Mode of Peroxisome Proliferator-Activated Receptor gamma Activation by Luteolin

The peroxisome proliferator-activated receptor gamma (PPAR gamma) is a target for treatment of type II diabetes and other conditions. PPAR gamma full agonists, such as thiazolidinediones (TZDs), are effective insulin sensitizers and anti-inflammatory agents, but their use is limited by adverse side effects. Luteolin is a flavonoid with anti-inflammatory actions that binds PPAR gamma but, unlike TZDs, does not promote adipocyte differentiation. However, previous reports suggested variously that luteolin is a PPAR gamma agonist or an antagonist. We show that luteolin exhibits weak partial agonist/antagonist activity in transfections, inhibits several PPAR gamma target genes in 3T3-L1 cells (LPL, ORL1, and CEBP alpha) and PPAR gamma-dependent adipogenesis, but activates GLUT4 to a similar degree as rosiglitazone, implying gene-specific partial agonism. The crystal structure of the PPAR gamma ligand-binding domain (LBD) reveals that luteolin occupies a buried ligand-binding pocket (LBP) but binds an inactive PPAR gamma LBD conformer and occupies a space near the beta-sheet region far from the activation helix (H12), consistent with partial agonist/antagonist actions. A single myristic acid molecule simultaneously binds the LBP, suggesting that luteolin may cooperate with other ligands to bind PPAR gamma, and molecular dynamics simulations show that luteolin and myristic acid cooperate to stabilize the Omega-loop among H2', H3, and the beta-sheet region. It is noteworthy that luteolin strongly suppresses hypertonicity-induced release of the pro-inflammatory interleukin-8 from human corneal epithelial cells and reverses reductions in transepithelial electrical resistance. This effect is PPAR gamma-dependent. We propose that activities of luteolin are related to its singular binding mode, that anti-inflammatory activity does not require H12 stabilization, and that our structure can be useful in developing safe selective PPAR gamma modulators.

Switching the mode of sucrose utilization by Saccharomyces cerevisiae

Abstract Background Overflow metabolism is an undesirable characteristic of aerobic cultures of Saccharomyces cerevisiae during biomass-directed processes. It results from elevated sugar consumption rates that cause a high substrate conversion to ethanol and other bi-products, severely affecting cell physiology, bioprocess performance, and biomass yields. Fed-batch culture, where sucrose consumption rates are controlled by the external addition of sugar aiming at its low concentrations in the fermentor, is the classical bioprocessing alternative to prevent sugar fermentation by yeasts. However, fed-batch fermentations present drawbacks that could be overcome by simpler batch cultures at relatively high (e.g. 20 g/L) initial sugar concentrations. In this study, a S. cerevisiae strain lacking invertase activity was engineered to transport sucrose into the cells through a low-affinity and low-capacity sucrose-H+ symport activity, and the growth kinetics and biomass yields on sucrose analyzed using simple batch cultures. Results We have deleted from the genome of a S. cerevisiae strain lacking invertase the high-affinity sucrose-H+ symporter encoded by the AGT1 gene. This strain could still grow efficiently on sucrose due to a low-affinity and low-capacity sucrose-H+ symport activity mediated by the MALx1 maltose permeases, and its further intracellular hydrolysis by cytoplasmic maltases. Although sucrose consumption by this engineered yeast strain was slower than with the parental yeast strain, the cells grew efficiently on sucrose due to an increased respiration of the carbon source. Consequently, this engineered yeast strain produced less ethanol and 1.5 to 2 times more biomass when cultivated in simple batch mode using 20 g/L sucrose as the carbon source. Conclusion Higher cell densities during batch cultures on 20 g/L sucrose were achieved by using a S. cerevisiae strain engineered in the sucrose uptake system. Such result was accomplished by effectively reducing sucrose uptake by the yeast cells, avoiding overflow metabolism, with the concomitant reduction in ethanol production. The use of this modified yeast strain in simpler batch culture mode can be a viable option to more complicated traditional sucrose-limited fed-batch cultures for biomass-directed processes of S. cerevisiae.

