Naturally Occurring Variants of Human Α9 Nicotinic Receptor Differentially Affect Bronchial Cell Proliferation and Transformation

Isolation of polyadenilated mRNA from human immortalized bronchial epithelial cell line BEP2D revealed the presence of multiple isoforms of RNA coded by the CHRNA9 gene for α9 nicotinic acetylcholine receptor (nAChR). BEP2D cells were homozygous for the rs10009228 polymorphism encoding for N442S amino acid substitution, and also contained mRNA coding for several truncated isoforms of α9 protein. To elucidate the biologic significance of the naturally occurring variants of α9 nAChR, we compared the biologic effects of overexpression of full-length α9 N442 and S442 proteins, and the truncated α9 variant occurring due to a loss of the exon 4 sequence that causes frame shift and early termination of the translation. These as well as control vector were overexpressed in the BEP2D cells that were used in the assays of proliferation rate, spontaneous vs. tobacco nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced cellular transformation, and tumorigenicity in cell culture and mice. Overexpression of the S442 variant significantly increased cellular proliferation, and spontaneous and NNK-induced transformation. The N442 variant significantly decreased cellular transformation, without affecting proliferation rate. Overexpression of the truncated α9 significantly decreased proliferation and suppressed cellular transformation. These results suggested that α9 nAChR plays important roles in regulation of bronchial cell growth by endogenous acetylcholine and exogenous nicotine, and susceptibility to NNK-induced carcinogenic transformation. The biologic activities of α9 nAChR may be regulated at the splicing level, and genetic polymorphisms in CHRNA9 affecting protein levels, amino acid sequence and RNA splicing may influence the risk for lung cancer

SOCS2-Induced Proteasome-Dependent TRAF6 Degradation: A Common Anti-Inflammatory Pathway for Control of Innate Immune Responses

Pattern recognition receptors and receptors for pro-inflammatory cytokines provide critical signals to drive the development of protective immunity to infection. Therefore, counter-regulatory pathways are required to ensure that overwhelming inflammation harm host tissues. Previously, we showed that lipoxins modulate immune response during infection, restraining inflammation during infectious diseases in an Aryl hydrocarbon receptor (AhR)/suppressors of cytokine signaling (SOCS)2-dependent-manner. Recently, Indoleamine-pyrrole 2,3- dioxygenase (IDO)-derived tryptophan metabolites, including L-kynurenine, were also shown to be involved in several counter-regulatory mechanisms. Herein, we addressed whether the intracellular molecular events induced by lipoxins mediating control of innate immune signaling are part of a common regulatory pathway also shared by L-kynurenine exposure. We demonstrate that Tumor necrosis factor receptor-associated factor (TRAF)6 – member of a family of adapter molecules that couple the TNF receptor and interleukin-1 receptor/Toll-like receptor families to intracellular signaling events essential for the development of immune responses – is targeted by both lipoxins and L-kynurenine via an AhR/SOCS2-dependent pathway. Furthermore, we show that LXA4- and L-kynurenine-induced AhR activation, its subsequent nuclear translocation, leading SOCS2 expression and TRAF6 Lys47-linked poly-ubiquitination and proteosome-mediated degradation of the adapter proteins. The in vitro consequences of such molecular interactions included inhibition of TLR- and cytokine receptor-driven signal transduction and cytokine production. Subsequently, in vivo proteosome inhibition led to unresponsiveness to lipoxins, as well as to uncontrolled pro-inflammatory reactions and elevated mortality during toxoplasmosis. In summary, our results establish proteasome degradation of TRAF6 as a key molecular target for the anti-inflammatory pathway triggered by lipoxins and L-kynurenine, critical counter-regulatory mediators in the innate and adaptive immune systems

Antimalarial Therapy Selection for Quinolone Resistance among Escherichia coli in the Absence of Quinolone Exposure, in Tropical South America

