Functional and structural characterization of anti-β1-adrenoceptor autoantibodies of spontaneously hypertensive rats

Eighteen month old spontaneously hypertensive rats (SHR-rats) showed myocardial dysfunction and autoantibodies directed against the ₁-adrenoceptor similarly as known in human dilated cardiomyopathy or Chagas' disease. The agonist-like antibodies were able to activate the ₁-adrenoceptor mediated signal transduction cascade in cultured rat cardiomyocytes and induced a long-lasting stimulatory effect resulting in a harmful adrenergic overdrive. The antibodies recognized an epitope of the second extracellular loop of the ₁-adrenoceptor identical to that epitope identified in Chagas' disease. In conclusion, our assumption is supported that old SHR-rat are an useful animal model for investigating the role of anti-₁-adrenoceptor antibodies in the induction of human cardiomyopathy.     

Distinct SNP Combinations Confer Susceptibility to Urinary Bladder Cancer in Smokers and Non-Smokers

Recently, genome-wide association studies have identified and validated genetic variations associated with urinary bladder cancer (UBC). However, it is still unknown whether the high-risk alleles of several SNPs interact with one another, leading to an even higher disease risk. Additionally, there is no information available on how the UBC risk due to these SNPs compare to the risk of cigarette smoking and to occupational exposure to urinary bladder carcinogens, and whether the same or different SNP combinations are relevant in smokers and non-smokers. To address these questions, we analyzed the genotypes of six SNPs, previously found to be associated with UBC, together with the GSTM1 deletion, in 1,595 UBC cases and 1,760 controls, stratified for smoking habits. We identified the strongest interactions of different orders and tested the stability of their effect by bootstrapping. We found that different SNP combinations were relevant in smokers and non-smokers. In smokers, polymorphisms involved in detoxification of cigarette smoke carcinogens were most relevant (GSTM1, rs11892031), in contrast to those in non-smokers with MYC and APOBEC3A near polymorphisms (rs9642880, rs1014971) being the most influential. Stable combinations of up to three high-risk alleles resulted in higher odds ratios (OR) than the individual SNPs, although the interaction effect was less than additive. The highest stable combination effects resulted in an OR of about 2.0, which is still lower than the ORs of cigarette smoking (here, current smokers'' OR: 3.28) and comparable to occupational carcinogen exposure risks which, depending on the workplace, show mostly ORs up to 2.0.     

Expression and purification of recombinant human receptor for advanced glycation endproducts in Escherichia coli

The receptor for advanced glycation endproducts (RAGE) is a multiligand receptor that binds a variety of structurally and functionally unrelated ligands, including advanced glycation endproducts (AGEs), amyloid fibrils, amphoterin, and members of the S100 family of proteins. The receptor has been implicated in the pathology of diabetes as well as in inflammatory processes and tumor cell metastasis. For the present study, the extracellular region of RAGE (exRAGE) was expressed as a soluble, C-terminal hexahistidine-tagged fusion protein in the periplasmic space of Escherichia coli. Proper processing and folding of the purified protein, predicted to contain three immunoglobulin-type domains, was supported by the results of electrospray mass spectroscopy and circular dichroism experiments. Sedimentation velocity experiments showed that exRAGE was primarily monomeric in solution. Binding to several RAGE ligands, including AGE-BSA, immunoglobulin light chain amyloid fibrils, and glycosaminoglycans, was demonstrated using pull-down, dot-blot, or enzyme-linked microplate assays. Using surface plasmon resonance, the interaction of exRAGE with AGE-BSA was shown to fit a two-site model, with KD values of 88 nM and 1.4 microM. The E. coli-derived exRAGE did not bind the advanced glycation endproduct Nepsilon-(carboxymethyl)lysine, as reported for the cellular receptor, and the possible role of RAGE glycosylation in recognition of this ligand is discussed. This new RAGE construct will facilitate detailed studies of RAGE-ligand interactions and provides a platform for preparation of site-directed mutants for future structure/function studies.     

Metabolic syndrome in Yup'ik Eskimos: the Center for Alaska Native Health Research (CANHR) Study

Objective: This study investigated the prevalence of metabolic syndrome and its defining components among Yup'ik Eskimos. Research Methods and Procedures: A cross‐sectional study design that included 710 adult Yup'ik Eskimos ≥18 years of age residing in 8 communities in Southwest Alaska. The prevalence of metabolic syndrome was determined using the recently updated Adult Treatment Panel III criteria. Results: The prevalence of metabolic syndrome in this study cohort was 14.7%, and varied by sex with 8.6% of the men and 19.8% of the women having metabolic syndrome. This is lower than the prevalence of 23.9% in the general U.S. adult population. The most common metabolic syndrome components/risk factors were increased waist circumference and elevated blood glucose. High‐density lipoprotein (HDL) cholesterol levels in Yup'ik Eskimos were significantly higher, and triglycerides lower than levels reported in National Health and Nutritional Examination III. Discussion: Compared with other populations, metabolic syndrome is relatively uncommon in Yup'ik Eskimos. The higher prevalence among Yup'ik women is primarily explained by their large waist circumference, suggesting central body fat accumulation. Further increases in metabolic syndrome risk factors among Yup'ik Eskimos could lead to increases in the prevalence of type 2 diabetes and cardiovascular disease, once rare in this population.     

