Proteomic analysis of urine in rats chronically exposed to fluoride

Urine is an ideal source of materials to search for potential disease‐related biomarkers as it is produced by the affected tissues and can be easily obtained by noninvasive methods. 2‐DE‐based proteomic approach was used to better understand the molecular mechanisms of injury induced by fluoride (F^?) and define potential biomarkers of dental fluorosis. Three groups of weanling male Wistar rats were treated with drinking water containing 0 (control), 5, or 50 ppm F^? for 60 days (n = 15/group). During the experimental period, the animals were kept individually in metabolic cages, to analyze the water and food consumption, as well as fecal and urinary F^? excretion. Urinary proteome profiles were examined using 2‐DE and Colloidal Coomassie Brilliant Blue staining. A dose‐response regarding F^? intake and excretion was detected. Quantitative intensity analysis revealed 8, 11, and 8 significantly altered proteins between control vs. 5 ppm F^?, control vs. 50 ppm F^? and 5 ppm F^? vs. 50 ppm F^? groups, respectively. Two proteins regulated by androgens (androgen‐regulated 20‐KDa protein and α‐2μ‐globulin) and one related to detoxification (aflatoxin‐B1‐aldehyde‐reductase) were identified by MALDI‐TOF‐TOF MS/MS. Thus, proteomic analysis can help to better understand the mechanisms underlying F^? toxicity, even in low doses. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 25:8–14 2011; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.20353     

Caucasian and Asian specific rheumatoid arthritis risk loci reveal limited replication and apparent allelic heterogeneity in north Indians

Genome-wide association studies and meta-analysis indicate that several genes/loci are consistently associated with rheumatoid arthritis (RA) in European and Asian populations. To evaluate the transferability status of these findings to an ethnically diverse north Indian population, we performed a replication analysis. We investigated the association of 47 single-nucleotide polymorphisms (SNPs) at 43 of these genes/loci with RA in a north Indian cohort comprising 983 RA cases and 1007 age and gender matched controls. Genotyping was done using Infinium human 660w-quad. Association analysis by chi-square test implemented in plink was carried out in two steps. Firstly, association of the index or surrogate SNP (r2>0.8, calculated from reference GIH Hap-Map population) was tested. In the second step, evidence for allelic/locus heterogeneity at aforementioned genes/loci was assessed for by testing additional flanking SNPs in linkage equilibrium with index/surrogate marker.Of the 44 European specific index SNPs, neither index nor surrogate SNPs were present for nine SNPs in the genotyping array. Of the remaining 35, associations were replicated at seven genes namely PTPN22 (rs1217407, p = 3×10⁻³); IL2–21 (rs13119723, p = 0.008); HLA-DRB1 (rs660895, p = 2.56×10⁻⁵; rs6457617, p = 1.6×10⁻⁰⁹; rs13192471, p = 6.7×10⁻¹⁶); TNFA1P3 (rs9321637, p = 0.03); CCL21 (rs13293020, p = 0.01); IL2RA (rs2104286, p = 1.9×10⁻⁴) and ZEB1 (rs2793108, p = 0.006). Of the three Asian specific loci tested, rs2977227 in PADI4 showed modest association (p<0.02). Further, of the 140 SNPs (in LE with index/surrogate variant) tested, association was observed at 11 additional genes: PTPRC, AFF3, CD28, CTLA4, PXK, ANKRD55, TAGAP, CCR6, BLK, CD40 and IL2RB. This study indicates limited replication of European and Asian index SNPs and apparent allelic heterogeneity in RA etiology among north Indians warranting independent GWAS in this population. However, replicated associations of HLA-DRB1, PTPN22 (which confer ∼50% of the heritable risk to RA) and IL2RA suggest that cross-ethnicity fine mapping of such loci is apposite for identification of causal variants.     

Discovery of 7-arylsulfonyl-1,2,3,4, 4a,9a-hexahydro-benzo[4,5]furo[2,3-c]pyridines: identification of a potent and selective 5-HT₆ receptor antagonist showing activity in rat social recognition test

Serotoninergic neurotransmission has been implicated in modulation of learning and memory. It has been demonstrated that 5-hydroxytryptamine(6) (5-HT(6)) receptor antagonists show beneficial effect on cognition in several animal models. Based on a pharmacophore model reported in the literature, we have designed and successfully identified a 7-benzenesulfonyl-1,2,3,4-tetrahydro-benzo[4,5]furo[2,3-c]pyridine (3a) scaffold as a novel class of 5-HT(6) receptor antagonists. Despite good activity against 5-HT(6) receptor, 3a exhibited poor liver microsome stability in mouse, rat and dog. It was demonstrated that the saturation of the double bond of the tetrahydropyridine ring of 3a enhanced metabolic stability. However the resulting compound, 4a (7-phenylsulfonyl-1,2,3,4,4a,9a-hexahydro-benzo[4,5]furo[2,3-c] pyridine-HCl salt) exhibited ～30-fold loss in potency along with introduction of two chiral centers. In our optimization process for this series, we found that substituents at the 2 or 3 positions on the distal aryl group are important for enhancing activity against 5-HT(6). Separation of enantiomers and subsequent optimization and SAR with bis substituted phenyl sulfone provided potent 5-HT(6) antagonists with improved PK profiles in rat. A potent, selective 5-HT(6)R antagonist (15k) was identified from this study which showed good oral bioavailability (F=39%) in rat with brain penetration (B/P=2.76) and in vivo activity in a rat social recognition test.     