BACKGROUND: Bacterial resistance to antibiotics is thought to develop only in the presence of antibiotic pressure. Here we show evidence to suggest that fluoroquinolone resistance in Escherichia coli has developed in the absence of fluoroquinolone use. METHODS: Over 4 years, outreach clinic attendees in one moderately remote and five very remote villages in rural Guyana were surveyed for the presence of rectal carriage of ciprofloxacin-resistant gram-negative bacilli (GNB). Drinking water was tested for the presence of resistant GNB by culture, and the presence of antibacterial agents and chloroquine by HPLC. The development of ciprofloxacin resistance in E. coli was examined after serial exposure to chloroquine. Patient and laboratory isolates of E. coli resistant to ciprofloxacin were assessed by PCR-sequencing for quinolone-resistance-determining-region (QRDR) mutations. RESULTS: In the very remote villages, 4.8% of patients carried ciprofloxacin-resistant E. coli with QRDR mutations despite no local availability of quinolones. However, there had been extensive local use of chloroquine, with higher prevalence of resistance seen in the villages shortly after a Plasmodium vivax epidemic (p<0.01). Antibacterial agents were not found in the drinking water, but chloroquine was demonstrated to be present. Chloroquine was found to inhibit the growth of E. coli in vitro. Replica plating demonstrated that 2-step QRDR mutations could be induced in E. coli in response to chloroquine. CONCLUSIONS: In these remote communities, the heavy use of chloroquine to treat malaria likely selected for ciprofloxacin resistance in E. coli. This may be an important public health problem in malarious areas

From Sea to Sea: Canada's Three Oceans of Biodiversity

Evaluating and understanding biodiversity in marine ecosystems are both necessary and challenging for conservation. This paper compiles and summarizes current knowledge of the diversity of marine taxa in Canada's three oceans while recognizing that this compilation is incomplete and will change in the future. That Canada has the longest coastline in the world and incorporates distinctly different biogeographic provinces and ecoregions (e.g., temperate through ice-covered areas) constrains this analysis. The taxonomic groups presented here include microbes, phytoplankton, macroalgae, zooplankton, benthic infauna, fishes, and marine mammals. The minimum number of species or taxa compiled here is 15,988 for the three Canadian oceans. However, this number clearly underestimates in several ways the total number of taxa present. First, there are significant gaps in the published literature. Second, the diversity of many habitats has not been compiled for all taxonomic groups (e.g., intertidal rocky shores, deep sea), and data compilations are based on short-term, directed research programs or longer-term monitoring activities with limited spatial resolution. Third, the biodiversity of large organisms is well known, but this is not true of smaller organisms. Finally, the greatest constraint on this summary is the willingness and capacity of those who collected the data to make it available to those interested in biodiversity meta-analyses. Confirmation of identities and intercomparison of studies are also constrained by the disturbing rate of decline in the number of taxonomists and systematists specializing on marine taxa in Canada. This decline is mostly the result of retirements of current specialists and to a lack of training and employment opportunities for new ones. Considering the difficulties encountered in compiling an overview of biogeographic data and the diversity of species or taxa in Canada's three oceans, this synthesis is intended to serve as a biodiversity baseline for a new program on marine biodiversity, the Canadian Healthy Ocean Network. A major effort needs to be undertaken to establish a complete baseline of Canadian marine biodiversity of all taxonomic groups, especially if we are to understand and conserve this part of Canada's natural heritage

Subspecific Relationships and Genetic Structure in the Spotted Owl

Hierarchical genetic structure was examined in the three geographically-defined subspecies of spotted owl (Strix occidentalis) to define relationships among subspecies and quantify variation within and among regional and local populations. Sequences (522 bp) from domains I and II of the mitochondrial control region were analyzed for 213 individuals from 30 local breeding areas. Results confirmed significant differences between northern spotted owls and the other traditional geographically defined subspecies but did not provide support for subspecific level differences between California and Mexican spotted owls. Divergence times among subspecies estimated with a 936 bp portion of the cytochrome b gene dated Northern and California/Mexican spotted owl divergence time to 115,000–125,000 years ago, whereas California/Mexican spotted owl divergence was estimated at 15,000 years ago. Nested clade analyses indicated an association between California spotted owl and Mexican spotted owl haplotypes, implying historical contact between the two groups. Results also identified a number of individuals geographically classified as northern spotted owls (S. o. caurina) that contained haplotypes identified as California spotted owls (S. o. occidentalis). Among all northern spotted owls sampled (n = 131), 12.9% contained California spotted owl haplotypes. In the Klamath region, which is the contact zone between the two subspecies, 20.3% (n = 59) of owls were classified as California spotted owls. The Klamath region is a zone of hybridization and speciation for many other taxa as well. Analyses of population structure indicated gene flow among regions within geographically defined subspecies although there was significant differentiation among northern and southern regions of Mexican spotted owls. Among all areas examined, genetic diversity was not significantly reduced except in California spotted owls where the southern region consists of one haplotype. Our results indicate a stable contact zone between northern and California spotted owls, maintaining distinct subspecific haplotypes within their traditional ranges. This supports recovery efforts based on the traditional subspecies designation for the northern spotted owl. Further, although little variation was found between California and Mexican spotted owls, we suggest they should be managed separately because of current isolation between groups