Caffeic acid, tyrosol and p-coumaric acid are potent inhibitors of 5-S-cysteinyl-dopamine induced neurotoxicity

Parkinson's disease is characterized by a progressive and selective loss of dopaminergic neurons in the substantia nigra. Recent investigations have shown that conjugates such as the 5-S-cysteinyl-dopamine, possess strong neurotoxicity and may contribute to the underlying progression of the disease pathology. Although the neuroprotective actions of flavonoids are well reported, that of hydroxycinnamates and other phenolic acids is less established. We show that the hydroxycinnamates caffeic acid and p-coumaric acid, the hydroxyphenethyl alcohol, tyrosol, and a Champagne wine extract rich in these components protect neurons against injury induced by 5-S-cysteinyl-dopamine in vitro. The protection induced by these polyphenols was equal to or greater than that observed for the flavonoids, (+)-catechin, (-)-epicatechin and quercetin. For example, p-coumaric acid evoked significantly more protection at 1muM (64.0+/-3.1%) than both (-)-epicatechin (46.0+/-4.1%, p<0.05) and (+)-catechin (13.1+/-3.0%, p<0.001) at the same concentration. These data indicate that hydroxycinnamates, phenolic acids and phenolic alcohol are also capable of inducing neuroprotective effects to a similar extent to that seen with flavonoids.

Glycogen phosphorylase inhibitors: a free energy perturbation analysis of glucopyranose spirohydantoin analogues

GP catalyzes the phosphorylation of glycogen to Glc-1-P. Because of its fundamental role in the metabolism of glycogen, GP has been the target for a systematic structure-assisted design of inhibitory compounds, which could be of value in the therapeutic treatment of type 2 diabetes mellitus. The most potent catalytic-site inhibitor of GP identified to date is spirohydantoin of glucopyranose (hydan). In this work, we employ MD free energy simulations to calculate the relative binding affinities for GP of hydan and two spirohydantoin analogues, methyl-hydan and n-hydan, in which a hydrogen atom is replaced by a methyl- or amino group, respectively. The results are compared with the experimental relative affinities of these ligands, estimated by kinetic measurements of the ligand inhibition constants. The calculated binding affinity for methyl-hydan (relative to hydan) is 3.75 +/- 1.4 kcal/mol, in excellent agreement with the experimental value (3.6 +/- 0.2 kcal/mol). For n-hydan, the calculated value is 1.0 +/- 1.1 kcal/mol, somewhat smaller than the experimental result (2.3 +/- 0.1 kcal/mol). A free energy decomposition analysis shows that hydan makes optimum interactions with protein residues and specific water molecules in the catalytic site. In the other two ligands, structural perturbations of the active site by the additional methyl- or amino group reduce the corresponding binding affinities. The computed binding free energies are sensitive to the preference of a specific water molecule for two well-defined positions in the catalytic site. The behavior of this water is analyzed in detail, and the free energy profile for the translocation of the water between the two positions is evaluated. The results provide insights into the role of water molecules in modulating ligand binding affinities. A comparison of the interactions between a set of ligands and their surrounding groups in X-ray structures is often used in the interpretation of binding free energy differences and in guiding the design of new ligands. For the systems in this work, such an approach fails to estimate the order of relative binding strengths, in contrast to the rigorous free energy treatment.

Kinetic and crystallographic studies of glucopyranose spirohydantoin and glucopyranosylamine analogs inhibitors of glycogen phosphorylase

Glycogen phosphorylase (GP) is currently exploited as a target for inhibition of hepatic glycogenolysis under high glucose conditions. Spirohydantoin of glucopyranose and N-acetyl-beta-D-glucopyranosylamine have been identified as the most potent inhibitors of GP that bind at the catalytic site. Four spirohydantoin and three beta-D-glucopyranosylamine analogs have been designed, synthesized and tested for inhibition of GP in kinetic experiments. Depending on the functional group introduced, the K(i) values varied from 16.5 microM to 1200 microM. In order to rationalize the kinetic results, we determined the crystal structures of the analogs in complex with GP. All the inhibitors bound at the catalytic site of the enzyme, by making direct and water-mediated hydrogen bonds with the protein and by inducing minor movements of the side chains of Asp283 and Asn284, of the 280s loop that blocks access of the substrate glycogen to the catalytic site, and changes in the water structure in the vicinity of the site. The differences observed in the Ki values of the analogs can be interpreted in terms of variations in hydrogen bonding and van der Waals interactions, desolvation effects, ligand conformational entropy, and displacement of water molecules on ligand binding to the catalytic site.