Pharmaceutical residues in aquatic systems: mode of action and effects on mussel physiology

Pharmaceutical residues contaminate aquatic ecosystems as a result of their widespread human and veterinary usage. Since continuously released and not efficiently removed, certain pharmaceuticals exhibit pseudo-persistence thus generating concerns for the health of aquatic wildlife. This work aimed at assessing on mussels Mytilus galloprovincialis, under laboratory conditions, the effects of three pharmaceuticals, carbamazepine (antiepileptic), propranolol (β-blocker) and oxytetracycline (antibiotic), to evaluate if the human-based mode of action of these molecules is conserved in invertebrates. Furthermore, in the framework of the European MEECE Programme, mussels were exposed to oxytetracycline and copper at increasing temperatures, simulating variations due to climate changes. The effects of these compounds were assessed evaluating a battery of biomarkers, the expression of HSP70 proteins and changes in cAMP-related parameters. A decrease in lysosomal membrane stability, induction of oxidative stress, alterations of cAMP-dependent pathway and the induction of defense mechanisms were observed indicating the development of a stress syndrome, and a worsening in mussels health status. Data obtained in MEECE Programme confirmed that the toxicity of substances can be enhanced following changes in temperature. The alterations observed were obtained after exposure to pharmaceuticals at concentrations sometimes lower than those detected in the aquatic environment. Hence, further research is advisable regarding subtle effects of pharmaceuticals on non-target organisms. Furthermore, results obtained during a research stay in the laboratories of Cádiz University (Spain) are presented. The project aimed at measuring possible effects of polluted sediments in Algeciras Bay (Spain) and in Cádiz Bay, by assessing different physiological parameters in caged crabs Carcinus maenas and clams Ruditapes decussatus exposed in situ for 28 days. The neutral red retention assay was adapted to these species and proved to be a sensitive screening tool for the assessment of sediment quality.

Methods and strategies to identify the mode of action of phytoactive compounds

Small molecules affecting biological processes in plants are widely used in agricultural practice as herbicides or plant growth regulators and in basic plant sciences as probes to study the physiology of plants. Most of the compounds were identified in large screens by the agrochemical industry, as phytoactive natural products and more recently, novel phytoactive compounds originated from academic research by chemical screens performed to induce specific phenotypes of interest. The aim of the present PhD thesis is to evaluate different approaches used for the identification of the primary mode of action (MoA) of a phytoactive compound. Based on the methodologies used for MoA identification, three approaches are discerned: a phenotyping approach, an approach based on a genetic screen and a biochemical screening approach.rnFour scientific publications resulting from my work are presented as examples of how a phenotyping approach can successfully be applied to describe the plant MoA of different compounds in detail.rnI. A subgroup of cyanoacrylates has been discovered as plant growth inhibitors. A set of bioassays indicated a specific effect on cell division. Cytological investigations of the cell division process in plant cell cultures, studies of microtubule assembly with green fluorescent protein marker lines in vivo and cross resistant studies with Eleusine indica plants harbouring a mutation in alpha-tubulin, led to the description of alpha-tubulin as a target site of cyanoacrylates (Tresch et al., 2005).rnII. The MoA of the herbicide flamprop-m-methyl was not known so far. The studies described in Tresch et al. (2008) indicate a primary effect on cell division. Detailed studies unravelled a specific effect on mitotic microtubule figures, causing a block in cell division. In contrast to other inhibitors of microtubule rearrangement such as dinitroanilines, flamprop-m-methyl did not influence microtubule assembly in vitro. An influence of flamprop-m-methyl on a target within the cytoskeleton signalling network could be proposed (Tresch et al., 2008).rnIII. The herbicide endothall is a protein phosphatase inhibitor structurally related to the natural product cantharidin. Bioassay studies indicated a dominant effect on dark-growing cells that was unrelated to effects observed in the light. Cytological characterisation of the microtubule cytoskeleton in corn tissue and heterotrophic tobacco cells showed a specific effect of endothall on mitotic spindle formation and ultrastructure of the nucleus in combination with a decrease of the proliferation index. The observed effects are similar to those of other protein phosphatase inhibitors such as cantharidin and the structurally different okadaic acid. Additionally, the observed effects show similarities to knock-out lines of the TON1 pathway, a protein phosphatase-regulated signalling pathway. The data presented in Tresch et al. (2011) associate endothall’s known in vitro inhibition of protein phosphatases with in vivo-effects and suggest an interaction between endothall and the TON1 pathway.rnIV. Mefluidide as a plant growth regulator induces growth retardation and a specific phenotype indicating an inhibition of fatty acid biosynthesis. A test of the cuticle functionality suggested a defect in the biosynthesis of very-long-chain fatty acids (VLCFA) or waxes. Metabolic profiling studies showed similarities with different groups of VLCFA synthesis inhibitors. Detailed analyses of VLCFA composition in tissues of duckweed (Lemna paucicostata) indicated a specific inhibition of the known herbicide target 3 ketoacyl-CoA synthase (KCS). Inhibitor studies using a yeast expression system established for plant KCS proteins verified the potency of mefluidide as an inhibitor of plant KCS enzymes. It could be shown that the strength of inhibition varied for different KCS homologues. The Arabidopsis Cer6 protein, which induces a plant growth phenotype similar to mefluidide when knocked out, was one of the most sensitive KCS enzymes (Tresch et al., 2012).rnThe findings of my own work were combined with other publications reporting a successful identification of the MoA and primary target proteins of different compounds or compound classes.rnA revised three-tier approach for the MoA identification of phytoactive compounds is proposed. The approach consists of a 1st level aiming to address compound stability, uniformity of effects in different species, general cytotoxicity and the effect on common processes like transcription and translation. Based on these findings advanced studies can be defined to start the 2nd level of MoA characterisation, either with further phenotypic characterisation, starting a genetic screen or establishing a biochemical screen. At the 3rd level, enzyme assays or protein affinity studies should show the activity of the compound on the hypothesized target and should associate the in vitro effects with the in vivo profile of the compound.

