic character of the reaction: 6[Cu(L')(C6H5S)] [Cu6(L')2(C6H5S)6] + 4L' ( G298 = -58.6 kJ mol-1, where L' and C6H5S- are simplified models for L and C6F5S-, respectively). The energetics pertinent to the ionic dissociation of the cluster in acetonitrile is computed using the polarizable continuum model (PCM) approach. J. Org. Chem., 72 (24), 9060 -9066, 2007 Synthesis of -Functionalized Porphyrins via Palladium-Catalyzed Carbon-Heteroatom Bond Formations: Expedient Entry into -Chiral Porphyrins Abstract A procedure was developed for the preparation of -monobromo-tetraphenylporphyrin (BrTPP) in a greatly improved yield from the selective bromination of tetraphenylpor-phyrin (TPP) by NBS. BrTPP was successfully employed as a versatile synthon for convenient synthesis of a wide range of -monofunctionalized porphyrins with various heteroatom carbon-heteroatom bond formations. functionalities via palladium-mediated Examples include -amino, -amido, -oxo, and -mercaptoporphyrins from reactions with amines, amides, alcohols, and thiols, respectively. Applying the synthetic approach to chiral amides, -chiral porphyrins were effectively constructed. Org. Lett., 9 (20), 4033 -4036, 2007. Synthesis of the Acutumine Spirocycle via a Radical-Polar Crossover Reaction Abstract A new radical-polar crossover reaction was developed that consists of intramolecular conjugate addition of an aryl radical followed by enolate formation and hydroxylation. A C-C bond, a C-O bond, and two congested stereocenters are created in the process. The product is obtained as a single isomer. The method was used to synthesize the spirocyclic subunit of the alkaloid acutumine. Journal of Chemical Physics; 11/14/2007, Vol. 127 Issue 18 Random-phase-approximation-based correlation energy functionals: Benchmark results for atoms. Jiang, Hong; Engel, Eberhard The random phase approximation for the correlation energy functional of the density functional theory has recently attracted renewed interest. Formulated in terms of the Kohn-Sham orbitals and eigenvalues, it promises to resolve some of the fundamental limitations of the local density and generalized gradient approximations, as, for instance, their inability to account for dispersion forces. First results for atoms, however, indicate that the random phase approximation overestimates correlation effects as much as the orbital-dependent functional obtained by a second order perturbation expansion on the basis of the Kohn-Sham Hamiltonian. In this contribution, three simple extensions of the random phase approximation are examined; (a) its augmentation by a local density approximation for short-range correlation, (b) its combination with the second order exchange term, and (c) its combination with a partial resummation of the perturbation series including the second order exchange. It is found that the ground state and correlation energies as well as the ionization potentials resulting from the extensions (a) and (c) for closed subshell atoms are clearly superior to those obtained with the unmodified random phase approximation. Quite some effort is made to ensure highly converged data, so that the results may serve as benchmark data. The numerical techniques developed in this context, in particular, for the inherent frequency integration, should also be useful for applications of random phase approximation-type functionals to more complex systems. Journal of Chemical Physics; 11/14/2007, Vol. 127 Issue 18 Adsorption, desorption, and diffusion of nitrogen in a model nanoporous material. II. Diffusion limited kinetics in amorphous solid water. By: Zubkov, Tykhon; Smith, R. Scott; Engstrom, Todd R.; Kay, Bruce D. The adsorption, desorption, and diffusion kinetics of N2 on thick (up to ∼9 μm) porous films of amorphous solid water (ASW) films were studied using molecular beam techniques and temperature programmed desorption. Porous ASW films were grown on Pt (111) at low temperature (<30 K) from a collimated H2O beam at glancing incident angles. In thin films (<1 μm), the desorption kinetics are well described by a model that assumes rapid and uniform N2 distribution throughout the film. In thicker films (>1 μm), N2 adsorption at 27 K results in a nonuniform distribution, where most of N2 is trapped in the outer region of the film. Redistribution of N2 can be induced by thermal annealing. The apparent activation energy for this process is ∼7 kJ/mol, which is approximately half of the desorption activation energy at