Investigating flavonoids as molecular templates for the design of small-molecule inhibitors of cell signaling

Epidemiological and clinical trials reveal compelling evidence for the ability of dietary flavonoids to lower cardiovascular disease risk. The mechanisms of action of these polyphenolic compounds are diverse, and of particular interest is their ability to function as protein and lipid kinase inhibitors. We have previously described structure-activity studies that reinforce the possibility for using flavonoid structures as templates for drug design. In the present study, we aim to begin constructing rational screening strategies for exploiting these compounds as templates for the design of clinically relevant, antiplatelet agents. We used the platelet as a model system to dissect the structural influence of flavonoids, stilbenes, anthocyanidins, and phenolic acids on inhibition of cell signaling and function. Functional groups identified as relevant for potent inhibition of platelet function included at least 2 benzene rings, a hydroxylated B ring, a planar C ring, a C ring ketone group, and a C-2 positioned B ring. Hydroxylation of the B ring with either a catechol group or a single C-4' hydroxyl may be required for efficient inhibition of collagen-stimulated tyrosine phosphorylated proteins of 125 to 130 kDa, but may not be necessary for that of phosphotyrosine proteins at approximately 29 kDa. The removal of the C ring C-3 hydroxyl together with a hydroxylated B ring (apigenin) may confer selectivity for 37 to 38 kDa phosphotyrosine proteins. We conclude that this study may form the basis for construction of maps of flavonoid inhibitory activity on kinase targets that may allow a multitargeted therapeutic approach with analogue counterparts and parent compounds.

The imminent availability of a potent weight-loss medicine in pharmacies: public perception of effectiveness

Orlistat is an effective weight-loss medicine, which will soon be available for purchase in pharmacies. We used a factorial experiment and found that informing people about the availability for purchase of this medicinal product previously only available on prescription resulted in higher ratings of perceived value and effectiveness compared to a natural health supplement even though we used the same statement about effectiveness. This positive perception of orlistat was not impaired by the provision of side-effect information. Orlistat will soon be available in pharmacies. Health professionals must act to prevent its misuse by those not overweight.

Dry matter intake is decreased more by abomasal infusion of unsaturated free fatty acids than by unsaturated Triglycerides

Previous experiments from our group have demonstrated that abomasal infusion of unsaturated free fatty acids (FFA) markedly decreases dry matter intake (DMI) in dairy cows. In contrast, experiments from other groups have noted smaller decreases in DMI when unsaturated triglycerides (TG) were infused postruminally. Our hypothesis was that unsaturated FFA would be more potent inhibitors of DMI than an equivalent amount of unsaturated TG. Four Holstein cows in late lactation were used in a single reversal design. Cows were fed a total mixed ration containing (DM basis) 23% alfalfa silage, 23% corn silage, 40.3% ground shelled corn, and 10.5% soybean meal. Two cows received soy FFA (UFA; 0, 200, 400, 600 g/d) and 2 received soy oil (TG) in the same amounts; cows then were switched to the other lipid source. Cows were abomasally infused with each amount for 5-d periods. The daily amount of lipid was pulse-dosed in 4 equal portions at 0600, 1000, 1700, and 2200 h; no emulsifiers were used and there was no sign of digestive disturbance. Both lipid sources linearly decreased DMI, with a significant interaction between lipid source and amount. Slope-ratio analysis indicated that UFA were about 2 times more potent in decreasing DMI than were TG. Decreased DMI led to decreased milk production. Milk fat content was increased linearly by lipid infusion. Milk fat yield decreased markedly for UFA infusion but was relatively unaffected by infusion of TG. Contents of short- and medium-chain fatty acids in milk fat decreased as the amount of either infusate increased. Contents of C-18:2 and C18: 3 in milk fat were increased linearly by abomasal infusion of either fat source; cis-9 C-18:1 was unaffected. Transfer of infused C18: 2 to milk fat was 35.6, 42.5, and 27.8% for 200, 400, and 600 g/d of UFA, and 34.3, 39.6, and 34.0% for respective amounts of TG. Glucagon-like peptide-1 (7-36) amide (GLP-1) concentration in plasma significantly increased as DMI decreased with increasing infusion amount of UFA or TG. Plasma concentration of cholecystokinin-octapeptide (CCK-8) was unaffected by lipid infusion. These results indicate that unsaturated FFA reaching the duodenum are more potent inhibitors of DMI than are unsaturated TG; the effect may be at least partially mediated by GLP-1.

