The putative NTPase/helicase protein from severe acute respiratory syndrome coronavirus (SARS-CoV) is postulated to play a number of crucial roles in the viral life cycle, making it an attractive target for anti-SARS therapy. We have cloned, expressed, and purified this protein as an N-terminal hexahistidine fusion in Escherichia coli and have characterized its helicase and NTPase activities. The enzyme unwinds doublestranded DNA, dependent on the presence of a 5 singlestranded overhang, indicating a 5 to 3 polarity of activity, a distinct characteristic of coronaviridae helicases. We provide the first quantitative analysis of the polynucleic acid binding and NTPase activities of a Nidovirus helicase, using a high throughput phosphate release assay that will be readily adaptable to the future testing of helicase inhibitors. All eight common NTPs and dNTPs were hydrolyzed by the SARS helicase in a magnesium-dependent reaction, stimulated by the presence of either single-stranded DNA or RNA. The enzyme exhibited a preference for ATP, dATP, and dCTP over the other NTP/dNTP substrates. Homopolynucleotides significantly stimulated the ATPase activity (15-25-fold) with the notable exception of poly(G) and poly(dG), which were non-stimulatory. We found a large variation in the apparent strength of binding of different homopolynucleotides, with dT 24 binding over 10 times more strongly than dA 24 as observed by the apparent K m .

A genome-wide association (GWA) study of treatment outcomes (response and remission) of selective serotonin reuptake inhibitors (SSRIs) was conducted using 529 subjects with major depressive disorder (MDD). While no SNP associations reached the genome-wide level of significance, 14 SNPs of interest were identified for functional analysis. The rs11144870 SNP in riboflavin kinase (RFK) gene on chromosome 9 was associated with eight week treatment response (OR = 0.42, p = 1.04×10−6). The rs915120 SNP in the G protein-coupled receptor kinase 5 (GRK5) gene on chromosome 10 was associated with eight week remission (OR = 0.50, p = 1.15×10−5). Both SNPs were shown to influence transcription by a reporter gene assay and to alter nuclear protein binding using an electrophoretic mobility shift assay. This report represents an example of joining functional genomics with traditional GWA study results derived from a GWA analysis of SSRI treatment outcomes. The goal of this analytic strategy is to provide insights into the potential relevance of biologically plausible observed associations.

Acetaminophen is the leading cause of acute hepatic failure in many developed nations. Acetaminophen hepatotoxicity is mediated by the reactive metabolite N-acetyl-p-benzoquinonimine (NAPQI). We performed a "discovery" genome-wide association study using a cell line-based model system to study the possible contribution of genomics to NAPQI-induced cytotoxicity. A total of 176 lymphoblastoid cell lines from healthy subjects were treated with increasing concentrations of NAPQI. Inhibiting concentration 50 values were determined and were associated with "glutathione pathway" gene single nucleotide polymorphisms (SNPs) and genome-wide basal messenger RNA expression, as well as with 1.3 million genome-wide SNPs. A group of SNPs in linkage disequilibrium on chromosome 3 was highly associated with NAPQI toxicity. The p value for rs2880961, the SNP with the lowest p value, was 1.88 × 10(-7). This group of SNPs mapped to a "gene desert," but chromatin immunoprecipitation assays demonstrated binding of several transcription factor proteins including heat shock factor 1 (HSF1) and HSF2, at or near rs2880961. These chromosome 3 SNPs were not significantly associated with variation in basal expression for any of the genome-wide genes represented on the Affymetrix U133 Plus 2.0 GeneChip. We have used a cell line-based model system to identify a SNP signal associated with NAPQI cytotoxicity. If these observations are validated in future clinical studies, this SNP signal might represent a potential biomarker for risk of acetaminophen hepatotoxicity. The mechanisms responsible for this association remain unclear.

IntroductionNatural herbal compounds with novel actions different from existing breast cancer (BCa) treatment modalities are attractive for improving therapeutic efficacy and safety. We have recently shown that penta-1,2,3,4,6-O-galloyl-β-D-glucose (PGG) induced S-phase arrest in prostate cancer (PCa) cells through inhibiting DNA replicative synthesis and G1 arrest, in addition to inducing cell death at higher levels of exposure. We and others have shown that PGG through intraperitoneal (i.p.) injection exerts a strong in vivo growth suppression of human PCa xenograft models in athymic nude mice. This study aims to test the hypothesis that the novel targeting actions of PGG are applicable to BCa cells, especially those lacking proven drugable targets.MethodsMono-layer cell culture models of p53-wild type estrogen receptor (ER)-dependent MCF-7 BCa cells and p53-mutant ER-/progesterone receptor (PR)- and Her2-regular (triple-negative) MDA-MB-231 BCa were exposed to PGG for a comprehensive investigation of cellular consequences and molecular targets/mediators. To test the in vivo efficacy, female athymic mice inoculated with MDA-MB-231 xenograft were treated with 20 mg PGG/kg body weight by daily gavage starting 4 days after cancer cell inoculation.ResultsExposure to PGG induced S-phase arrest in both cell lines as indicated by the lack of 5-bromo2'-deoxy-uridine (BrdU) incorporation into S-phase cells as well as G1 arrest. Higher levels of PGG induced more caspase-mediated apoptosis in MCF-7, in strong association with induction of P53 Ser15 phosphorylation, than in MDA-MB-231 cells. The cell cycle arrests were achieved without an induction of cyclin dependent kinase (CDK) inhibitory proteins P21Cip1 and P27Kip1. PGG treatment led to decreased cyclin D1 in both cell lines and over-expressing cyclin D1 attenuated G1 arrest and hastened S arrest. In serum-starvation synchronized MCF-7 cells, down-regulation of cyclin D1 was associated with de-phosphorylation of retinoblastoma (Rb) protein by PGG shortly before G1-S transition. In vivo, oral administration of PGG led to a greater than 60% inhibition of MDA-MB231 xenograft growth without adverse effect on host body weight.ConclusionsOur in vitro and in vivo data support PGG as a potential drug candidate for breast cancer with novel targeting actions, especially for a triple negative BCa xenograft model.