Binding model for eriodictyol to Jun-N terminal kinase and its anti-inflammatory signaling pathway

The anti-inflammatory activity of eriodictyol and its mode of action were investigated. Eriodictyol suppressed tumor necrosis factor (mTNF)-α, inducible nitric oxide synthase (miNOS), interleukin (mIL)-6, macrophage inflammatory protein (mMIP)-1, and mMIP-2 cytokine release in LPS-stimulated macrophages. We found that the anti-inflammatory cascade of eriodictyol is mediated through the Toll-like Receptor (TLR)4/CD14, p38 mitogen-activated protein kinases (MAPK), extracellular-signalregulated kinase (ERK), Jun-N terminal kinase (JNK), and cyclooxygenase (COX)-2 pathway. Fluorescence quenching and saturation-transfer difference (STD) NMR experiments showed that eriodictyol exhibits good binding affinity to JNK, 8.79 × 10⁵ M^-1. Based on a docking study, we propose a model of eriodictyol and JNK binding, in which eriodictyol forms 3 hydrogen bonds with the side chains of Lys55, Met111, and Asp169 in JNK, and in which the hydroxyl groups of the B ring play key roles in binding interactions with JNK. Therefore, eriodictyol may be a potent anti-inflammatory inhibitor of JNK. [BMB Reports 2013; 46(12): 594-599]     

Association study of 37 genes related to serotonin and dopamine neurotransmission and neurotrophic factors in cocaine dependence

Cocaine dependence is a neuropsychiatric disorder in which both environmental and genetic factors are involved. Several processes, that include reward and neuroadaptations, mediate the transition from use to dependence. In this regard, dopamine and serotonin neurotransmission systems are clearly involved in reward and other cocaine‐related effects, whereas neurotrophic factors may be responsible for neuroadaptations associated with cocaine dependence. We examined the contribution to cocaine dependence of 37 genes related to the dopaminergic and serotoninergic systems, neurotrophic factors and their receptors through a case–control association study with 319 single nucleotide polymorphisms selected according to genetic coverage criteria in 432 cocaine‐dependent patients and 482 sex‐matched unrelated controls. Single marker analyses provided evidence for association of the serotonin receptor HTR2A with cocaine dependence [rs6561333; nominal P‐value adjusted for age = 1.9e?04, odds ratio = 1.72 (1.29–2.30)]. When patients were subdivided according to the presence or absence of psychotic symptoms, we confirmed the association between cocaine dependence and HTR2A in both subgroups of patients. Our data show additional evidence for the involvement of the serotoninergic system in the genetic susceptibility to cocaine dependence.     

A study of the possible association between adenosine A2A receptor gene polymorphisms and attention‐deficit hyperactivity disorder traits

The adenosine A_2A receptor (ADORA2A) is linked to the dopamine neurotransmitter system and is also implicated in the regulation of alertness, suggesting a potential association with attention‐deficit hyperactivity disorder (ADHD) traits. Furthermore, animal studies suggest that the ADORA2A may influence ADHD‐like behavior. For that reason, the ADORA2A gene emerges as a promising candidate for studying the etiology of ADHD traits. The aim of this study was to examine the relationship between ADORA2A gene polymorphisms and ADHD traits in a large population‐based sample. This study was based on the Child and Adolescent Twin Study in Sweden (CATSS), and included 1747 twins. Attention‐deficit hyperactivity disorder traits were assessed through parental reports, and samples of DNA were collected. Associations between six single nucleotide polymorphisms (SNPs) and ADHD traits were examined, and results suggested a nominal association between ADHD traits and three of these SNPs: rs3761422, rs5751876 and rs35320474. For one of the SNPs, rs35320474, results remained significant after correction for multiple comparisons. These results indicate the possibility that the ADORA2A gene may be involved in ADHD traits. However, more studies replicating the present results are warranted before this association can be confirmed .     