the corresponding coverage. Preadsorption of Kr preferentially adsorbs onto the highest energy binding sites, thereby preventing N2 from trapping in the outer region of the film which facilitates N2 transport deeper into the porous film. Despite the onset of limited diffusion, the adsorption kinetics are efficient, precursor mediated, and independent of film thickness. An adsorption mechanism is proposed, in which a high-coverage N<sub>2</sub> front propagates into a pore by the rapid transport of physisorbed second layer N2 species on top of the first surface bound layer. Inorg. Chem., 46 (24), 10229 -10240. Influence of Steric Hindrance on the Core Geometry and Sulfoxidation Chemistry of Carboxylate-Rich Diiron(II) Complexes Abstract: The asymmetric terphenyl-2'-carboxylate ligand 3,5-dimethyl-1,1':3',1' Ph,Xyl '-terphenyl-2'-carboxylate, O2CAr , was prepared in high yield. This ligand facilitates the assembly of the diiron(II) complexes [Fe2( -O2CArTol)2(O2CArPh,Xyl)2(THF)2] [2, -O2CArTol = 2,6-di-p-tolylbenzoate], [Fe2( -O2CArTol)2(O2CArPh,Xyl)2(pyridine)2] (5), [Fe2( -O2CArPh,Xyl)2-(O2CArPh,Xyl)2(THF)2] (3), and [Fe2( -O2CArPh,Xyl)2(O2CArPh,Xyl)2(pyridine)2] (6), all of which have a windmill geometry. The iron-iron distance of 3.355[10] Å in 6 is ~1 Å shorter than that in the analogue [Fe2( -O2CArTol)2(O2CArTol)2(pyridine)2] (4) and similar to the ~3.3 Å metal-metal separation at the active site of the reduced diiron(II) form of the soluble methane monooxygenase hydroxylase enzyme (MMOHred). A series of t ortho-substituted picolyl-based ligands, 2-picSMe, 2-picSEt, 2-picS Bu, 2-picSPh, 2-picSPh(Me3) (Ph(Me3) = mesityl), and 2-picSPh(iPr3) (Ph(iPr3) = 2,4,6-triisopropylphenyl), were prepared and allowed to react with [Fe2( -O2CAr)2(O2CAr)2(THF)2] to produce [Fe2( -O2CAr)3(O2CAr)(picSR)] (7-13, Ar = ArTol or ArPh,Xyl) complexes in 45-87% yields. The substrates tethered to the pyridine N-donor ligands picSR, where R = Me, Et, tBu, or Ph, coordinate to one iron atom of the diiron(II) center by the nitrogen and sulfur atoms to form a five-membered chelate ring. The Fe-S distance becomes elongated with increasing steric hindrance imparted by the R group. The most sterically hindered ligands, 2-picSPh(Me3) and 2-picSPh(iPr3), bind to the metal only through the pyridine nitrogen atom. The reactions of several of these complexes with dioxygen were investigated, and the oxygenated products were analyzed by 1H NMR spectroscopy and GC/MS measurements following decomposition on a Chelex resin. The amount of sulfoxidation product is correlated with the Fe···S distance. The ratio of oxidized to unoxidized thioether substrate varies from 3.5, obtained upon oxygenation of the weakly coordinated 2-picSPh ligand in 10, to 1.0, obtained for the bulky 2-picSPh(iPr3) ligand in 12, for which the iron-sulfur distance is >4 Å. External thioether substrates were not oxidized when present in oxygenated solutions of paddlewheel and windmill diiron(II) complexes containing 1-methylimidazole or pyridine ligands, respectively. biochemical pharmacology 71 ( 2006 ) 540 – 549 Structure activity and molecular modeling analyses of ribose- and base-modified uridine 50-triphosphate analogues at the human P2Y2 and P2Y4 receptors Kenneth A. Jacobson a,*, Stefano Costanzi b, Andrei A. Ivanov a, Susanna Tchilibon a, Pedro Besada a, Zhan-Guo Gao a, Savitri Maddileti c, T. Kendall Harden c a Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA b Computational Chemistry Core Laboratory, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA c Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, NC 27599, USA abstract With the long-term goal of developing receptor subtype-selective high affinity agonists for the uracil nucleotide-activated P2Y receptors, we have carried out a series of structure activity and molecular modeling studies of the human P2Y2 and P2Y4 receptors. UTP analogues with substitutions in the 20-position of the ribose moiety retained capacity to activate both P2Y2 and P2Y4 receptors. Certain of these analogues were equieffective for activation of both receptors whereas 20-amino-20-deoxy-UTP exhibited higher potency for the P2Y2 receptor and 20-azido-UTP exhibited higher potency for the P2Y4 receptor. 4-Thio substitution of the uracil base resulted in a UTP analogue with increased potency relative to UTP for activation of both the P2Y2 and P2Y4 receptors. In contrast, 2-thio substitution and halo- or alkyl substitution in the 5-position of the uracil base resulted in molecules that were 3–30-fold more potent at the P2Y2 receptor than P2Y4 receptor. 