Novel brain penetrant benzofuropiperidine 5-HT₆ receptor antagonists

7-Arylsulfonyl substituted benzofuropiperidine was discovered as a novel scaffold for 5HT(6) receptor antagonists. Optimization by substitution at C-1 position led to identification of selective, orally bioavailable, brain penetrant antagonists with reduced hERG liability. An advanced analog tested in rat social recognition model showed significant activity suggesting potential utility in the enhancement of short-term memory.     

Genetic profiling of genes from the oxidative stress pathway among North and South Indians

The case-control association study design has been extensively used for elucidating the genetic basis of complex traits. Considerable variation in frequencies of various gene polymorphisms has been reported across different populations and ethnic groups. Thus before beginning such studies, one must know the gene variants that exist in the population. Such information is not available for the ethnically distinct Indian population, which, on the basis of the languages spoken, can be further subdivided into Indo-Europeans (North Indians) and Dravidians (South Indians). In this study we provide information on allele and genotype frequencies, pairwise linkage disequilibrium, and predominant haplotypes in two populations (North India, n=96; South India, n=96) for several of the commonly investigated polymorphisms in the oxidative stress pathway genes. Of the 33 polymorphisms in 19 genes tested, significant differences in allele and genotype frequencies between the two populations were observed for SOD3 Ala58Thr, UCP1-3826 C/T, NOS3-786 T/C, and TNFA-308 G/A polymorphisms.     

Solution structure of the phytotoxic protein PcF: The first characterized member of the Phytophthora PcF toxin family

The PcF protein from Phytophthora cactorum is the first member of the “PcF toxin family” from the plant pathogens Phytophthora spp. It is able to induce withering in tomato and strawberry leaves. The lack of sequence similarity with other proteins hampers the identification of the molecular mechanisms responsible for its toxicity. Here, we show that the six cysteines form a disulphide pattern that is exclusive for PcF and essential for the protein withering activity. The NMR solution structure identifies a novel fold among protein effectors: a helix‐loop‐helix motif. The presence of a negatively charged surface suggests that it might act as a site of electrostatic interaction. Interestingly, a good fold match with Ole e 6, a plant protein with allergenic activity, highlighted the spatial superimposition of a stretch of identical residues. This finding suggests a possible biological activity based on molecular mimicry.     

TrkA kinase inhibitors from a library of modified and isosteric Staurosporine aglycone

An immobilized Staurosporine aglycone isostere where one of the indole nitrogen atoms was replaced by carbon has been sequentially functionalized to generate compounds inhibiting TrkA kinase. In the first phase, initial screening of a library of C13-hydroxymethyl-7-oxo-indenopyrrolocarbazoles resulted in several potent compounds, one of which was further optimized to generate the corresponding carbamates on solid phase. Some of the major carbamate diastereomers were found to be several-fold more potent than their alcohol parents. Synthesis, SAR analysis, kinase selectivity, and anti-tumor properties of a TrkA inhibitor (12a) are discussed.     

A new species of Dactylostomum Woolcock, 1935 (Digenea: Opecoelidae) from the goatfish Upeneichthys lineatus (Bloch & Schneider) (Mullidae) in Western Australian waters

Dactylostomum nicolli n. sp. is described from the intestine of the goatfish Upeneichthys lineatus (Bloch & Schneider) (Mullidae) caught off Point Peron, Western Australia. The problem of assigning the new species to the appropriate genus, especially with regard to its close resemblance to Paropecoelus Pritchard, 1966, which also infects goatfishes, is discussed. This is the second report of Dactylostomum Woolcock, 1935 from an Australian marine fish. The new species is distinguished by its elongate form, simple ventral sucker papillae and irregularly shaped ovary.     

Stimulation and inhibition of fibril formation by a peptide in the presence of different concentrations of SDS

Sodium dodecyl sulphate (SDS), a detergent that mimics some characteristics of biological membranes, has been found to affect significantly fibril formation by a peptide from human complement receptor 1. In aqueous solution the peptide is unfolded but slowly aggregates to form fibrils. In sub-micellar concentrations of SDS the peptide is initially alpha-helical but converts rapidly to a beta-sheet structure and large quantities of fibrils form. In SDS above the critical micellar concentration the peptide adopts a stable alpha-helical structure and no fibrils are observed. These findings demonstrate the sensitivity of fibril formation to solution conditions and suggest a possible role for membrane components in amyloid fibril formation in living systems.