Apolar distal pocket mutants of yeast cytochrome c peroxidase: Hydrogen peroxide reactivity and cyanide binding of the TriAla,TriVal, and TriLeu variants

Three yeast cytochrome c peroxidase (CcP) variants with apolar distal heme pockets have been constructed. The CcP variants have Arg48, Trp51, and His52 mutated to either all alanines, CcP(triAla), all valines, CcP(triVal), or all leucines, CcP(triLeu). The triple mutants have detectable enzymatic activity at pH 6 but the activity is less than 0.02% that of wild-type CcP. The activity loss is primarily due to the decreased rate of reaction between the triple mutants and H₂O₂ compared to wild-type CcP. Spectroscopic properties and cyanide binding characteristics of the triple mutants have been investigated over the pH stability region of CcP, pH 4 to 8. The absorption spectra indicate that the CcP triple mutants have hemes that are predominantly five-coordinate, high-spin at pH 5 and six-coordinate, low-spin at pH 8. Cyanide binding to the triple mutants is biphasic indicating that the triple mutants have two slowly-exchanging conformational states with different cyanide affinities. The binding affinity for cyanide is reduced at least two orders of magnitude in the triple mutants compared to wild-type CcP and the rate of cyanide binding is reduced by four to five orders of magnitude. Correlation of the reaction rates of CcP and 12 distal pocket mutants with H₂O₂ and HCN suggests that both reactions require ionization of the reactants within the distal heme pocket allowing the anion to bind the heme iron. Distal pocket features that promote substrate ionization (basic residues involved in base-catalyzed substrate ionization or polar residues that can stabilize substrate anions) increase the overall rate of reaction with H₂O₂ and HCN while features that inhibit substrate ionization slow the reactions.     

Identification and characterization of bi-thiazole-2,2′-diamines as kinase inhibitory scaffolds

Based on bioinformatics interrogation of the genome, > 500 mammalian protein kinases can be clustered within seven different groups. Of these kinases, the mitogen-activated protein kinase (MAPK) family forms part of the CMGC group of serine/threonine kinases that includes extracellular signal regulated kinases (ERKs), cJun N-terminal kinases (JNKs), and p38 MAPKs. With the JNKs considered attractive targets in the treatment of pathologies including diabetes and stroke, efforts have been directed to the discovery of new JNK inhibitory molecules that can be further developed as new therapeutics. Capitalizing on our biochemical understanding of JNK, we performed in silico screens of commercially available chemical databases to identify JNK1-interacting compounds and tested their in vitro JNK inhibitory activity. With in vitro and cell culture studies, we showed that the compound, 4′-methyl-N²-3-pyridinyl-4,5′-bi-1,3-thiazole-2,2′-diamine (JNK Docking (JD) compound 123, but not the related compound (4′-methyl-N ~ 2 ~ -(6-methyl-2-pyridinyl)-4,5′-bi-1,3-thiazole-2,2′-diamine (JD124), inhibited JNK1 activity towards a range of substrates. Molecular docking, saturation transfer difference NMR experiments and enzyme kinetic analyses revealed both ATP- and substrate-competitive inhibition of JNK by JD123. In characterizing JD123 further, we noted its ATP-competitive inhibition of the related p38-γ MAPK, but not ERK1, ERK2, or p38-α, p38-β or p38-δ. Further screening of a broad panel of kinases using 10 μM JD123, identified inhibition of kinases including protein kinase Bβ (PKBβ/Aktβ). Appropriately modified thiazole diamines, as typified by JD123, thus provide a new chemical scaffold for development of inhibitors for the JNK and p38-γ MAPKs as well as other kinases that are also potential therapeutic targets such as PKBβ/Aktβ.     

Genome-wide association study for feed efficiency in collective cage-raised rabbits under full and restricted feeding

Feed efficiency (FE) is one of the most economically and environmentally relevant traits in the animal production sector. The objective of this study was to gain knowledge about the genetic control of FE in rabbits. To this end, GWASs were conducted for individual growth under two feeding regimes (full feeding and restricted) and FE traits collected from cage groups, using 114 604 autosome SNPs segregating in 438 rabbits. Two different models were implemented: (1) an animal model with a linear regression on each SNP allele for growth trait; and (2) a two-trait animal model, jointly fitting the performance trait and each SNP allele content, for FE traits. This last modeling strategy is a new tool applied to GWAS and allows information to be considered from non-genotyped individuals whose contribution is relevant in the group average traits. A total of 189 SNPs in 17 chromosomal regions were declared to be significantly associated with any of the five analyzed traits at a chromosome-wide level. In 12 of these regions, 20 candidate genes were proposed to explain the variation of the analyzed traits, including genes such as FTO, NDUFAF6 and CEBPA previously associated with growth and FE traits in monogastric species. Candidate genes associated with behavioral patterns were also identified. Overall, our results can be considered as the foundation for future functional research to unravel the actual causal mutations regulating growth and FE in rabbits.     