6-Aza-UTP was a P2Y2 receptor agonist that exhibited no activity at the P2Y4 receptor. Stereoisomers of UTPaS and 20-deoxy-UTPaS were more potent at the P2Y2 than P2Y4 receptor, and the R-configuration was favored at both receptors. Molecular docking studies revealed that the binding mode of UTP is similar for both the P2Y2 and P2Y4 receptor binding pockets with the most prominent dissimilarities of the two receptors located in the second transmembrane domain (V90 in the P2Y2 receptor and I92 in the P2Y4 receptor) and the second extracellular loop (T182 in the P2Y2 receptor and L184 in the P2Y4 receptor). In summary, this work reveals substitutions in UTP that differentially affect agonist activity at P2Y2 versus P2Y4 receptors and in combination with molecular modeling studies should lead to chemical synthesis of new receptor subtype-selective drugs. Free Radical Research, July 2007; 41(7): 812–822 Cell adhesion and integrin expression are modulated by oxidative stress in EA.hy 926 cells Abstract The effects of oxidative stress on integrin-mediated cell adhesion to the extracellular matrix (ECM) and related apoptosis were investigated using the EA.hy926 endothelial cells treated (or not) with two oxidants: the hypoxanthine/xanthine oxidase system (HX/XO) or the tert-butyl hydroperoxide (t-BHP) which both increased cell apoptosis. Cell adhesion onto vitronectin (Vn) and fibronectin (Fn) was increased at low concentrations of HX/XO (up to 5 mU/ml) or t-BHP (up to 125mM) and prevented ROS-induced apoptosis. Flow cytometry analysis of integrin expression showed that the expression of integrin av and a5 subunits was, respectively, increased and decreased. Cell adhesion inhibition experiments using function-blocking monoclonal antibodies against integrin subunits indicated that avb1 and avb3 integrins were involved in adhesion of cells to Vn, and avb3 integrin played a major role in oxidant-treated cells. For adhesion to Fn, a5b1 and avb1 integrins were required for oxidant-treated cells. Taken together, the results suggest that reactive oxygen species (ROS) produced either by HX/XO or t-BHP could affect expression and/or activation of specific integrins in the interaction of EA.hy926 cells with ECM. Keywords: Reactive oxygen species, apoptosis, endothelial cell adhesion, extracellular matrix, integrins J. Agric. Food Chem. 2007, 55, 36863691 Reactivity Relationships of Flavan-3-ols on Product Generation in Aqueous Glucose/Glycine Model Systems YUKO NODA AND DEVIN G. PETERSON* Department of Food Science, The Pennsylvania State University, 327 Food Science Building, University Park, Pennsylvania 16802-2504 Abstract Ring structure-reactivity relationships of three flavan-3-ols [epicatechin (EC), epicatechin gallate (ECG), and epigallocatechin gallate (EGCG)] and three simple phenolic compounds (1,3,5-trihydroxybenzene, 1,2,3-trihydroxybenzene, and methylgallate as the analogous individual A, B, and C benzene rings of EGCG) on product generation in an aqueous glucose-glycine reaction model system (125 °C and 30 min) were investigated. The addition of EC, ECG, or EGCG to a glucoseglycine model was reported to similarly significantly reduce the formation of pyrazine, methylsubstituted pyrazines, and cyclotene. All three flavan-3-ols were also reported to generate phenolic-C2, C3, C4, and C6 sugar fragment adducts and to statistically reduce the concentration of glyoxal, glycolaldehyde, methylglyoxal, hydroxyacetone, diacetyl, acetoin, and 3-deoxyglucosone during the reaction time course, except for the EGCG reaction where 3-deoxyglucosone was not statistically different from the control after 20 min. For the simple phenolic compounds, methylgallate followed by 1,2,3-trihydroxybenzene was the least reactive, while 1,3,5-trihydroxybenzene was reported as the most reactive phenolic structure for quenching or reducing the concentration of the R-hydroxyand R-dicarbonyl sugar fragments during the reaction time course. These results imply that the main mechanism flavan-3-ols reduced product generation was phenolic-sugar fragment carbonyl trapping reactions primarily on the A ring (the meta-polyhydroxylated benzene ring) or not due to the alteration of the reaction reduction potential. KEYWORDS: Carbonyl reactions; flavan-3-ols; Maillard reaction; sugar fragments; structure-reactivity; trapping; aroma generation; polyhydroxybenzene