The effect of a slow mode of BMP-2 delivery on the inflammatory response provoked by bone-defect-filling polymeric scaffolds

We investigated the inflammatory response to, and the osteoinductive efficacies of, four polymers (collagen, Ethisorb, PLGA and Polyactive) that bore either an adsorbed (fast-release kinetics) or a calcium-phosphate-coating-incorporated (slow-release kinetics) depot of BMP-2. Titanium-plate-supported discs of each polymer (n = 6 per group) were implanted at an ectopic (subcutaneous) ossification site in rats (n = 48). Five weeks later, they were retrieved for a histomorphometric analysis of the volumes of ectopic bone and foreign-body giant cells (a gauge of inflammatory reactivity), and the degree of polymer degradation. For each polymer, the osteoinductive efficacy of BMP-2 was higher when it was incorporated into a coating than when it was directly adsorbed onto the material. This mode of BMP-2 carriage was consistently associated with an attenuation of the inflammatory response. For coated materials, the volume density of foreign-body giant cells was inversely correlated with the volume density of bone (r(2) = 0.96), and the volume density of bone was directly proportional to the surface-area density of the polymer (r(2) = 0.97). Following coating degradation, other competitive factors, such as the biocompatibility and the biodegradability of the polymer itself, came into play.

A novel mode of action of the putative sphingosine kinase inhibitor 2-(p-hydroxyanilino)-4-(p-chlorophenyl) thiazole (SKI II): induction of lysosomal sphingosine kinase 1 degradation

Sphingosine kinase 1 (SK1) is a key enzyme in the generation of sphingosine 1-phosphate (S1P) which critically regulates a variety of important cell responses such as proliferation and migration. Therefore, inhibition of SK-1 has been suggested to be an attractive approach to treat tumor growth and metastasis formation.