Synthesis,DNA/BSA binding studies and in vitro biological assay of nickel(II) complexes incorporating tridentate aroylhydrazone and triphenylphosphine ligands

Abstract

Two new nickel (II) triphenylphosphine complexes derived from tridentate aroylhydrazone ligands [H₂L¹ = 2-hydroxy-3-methoxybenzylidene)benzohydrazone and H₂L² = N′-(2-hydroxy-3-methoxybenzylidene)-2-hydroxybenzoylhydrazone] and triphenylphosphine were prepared and their molecular structures were determined by single crystal X-ray diffraction analysis. Both nickel(II) complexes showed slightly distorted square planar geometry with one tridentate aroylhydrazone ligand coordinated through ONO donor atoms and one triphenylphosphine ligand coordinated to the nickel center through the phosphorus atom. DNA interaction studies indicated that both complexes possessed higher affinity to herring sperm DNA (HS-DNA) than the corresponding free aroylhydrazone ligand. Molecular docking investigations showed that both complexes could bind to DNA through intercalation of the phenyl rings between adjacent base pairs in the double helix. Meanwhile, bovine serum albumin (BSA) binding studies revealed the complexes could effectively interact with BSA and change the secondary structure of BSA. Further pharmacological evaluations of the synthesized complexes by in vitro antioxidant assays demonstrated high antioxidant activity against NO· and O₂˙^? radicals. The anticancer activity of each complex was assessed through in vitro cytotoxicity assays (CCK-8 kit) toward A549 and MCF-7 cancer cell and normal L-02 cell lines. Significantly, the Ni(II) complex derived from H₂L¹ ligand was found to be more effective cytotoxic toward MCF-7cancerous cell with the IC₅₀ value equaled 9.7?μM, which showed potent cytotoxic activity over standard drug cisplatin.

Abbreviations A549 human lung carcinoma cell

BSA bovine serum albumin

CCK-8 Cell Counting Kit-8

DFT density functional theory

DNA deoxyribonucleic acid

DPPH˙ 2,2-diphenyl-1-picrylhydrazyl

H₂L¹ 2-hydroxy-3-methoxybenzylidene)benzohydrazone N′-(2-hydroxy-3-methoxybenzylidene)-2-hydroxybenzoylhydrazone

H₂L² N′-(2-hydroxy-3-methoxybenzylidene)-2-hydroxybenzoylhydrazone

HOMO highest occupied molecular orbital

IC₅₀ the 50% activity

L-02 human normal liver cell

LOMO lowest unoccupied molecular orbital (LUMO)

MCF-7 human breast carcinoma cell

NO˙ nitric oxide

O₂˙^? superoxide anion

SOD superoxide dismutase

Communicated by Ramaswamy H. Sarma     

Sample size recalculation in multicenter randomized controlled clinical trials based on noncomparative data

Many late-phase clinical trials recruit subjects at multiple study sites. This introduces a hierarchical structure into the data that can result in a power-loss compared to a more homogeneous single-center trial. Building on a recently proposed approach to sample size determination, we suggest a sample size recalculation procedure for multicenter trials with continuous endpoints. The procedure estimates nuisance parameters at interim from noncomparative data and recalculates the sample size required based on these estimates. In contrast to other sample size calculation methods for multicenter trials, our approach assumes a mixed effects model and does not rely on balanced data within centers. It is therefore advantageous, especially for sample size recalculation at interim. We illustrate the proposed methodology by a study evaluating a diabetes management system. Monte Carlo simulations are carried out to evaluate operation characteristics of the sample size recalculation procedure using comparative as well as noncomparative data, assessing their dependence on parameters such as between-center heterogeneity, residual variance of observations, treatment effect size and number of centers. We compare two different estimators for between-center heterogeneity, an unadjusted and a bias-adjusted estimator, both based on quadratic forms. The type 1 error probability as well as statistical power are close to their nominal levels for all parameter combinations considered in our simulation study for the proposed unadjusted estimator, whereas the adjusted estimator exhibits some type 1 error rate inflation. Overall, the sample size recalculation procedure can be recommended to mitigate risks arising from misspecified nuisance parameters at the planning stage.