Dissection of β‐barrel outer membrane protein assembly pathways through characterizing BamA POTRA 1 mutants of Escherichia coli

BamA of Escherichia coli is an essential component of the hetero‐oligomeric machinery that mediates β‐barrel outer membrane protein (OMP) assembly. The C‐ and N‐termini of BamA fold into trans‐membrane β‐barrel and five soluble POTRA domains respectively. Detailed characterization of BamA POTRA 1 missense and deletion mutants revealed two competing OMP assembly pathways, one of which is followed by the archetypal trimeric β‐barrel OMPs, OmpF and LamB, and is dependent on POTRA 1. Interestingly, our data suggest that BamA also requires its POTRA 1 domain for proper assembly. The second pathway is independent of POTRA 1 and is exemplified by TolC. Site‐specific cross‐linking analysis revealed that the POTRA 1 domain of BamA interacts with SurA, a periplasmic chaperone required for the assembly of OmpF and LamB, but not that of TolC and BamA. The data suggest that SurA and BamA POTRA 1 domain function in concert to assist folding and assembly of most β‐barrel OMPs except for TolC, which folds into a unique soluble α‐helical barrel and an OM‐anchored β‐barrel. The two assembly pathways finally merge at some step beyond POTRA 1 but presumably before membrane insertion, which is thought to be catalysed by the trans‐membrane β‐barrel domain of BamA.     

Novel benzothiophene H1-antihistamines for the treatment of insomnia

SAR of lead benzothiophene H₁-antihistamine 2 was explored to identify backup candidates with suitable pharmacokinetic profiles for an insomnia program. Several potent and selective H₁-antihistamines with a range of projected half-lives in humans were identified. Compound 16d had a suitable human half-life as demonstrated in a human microdose study, but variability in pharmacokinetic profile, attributed to metabolic clearance, prevented further development of this compound. Compound 28b demonstrated lower predicted clearance in preclinical studies, and may represent a more suitable backup compound.     

Identification of a novel selective H1-antihistamine with optimized pharmacokinetic properties for clinical evaluation in the treatment of insomnia

Analogs of the known H₁-antihistamine R-dimethindene with suitable selectivity for key GPCRs, P450 enzymes and hERG channel were assessed for metabolism profile and in vivo properties. Several analogs were determined to exhibit diverse metabolism. One of these compounds, 10a, showed equivalent efficacy in a rat EEG/EMG model to a previously identified clinical candidate and a potentially superior pharmacokinetic profile as determined from a human microdose study.     

Identification of a novel risk locus for progressive supranuclear palsy by a pooled genomewide scan of 500,288 single-nucleotide polymorphisms

To date, only the H1 MAPT haplotype has been consistently associated with risk of developing the neurodegenerative disease progressive supranuclear palsy (PSP). We hypothesized that additional genetic loci may be involved in conferring risk of PSP that could be identified through a pooling-based genomewide association study of >500,000 SNPs. Candidate SNPs with large differences in allelic frequency were identified by ranking all SNPs by their probe-intensity difference between cohorts. The MAPT H1 haplotype was strongly detected by this methodology, as was a second major locus on chromosome 11p12-p11 that showed evidence of association at allelic (P<.001), genotypic (P<.001), and haplotypic (P<.001) levels and was narrowed to a single haplotype block containing the DNA damage-binding protein 2 (DDB2) and lysosomal acid phosphatase 2 (ACP2) genes. Since DNA damage and lysosomal dysfunction have been implicated in aging and neurodegenerative processes, both genes are viable candidates for conferring risk of disease.     

Trifolium pratense isoflavones in the treatment of menopausal hot flushes: A systematic review and meta-analysis

