High-grade transgenic somatic chimeras from chicken embryonic stem cells

Male and female embryonic stem (ES) cell lines were derived from the area pellucidae of Stage X (EG&K) chicken embryos. These ES cell lines were grown in culture for extended periods of time and the majority of the cells retained a diploid karyotype. When reintroduced into Stage VI-X (EG&K) recipient embryos, the cES cells were able to contribute to all somatic tissues. By combining irradiation of the recipient embryo with exposure of the cES cells to the embryonic environment in diapause, a high frequency and extent of chimerism was obtained. High-grade chimeras, indistinguishable from the donor phenotype by feather pigmentation, were produced. A transgene encoding GFP was incorporated into the genome of cES cells under control of the ubiquitous promoter CX and GFP was widely expressed in somatic tissues. Although cES cells made extensive contributions to the somatic tissues, contribution to the germline was not observed.     

Inter-organ proteomic analysis reveals insights into the molecular mechanisms underlying the anti-diabetic effects of cis-9, trans-11-conjugated linoleic acid in ob/ob mice

cis‐9, trans‐11‐Conjugated linoleic acid (c9 t11 CLA) exerts anti‐diabetic effects by improving systemic insulin sensitivity and inflammation. Levels of CLA in beef can be increased by feeding cattle on pasture. This study aimed to explore the efficacy of a CLA‐rich diet (0.6% w/w c9 t11 CLA), presented as beef enriched with CLA or beef supplemented with synthetic CLA (c9 t11 CLA), for 28 days on molecular biomarkers of the metabolic syndrome, and adipose, hepatic, and skeletal muscle proteome in male ob/ob mice. Despite equal weight gain, CLA‐fed mice had lower plasma glucose, insulin, non‐esterified fatty acid, triacylglycerol and interleukin‐6, and higher adiponectin concentrations than controls. c9 t11 CLA induced differential regulation of redox status across all tissues, and decreased hepatic and muscle endoplasmic reticulum stress. CLA also modulated mechanistic links between the actin cytoskeleton, insulin signalling, glucose transport and inflammation in the adipose tissue. In the liver and muscle, c9 t11 CLA improved metabolic flexibility through co‐ordination between carbohydrate and energy metabolism. c9 t11 CLA may ameliorate systemic insulin sensitivity in obesity‐induced diabetes by altering cellular stress and redox status, and modulating nutrient handling in key insulin‐sensitive tissues through complex biochemical interplay among representative proteomic signatures.     

M-cells: origin, morphology and role in mucosal immunity and microbial pathogenesis

M-cells are specialized cells found in the follicle-associated epithelium of intestinal Peyer's patches of gut-associated lymphoid tissue and in isolated lymphoid follicles, appendix and in mucosal-associated lymphoid tissue sites outside the gastrointestinal tract. In the gastrointestinal tract, M-cells play an important role in transport of antigen from the lumen of the small intestine to mucosal lymphoid tissues, where processing and initiation of immune responses occur. Thus, M-cells act as gateways to the mucosal immune system and this function has been exploited by many invading pathogens. Understanding the mechanism by which M-cells sample antigen will inform the design of oral vaccines with improved efficacy in priming mucosal and systemic immune responses. In this review, the origin and morphology of M-cells, and their role in mucosal immunity and pathogenesis of infections are discussed.     

Elafin, an elastase-specific inhibitor, is cleaved by its cognate enzyme neutrophil elastase in sputum from individuals with cystic fibrosis

Elafin is a neutrophil serine protease inhibitor expressed in lung and displaying anti-inflammatory and anti-bacterial properties. Previous studies demonstrated that some innate host defense molecules of the cystic fibrosis (CF) and chronic obstructive pulmonary disease airways are impaired due to increased proteolytic degradation observed during lung inflammation. In light of these findings, we thus focused on the status of elafin in CF lung. We showed in the present study that elafin is cleaved in sputum from individuals with CF. Pseudomonas aeruginosa-positive CF sputum, which was found to contain lower elafin levels and higher neutrophil elastase (NE) activity compared with P. aeruginosa-negative samples, was particularly effective in cleaving recombinant elafin. NE plays a pivotal role in the process as only NE inhibitors are able to inhibit elafin degradation. Further in vitro studies demonstrated that incubation of recombinant elafin with excess of NE leads to the rapid cleavage of the inhibitor. Two cleavage sites were identified at the N-terminal extremity of elafin (Val-5-Lys-6 and Val-9-Ser-10). Interestingly, purified fragments of the inhibitor (Lys-6-Gln-57 and Ser-10-Gln-57) were shown to still be active for inhibiting NE. However, NE in excess was shown to strongly diminish the ability of elafin to bind lipopolysaccharide (LPS) and its capacity to be immobilized by transglutamination. In conclusion, this study provides evidence that elafin is cleaved by its cognate enzyme NE present at excessive concentration in CF sputum and that P. aeruginosa infection promotes this effect. Such cleavage may have repercussions on the innate immune function of elafin.     

A trimeric quaternary structure is conserved in bacterial and human glutamate transporters

Neuronal and glial glutamate transporters play a central role in the termination of synaptic transmission and in extracellular glutamate homeostasis in the mammalian central nervous system. They are known to be multimers; however, the number of subunits forming a functional transporter is controversial. We studied the subunit stoichiometry of two distantly related glutamate transporters, the human glial glutamate transporter hEAAT2 and a bacterial glutamate transporter from Escherichia coli, ecgltP. Using blue native polyacrylamide gel electrophoresis, analysis of concatenated transporters, and chemical cross-linking, we demonstrated that human and prokaryotic glutamate transporters expressed in Xenopus laevis oocytes or in mammalian cells are assembled as trimers composed of three identical subunits. In an inducible mammalian cell line expressing hEAAT2 the glutamate uptake currents correlate to the amount of trimeric transporters. Overexpression and purification of ecgltP in E. coli resulted in a homogenous population of trimeric transporters that were functional after reconstitution in lipid vesicles. Our results indicate that an evolutionarily conserved trimeric quaternary structure represents the sole native and functional state of glutamate transporters.     

The unusual redox properties of flavocytochrome P450 BM3 flavodoxin domain

Flavocytochrome P450 BM3 FMN domain is unique among the family of flavodoxins and homologues, in not forming a stable neutral blue FMN semiquinone radical. Anaerobic, one-electron reduction of the isolated domain over the pH 7-9.5 range showed that it forms an anionic red semiquinone that disproportionates slowly (0.014s(-1) at pH 7). The rate of disproportionation decreased at higher pH, indicating that protonation of the anionic semiquinone is an important feature of the mechanism. The reduction potential for the oxidised-semiquinone couple was determined to be -240mV and was largely independent of pH. The semiquinone appears, therefore, to be kinetically trapped by a slow protonation event, enabling it to act as a low-potential electron donor to the P450 heme.