Modelling heterotachy in phylogenetic inference by reversible-jump Markov chain Monte Carlo

The rate at which a given site in a gene sequence alignment evolves over time may vary. This phenomenon-known as heterotachy-can bias or distort phylogenetic trees inferred from models of sequence evolution that assume rates of evolution are constant. Here, we describe a phylogenetic mixture model designed to accommodate heterotachy. The method sums the likelihood of the data at each site over more than one set of branch lengths on the same tree topology. A branch-length set that is best for one site may differ from the branch-length set that is best for some other site, thereby allowing different sites to have different rates of change throughout the tree. Because rate variation may not be present in all branches, we use a reversible-jump Markov chain Monte Carlo algorithm to identify those branches in which reliable amounts of heterotachy occur. We implement the method in combination with our 'pattern-heterogeneity' mixture model, applying it to simulated data and five published datasets. We find that complex evolutionary signals of heterotachy are routinely present over and above variation in the rate or pattern of evolution across sites, that the reversible-jump method requires far fewer parameters than conventional mixture models to describe it, and serves to identify the regions of the tree in which heterotachy is most pronounced. The reversible-jump procedure also removes the need for a posteriori tests of 'significance' such as the Akaike or Bayesian information criterion tests, or Bayes factors. Heterotachy has important consequences for the correct reconstruction of phylogenies as well as for tests of hypotheses that rely on accurate branch-length information. These include molecular clocks, analyses of tempo and mode of evolution, comparative studies and ancestral state reconstruction. The model is available from the authors' website, and can be used for the analysis of both nucleotide and morphological data.

Inference on microsatellite mutation processes in the invasive mite, Varroa destructor, using reversible jump Markov chain Monte Carlo

Varroa destructor is a parasitic mite of the Eastern honeybee Apis cerana. Fifty years ago, two distinct evolutionary lineages (Korean and Japanese) invaded the Western honeybee Apis mellifera. This haplo-diploid parasite species reproduces mainly through brother sister matings, a system which largely favors the fixation of new mutations. In a worldwide sample of 225 individuals from 21 locations collected on Western honeybees and analyzed at 19 microsatellite loci, a series of de novo mutations was observed. Using historical data concerning the invasion, this original biological system has been exploited to compare three mutation models with allele size constraints for microsatellite markers: stepwise (SMM) and generalized (GSM) mutation models, and a model with mutation rate increasing exponentially with microsatellite length (ESM). Posterior probabilities of the three models have been estimated for each locus individually using reversible jump Markov Chain Monte Carlo. The relative support of each model varies widely among loci, but the GSM is the only model that always receives at least 9% support, whatever the locus. The analysis also provides robust estimates of mutation parameters for each locus and of the divergence time of the two invasive lineages (67,000 generations with a 90% credibility interval of 35,000-174,000). With an average of 10 generations per year, this divergence time fits with the last post-glacial Korea Japan land separation. (c) 2005 Elsevier Inc. All rights reserved.

Bayesian analysis of correlated evolution of discrete characters by reversible-jump Markov chain Monte Carlo

We describe a Bayesian method for investigating correlated evolution of discrete binary traits on phylogenetic trees. The method fits a continuous-time Markov model to a pair of traits, seeking the best fitting models that describe their joint evolution on a phylogeny. We employ the methodology of reversible-jump ( RJ) Markov chain Monte Carlo to search among the large number of possible models, some of which conform to independent evolution of the two traits, others to correlated evolution. The RJ Markov chain visits these models in proportion to their posterior probabilities, thereby directly estimating the support for the hypothesis of correlated evolution. In addition, the RJ Markov chain simultaneously estimates the posterior distributions of the rate parameters of the model of trait evolution. These posterior distributions can be used to test among alternative evolutionary scenarios to explain the observed data. All results are integrated over a sample of phylogenetic trees to account for phylogenetic uncertainty. We implement the method in a program called RJ Discrete and illustrate it by analyzing the question of whether mating system and advertisement of estrus by females have coevolved in the Old World monkeys and great apes.

Cyclins, cyclin-dependent kinases, and cyclin-dependent kinase inhibitors: detection methods and activity measurements

The cyclin/cyclin-dependent kinase (Cdk) complexes and the Cdk inhibitors (CDKI) are crucial regulators of cell cycle progression in all eukaryotic cells. Using rat cardiac myocytes as a model system, this chapter provides a detailed account of methods that can be employed to measure both cyclin/Cdk activity in cells and the extent of CDKI inhibitory activity present in a particular cell type.

Reversible phase variation in the phnE gene, which is required for phosphonate metabolism in Escherichia coli K-12