OBJECTIVE: To critically assess the evidence of supplements containing Trifolium pratense (red clover) isoflavones in the reduction of hot flush frequency in menopausal women. DATA SOURCES: Systematic literature searches were performed in (Medline (1951 - April 2006), Embase (1974 - April 2006), CINAHL (1982 - April 2006), Amed (1985 - April 2006) and The Cochrane Library (Issue 2, 2006). Reference lists located were checked for further relevant publications. Experts in the field and manufacturers of identified products were contacted for unpublished material. No language restrictions were imposed. REVIEW METHODS: Studies were selected according to predefined inclusion and exclusion criteria. All randomized clinical trials of monopreparations containing T. pratense isoflavones for treating hot flushes were included. Study selection, data extraction and validation were performed by at least two reviewers with disagreements being settled by discussion. Weighted means and 95% confidence intervals were calculated and sensitivity analyses were performed. RESULTS: Seventeen potentially relevant articles were retrieved for further evaluation. Five were suitable for inclusion in the meta-analysis. The meta-analysis indicates a reduction in hot flush frequency in the active treatment group (40-82 mg daily) compared with the placebo group (weighted mean difference -1.5 hot flushes daily; 95% CI -2.94 to 0.03; p=0.05). CONCLUSION: There is evidence of a marginally significant effect of T. pratense isoflavones for treating hot flushes in menopausal women. Whether the size of this effect can be considered clinically relevant is unclear. Whereas there is no apparent evidence of adverse events during short-term use, there are no available data on the safety of long-term administration.     

COMPASS: a suite of pre- and post-search proteomics software tools for OMSSA

Here we present the Coon OMSSA Proteomic Analysis Software Suite (COMPASS): a free and open-source software pipeline for high-throughput analysis of proteomics data, designed around the Open Mass Spectrometry Search Algorithm. We detail a synergistic set of tools for protein database generation, spectral reduction, peptide false discovery rate analysis, peptide quantitation via isobaric labeling, protein parsimony and protein false discovery rate analysis, and protein quantitation. We strive for maximum ease of use, utilizing graphical user interfaces and working with data files in the original instrument vendor format. Results are stored in plain text comma-separated value files, which are easy to view and manipulate with a text editor or spreadsheet program. We illustrate the operation and efficacy of COMPASS through the use of two LC-MS/MS data sets. The first is a data set of a highly annotated mixture of standard proteins and manually validated contaminants that exhibits the identification workflow. The second is a data set of yeast peptides, labeled with isobaric stable isotope tags and mixed in known ratios, to demonstrate the quantitative workflow. For these two data sets, COMPASS performs equivalently or better than the current de facto standard, the Trans-Proteomic Pipeline.     

JNK2 promotes endothelial cell alignment under flow

Endothelial cells in straight, unbranched segments of arteries elongate and align in the direction of flow, a feature which is highly correlated with reduced atherosclerosis in these regions. The mitogen-activated protein kinase c-Jun N-terminal kinase (JNK) is activated by flow and is linked to inflammatory gene expression and apoptosis. We previously showed that JNK activation by flow is mediated by integrins and is observed in cells plated on fibronectin but not on collagen or basement membrane proteins. We now show thatJNK2 activation in response to laminar shear stress is biphasic, with an early peak and a later peak. Activated JNK localizes to focal adhesions at the ends of actin stress fibers, correlates with integrin activation and requires integrin binding to the extracellular matrix. Reducing JNK2 activation by siRNA inhibits alignment in response to shear stress. Cells on collagen, where JNK activity is low, align slowly. These data show that an inflammatory pathway facilitates adaptation to laminar flow, thereby revealing an unexpected connection between adaptation and inflammatory pathways.     

Upstream stimulatory factor 1 associated with familial combined hyperlipidemia, LDL cholesterol, and triglycerides

Positive evidence has been reported for linkage and association between the upstream stimulatory factor 1 gene (USF1) and familial combined hyperlipidemia (FCHL). We genotyped the two most positive single-nucleotide polymorphisms (SNPs) (usf1s1: rs3737787 and usf1s2: rs2073658) from previous studies in a large family sample. This sample included 2,195 subjects in 87 Utah pedigrees ascertained for early death due to coronary heart disease (CHD), early strokes, or early onset hypertension. There were a total of 262 relative pairs in these families with FCHL. In the full family sample, FCHL was associated with usf1s1 (P=0.02). Triglyceride and LDL cholesterol defined qualitatively or quantitatively were also associated with usf1s1 (P=0.02–0.05). Results were strengthened for qualitative and quantitative triglyceride and LDL cholesterol when data from males only was analyzed, revealing associations for usf1s1 (P=0.001–0.02), usf1s2 (P=0.02–0.05) and the haplotype of these two SNPs (P=0.01–0.04). The strongest results were in the subset of subjects from families ascertained for premature stroke or hypertension, rather than those ascertained for premature CHD. This study replicates the involvement of USF1 in FCHL and related lipid traits in a family sample not ascertained for FCHL.