How to Handle Speciose Clades? Mass Taxon-Sampling as a Strategy towards Illuminating the Natural History of Campanula (Campanuloideae)

Background

Speciose clades usually harbor species with a broad spectrum of adaptive strategies and complex distribution patterns, and thus constitute ideal systems to disentangle biotic and abiotic causes underlying species diversification. The delimitation of such study systems to test evolutionary hypotheses is difficult because they often rely on artificial genus concepts as starting points. One of the most prominent examples is the bellflower genus Campanula with some 420 species, but up to 600 species when including all lineages to which Campanula is paraphyletic. We generated a large alignment of petD group II intron sequences to include more than 70% of described species as a reference. By comparison with partial data sets we could then assess the impact of selective taxon sampling strategies on phylogenetic reconstruction and subsequent evolutionary conclusions.

Methodology/Principal Findings

Phylogenetic analyses based on maximum parsimony (PAUP, PRAP), Bayesian inference (MrBayes), and maximum likelihood (RAxML) were first carried out on the large reference data set (D680). Parameters including tree topology, branch support, and age estimates, were then compared to those obtained from smaller data sets resulting from “classification-guided” (D088) and “phylogeny-guided sampling” (D101). Analyses of D088 failed to fully recover the phylogenetic diversity in Campanula, whereas D101 inferred significantly different branch support and age estimates.

Conclusions/Significance

A short genomic region with high phylogenetic utility allowed us to easily generate a comprehensive phylogenetic framework for the speciose Campanula clade. Our approach recovered 17 well-supported and circumscribed sub-lineages. Knowing these will be instrumental for developing more specific evolutionary hypotheses and guide future research, we highlight the predictive value of a mass taxon-sampling strategy as a first essential step towards illuminating the detailed evolutionary history of diverse clades.     

Immunohistochemical method identifies lymphovascular invasion in a majority of oral squamous cell carcinomas and discriminates between blood and lymphatic vessel invasion.

Tumor invasion into blood and/or lymphatic channels is an important component of cancer staging and prognosis. Standard pathological methods do not provide sufficient contrast to discriminate between invasion into each type of vessel and are complicated by tissue retraction artifacts. We evaluated the ability of a triple-stain immunohistochemical method, combining cytokeratin, CD34, and podoplanin stains in a single section, to distinguish blood from lymphatic vascular invasion in oral squamous cell carcinoma and confirmed its results using multispectral analysis. The triple-stain method was significantly more sensitive in detecting invasive events than the standard hematoxylin and eosin staining method and easily discriminated between blood and lymphatic vessel invasion. Invasive events were present in blood and/or lymphatic vessels in the majority of patients with and without presentation of lymph node metastasis, indicating that vessel invasion in this cancer model is common and is not a rate-limiting step for lymph node metastasis.     

5-Aminomethylbenzimidazoles as potent ITK antagonists

Benzamide 1 demonstrated good potency as a selective ITK inhibitor, however the amide moiety was found to be hydrolytically labile in vivo, resulting in low oral exposure and the generation of mutagenic aromatic amine metabolites. Replacing the benzamide with a benzylamine linker not only addressed the toxicity issue, but also improved the cellular and functional potency as well as the drug-like properties. SAR studies around the benzylamines and the identification of 10n and 10o as excellent tools for proof-of-concept studies are described.     

Hfq Influences Multiple Transport Systems and Virulence in the Plant Pathogen Agrobacterium tumefaciens

The Hfq protein mediates gene regulation by small RNAs (sRNAs) in about 50% of all bacteria. Depending on the species, phenotypic defects of an hfq mutant range from mild to severe. Here, we document that the purified Hfq protein of the plant pathogen and natural genetic engineer Agrobacterium tumefaciens binds to the previously described sRNA AbcR1 and its target mRNA atu2422, which codes for the substrate binding protein of an ABC transporter taking up proline and γ-aminobutyric acid (GABA). Several other ABC transporter components were overproduced in an hfq mutant compared to their levels in the parental strain, suggesting that Hfq plays a major role in controlling the uptake systems and metabolic versatility of A. tumefaciens. The hfq mutant showed delayed growth, altered cell morphology, and reduced motility. Although the DNA-transferring type IV secretion system was produced, tumor formation by the mutant strain was attenuated, demonstrating an important contribution of Hfq to plant transformation by A. tumefaciens.