The binding mode of side chain- and C3-modified epothilones to tubulin

The tubulin-binding mode of C3- and C15-modified analogues of epothilone A (Epo A) was determined by NMR spectroscopy and computational methods and compared with the existing structural models of tubulin-bound natural Epo A. Only minor differences were observed in the conformation of the macrocycle between Epo A and the C3-modified analogues investigated. In particular, 3-deoxy- (compound 2) and 3-deoxy-2,3-didehydro-Epo A (3) were found to adopt similar conformations in the tubulin-binding cleft as Epo A, thus indicating that the 3-OH group is not essential for epothilones to assume their bioactive conformation. None of the available models of the tubulin-epothilone complex is able to fully recapitulate the differences in tubulin-polymerizing activity and microtubule-binding affinity between C20-modified epothilones 6 (C20-propyl), 7 (C20-butyl), and 8 (C20-hydroxypropyl). Based on the results of transferred NOE experiments in the presence of tubulin, the isomeric C15 quinoline-based Epo B analogues 4 and 5 show very similar orientations of the side chain, irrespective of the position of the nitrogen atom in the quinoline ring. The quinoline side chain stacks on the imidazole moiety of beta-His227 with equal efficiency in both cases, thus suggesting that the aromatic side chain moiety in epothilones contributes to tubulin binding through strong van der Waals interactions with the protein rather than hydrogen bonding involving the heteroaromatic nitrogen atom. These conclusions are in line with existing tubulin polymerization and microtubule-binding data for 4, 5, and Epo B.

Fluorides - mode of action and recommendations for use

Summary Various authors have shown that the caries decline in the industrialized countries during recent decades is based on the use of fluorides, of which local fluoride application in the form of fluoridated toothpastes is of primary importance. The caries-protective potential of fluorapatite is quite low; in contrast, dissolved fluorides in the vicinity of enamel are effective both in promoting remineralization and inhibiting demineralization. Considering the fact that the caries decline occurred at the same time that local fluoridation measures became widely used, the conclusion seems justified that regular application of F⁻ can inhibit caries.

First report about the mode of action of combined butafosfan and cyanocobalamin on hepatic metabolism in nonketotic early lactating cows

The primary aim was to investigate the effect of combined butafosfan and cyanocobalamin on liver metabolism in early lactating cows through mRNA expression measurements of genes encoding 31 enzymes and transport proteins of major metabolic processes in the liver using 16 multiparous early lactating dairy cows. The treatments included i.v. injection of 10 mL/100 kg of body weight combined butafosfan and cyanocobalamin (TG, n = 8) on 3 d consecutively at 25 +/- 3 d in milk or injection with physiological saline solution similarly applied (CG, n = 8). Results include a higher daily milk production for TG cows (41.1 +/- 0.9 kg, mean +/- SEM) compared with CG cows (39.5 +/- 0.7 kg). In plasma, the concentration of inorganic phosphorus was lower in the TG cows (1.25 +/- 0.08 mmol/L) after the treatment than in the CG cows (1.33 +/- 0.07 mmol/L). The plasma beta-hydroxybutyrate concentration was 0.65 +/- 0.13 mmol/L for all cows before the treatment, and remained unaffected post treatment. The unique result was that in the liver, the mRNA abundance of acyl-coenzyme A synthetase long-chain family member 1, involved in fatty acid oxidation and biosynthesis, was lower across time points after the treatment for TG compared with CG cows (17.5 +/- 0.15 versus 18.1 +/- 0.24 cycle threshold, log(2), respectively). In conclusion, certain effects of combined butafosfan and cyanocobalamin were observed on mRNA abundance of a gene in the liver of nonketotic early lactating cows.

The influence of BMP-2 and its mode of delivery on the osteoconductivity of implant surfaces during the early phase of osseointegration

Osteogenic agents, such as bone morphogenetic protein-2 (BMP-2), can stimulate the degradation as well as the formation of bone. Hence, they could impair the osteoconductivity of functionalized implant surfaces. We assessed the effects of BMP-2 and its mode of delivery on the osteoconductivity of dental implants with either a naked titanium surface or a calcium-phosphate-coated one. The naked titanium surface bore adsorbed BMP-2, whilst the coated one bore incorporated, adsorbed, or incorporated and adsorbed BMP-2. The implants were inserted into the maxillae of adult miniature pigs. The volume of bone deposited within a defined "osteoconductive" (peri-implant) space, and bone coverage of the implant surface delimiting this space, were estimated morphometrically 1-3 weeks later. After 3 weeks, the volume of bone deposited within the osteoconductive space was highest for coated and uncoated implants bearing no BMP-2, followed by coated implants bearing incorporated BMP-2; it was lowest for coated implants bearing only adsorbed BMP-2. Bone-interface coverage was highest for coated implants bearing no BMP-2, followed by coated implants bearing either incorporated, or incorporated and adsorbed BMP-2; it was lowest for uncoated implants bearing adsorbed BMP-2. Hence, the osteoconductivity of implant surfaces can be significantly modulated by BMP-2 and its mode of delivery.