It is known that Escherichia coli K-12 is cryptic (Phn(-)) for utilization of methyl phosphonate (MePn) and that Phn(+) variants can be selected for growth on MePn as the sole P source. Variants arise from deletion via a possible slip strand mechanism of one of three direct 8-bp repeat sequences in phnE, which restores function to a component of a putative ABC type transporter. Here we show that Phn(+) variants are present at the surprisingly high frequency of >10(-2) in K-12 strains. Amplified-fragment length polymorphism analysis was used to monitor instability in phnE in various strains growing under different conditions. This revealed that, once selection for growth on MePn is removed, Phn(+) revertants reappear and accumulate at high levels through reinsertion of the 8-bp repeat element sequence. It appears that, in K-12, phnE contains a high-frequency reversible gene switch, producing phase variation which either allows ("on" form) or blocks ("off" form) MePn utilization. The switch can also block usage of other metabolizable alkyl phosphonates, including the naturally occurring 2-aminoethylphosphonate. All K-12 strains, obtained from collections, appear in the "off" form even when bearing mutations in mutS, mutD, or dnaQ which are known to enhance slip strand events between repetitive sequences. The ability to inactivate the phnE gene appears to be unique to K-12 strains since the B strain is naturally Phn(+) and lacks the inactivating 8-bp insertion in phnE, as do important pathogenic strains for which genome sequences are known and also strains isolated recently from environmental sources.

The role of polyphenolic compounds in the diet as inhibitors of platelet function

Platelets play a substantial role in cardiovascular disease, and for many years there has been a search for dietary components that are able to inhibit platelet function and therefore decrease the risk of cardiovascular disease. Platelets can be inhibited by alcohol, dietary fats and some antioxidants, including a group of compounds, the polyphenols, found in fruits and vegetables. A number of these compounds have been shown to inhibit platelet function both in vitro and in vivo. In the present study the effects of the hydroxycinnamates and the flavonoid quercetin on platelet activation and cell signalling in vitro were investigated. The hydroxycinnamates inhibited platelet function, although not at levels that can be achieved in human plasma by dietary intervention. However, quercetin inhibited platelet aggregation at levels lower than those previously reported. Quercetin was also found to inhibit intracellular Ca mobilisation and whole-cell tyrosine protein phosphorylation in platelets, which are both processes essential for platelet activation. The effect of polyphenols on platelet aggregation in vivo was also investigated. Twenty subjects followed a low-polyphenol diet for 3 d before and also during supplementation. All subjects were supplemented with a polyphenol-rich meal every lunchtime for 5 d. Platelet aggregation and plasma flavonols were measured at baseline and after 5 d of dietary supplementation. Total plasma flavonoids increased significantly after the dietary intervention period (P = 0.001). However, no significant changes in ex vivo platelet aggregation were observed. Further investigation of the effects of individual polyphenolic compounds on platelet function, both in vitro and in vivo, is required in order to elucidate their role in the relationship between diet and the risk of cardiovascular disease.

Reversible, nondegradative conversion of crystalline aromatic poly(ether ketone)s into organo-soluble poly(ether dithioketal)s

Crystalline aromatic poly(ether ketone)s Such as PEEK and PEK may be cleanly and reversibly derivatized by dithioketalization of the carbonyl groups With 1,2-ethanedithiol or 1,3-propanedithiol under strong acid conditions. The resulting 1,3-dithiolane and 1,3-dithiane polymers are hydrolytically stable, amorphous, and readily soluble in organic solvents such as chloroform and THF and are thus (unlike their parent polymers) easily characterized by gel permeation chromatography (GPC). GPC analysis of a range of derivatized PEEK samples using light-scattering detection revealed, in some instances, a bimodal molecular weight distribution with a small but potentially significant (and previously undetected) very high-molecular-weight fraction.

Tin and tin-titanium as catalyst components for reversible hydrogen storage of sodium aluminium hydride

This paper is concerned with the effects of adding tin and/or titanium dopant to sodium aluminium hydride for both dehydrogenation and re-hydrogenation reactions during their reversible storage of molecular hydrogen. Temperature programmed decomposition (TPD) measurements show that the dehydrogenation kinetics of NaAlH4 are significantly enhanced upon doping the material with 2 mol% of tributyltin hydride, Sn(Bu)(3)H but the tin catalyst dopant is shown to be inferior than titanium. On the other hand, in this preliminary work, a significant synergetic catalytic effect is clearly revealed in material co-doped with both titanium and tin catalysts which shows the highest reversible rates of dehydrogenation and re-hydrogenation (after their hydrogen depletion). The re-hydrogenation rates of depleted Sn/Ti/NaAlH4 evaluated at both 9.5 and 140 bars hydrogen are also found to be favourable compared to the Ti/NaAlH4, which clearly suggest the importance of the catalyst choice. Basing on these results some mechanistic insights for the catalytic reversible dehydrogenation and re-hydrogenation processes of Sn/Ti/NaAlH4 are therefore made. (C) 2006 Elsevier Ltd. All rights reserved.

Application of the asymmetric hetero Diels-Alder reaction for synthesising carbohydrate derivatives and glycosidase inhibitors

This review provides a discussion of recent developments in the asymmetric hetero Diels-Alder reaction (AHDAR), with particular emphasis on the synthesis of carbohydrates, their derivatives, and inhibitors of carbohydrate processing enzymes.