The Social Dynamics of Communal Violence in India

Contrasting strands of explanation of the motives underlying collective action, as either culturally determined, as an attempt at compensation, point towards an understanding of identity politics as a reaction to given conditions. They pay little attention to the social dynamics that evolve in relation to the conflict within a group, and the possible motivation that can ensue from these. This article analyses the mobilisation among Hindu-nationalist organisations. Rather than seeking their attraction in their discursive outputs and the possible answers they might give in times of change, the contention is that they are to be sought in the specific internal dynamics and the possibilities they create within their historical context. These specific opportunities for action are inherent firstly in the mode of operation relying on participation and involvement, on their direct intervention, their localness and accessibility. Moreover, the dichotomisation inherent in violence makes possible the integration of different interests and different discontents under one banner and therefore contributes to the project of unification undertaken by Hindu-nationalism.

Do N-arachidonyl-glycine (NA-glycine) and 2-arachidonoyl glycerol (2-AG) share mode of action and the binding site on the β 2 subunit of GABA A receptors?

NA-glycine is an endogenous lipid molecule with analgesic properties, which is structurally similar to the endocannabinoids 2-AG and anandamide but does not interact with cannabinoid receptors. NA-glycine has been suggested to act at the G-protein coupled receptors GPR18 and GPR92. Recently, we have described that NA-glycine can also modulate recombinant α1β2γ2 GABAA receptors. Here we characterize in more detail this modulation and investigate the relationship of its binding site with that of the endocannabinoid 2-AG.

The Cytotoxic Mode of Action of the Venom of Cupiennius salei (Ctenidae)

The venom of the ctenid spider Cupiennius salei (Fig.16.1) is rich in components which belong to different functional groups. Besides low molecular mass compounds, the venom contains several disulphide-rich peptides, also called mini-proteins, which act as neurotoxins on ion channels or as enhancers of neurotoxins. Likewise, a variety of small cytolytic peptides, which destroy membranes very efficiently, and enzymes are present in the venom. Neurotoxins with cytolytic activity, cytolytic a-helical small cationic peptides and enzymes most probably attacking connective tissue and phospholipid membranes cause the overall cytotoxic effect of this venom. Synergistic and enhancing interactions between components enable the spider to achieve a maximum of toxicity with a minimum of venom quantity.

Hebbian analysis of the transformation of medial entorhinal grid-cell inputs to hippocampal place fields.

The discovery of grid cells in the medial entorhinal cortex (MEC) permits the characterization of hippocampal computation in much greater detail than previously possible. The present study addresses how an integrate-and-fire unit driven by grid-cell spike trains may transform the multipeaked, spatial firing pattern of grid cells into the single-peaked activity that is typical of hippocampal place cells. Previous studies have shown that in the absence of network interactions, this transformation can succeed only if the place cell receives inputs from grids with overlapping vertices at the location of the place cell's firing field. In our simulations, the selection of these inputs was accomplished by fast Hebbian plasticity alone. The resulting nonlinear process was acutely sensitive to small input variations. Simulations differing only in the exact spike timing of grid cells produced different field locations for the same place cells. Place fields became concentrated in areas that correlated with the initial trajectory of the animal; the introduction of feedback inhibitory cells reduced this bias. These results suggest distinct roles for plasticity of the perforant path synapses and for competition via feedback inhibition in the formation of place fields in a novel environment. Furthermore, they imply that variability in MEC spiking patterns or in the rat's trajectory is sufficient for generating a distinct population code in a novel environment and suggest that recalling this code in a familiar environment involves additional inputs and/or a different mode of operation of the network.