NADPH-oxidase but not inducible nitric oxide synthase contributes to resistance in a murine Staphylococcus aureus Newman pneumonia model

Staphylococcus aureus is a pathogen that often causes severe nosocomial infections including pneumonia. The present study was designed to examine innate phagocyte mediated immune mechanisms using a previously described murine S. aureus Newman pneumonia model. We found that BALB/c mice represent a more susceptible mouse strain compared to C57BL/6 mice after intranasal S. aureus Newman challenge. Depletion experiments revealed that neutrophils are a crucial determinant for resistance whereas depletion of alveolar macrophages protected mice to some degree from acute pulmonary S. aureus challenge. C57BL/6 mice lacking the subunit gp91phox of the NADPH-oxidase (gp91phox−/− mice) proved to be highly susceptible against the pathogen. In contrast, C57BL/6 inducible nitric oxidase synthase deficient (iNOS−/−) mice did not differ in their clinical outcome after infection. Neither bone marrow macrophages from iNOS−/− nor from gp91phox−/− mice were impaired in controlling intracellular persistence of S. aureus. Our data suggest that neutrophil and NADPH-oxidase mediated mechanisms are essential components in protecting the host against pulmonary S. aureus Newman challenge. On contrary, macrophages as well as NO mediated mechanisms do not seem to play a critical role for resistance in this model.     

Pleiotropic benefit of monomeric and oligomeric flavanols on vascular health--a randomized controlled clinical pilot study

Background

Cardiovascular diseases are expanding to a major social-economic burden in the Western World and undermine man''s deep desire for healthy ageing. Epidemiological studies suggest that flavanol-rich foods (e.g. grapes, wine, chocolate) sustain cardiovascular health. For an evidenced-based application, however, sound clinical data on their efficacy are strongly demanded.

Methods

In a double-blind, randomized, placebo-controlled intervention study we supplemented 28 male smokers with 200 mg per day of monomeric and oligomeric flavanols (MOF) from grape seeds. At baseline, after 4 and 8 weeks we measured macro- and microvascular function and a cluster of systemic biomarkers for major pathological processes occurring in the vasculature: disturbances in lipid metabolism and cellular redox balance, and activation of inflammatory cells and platelets.

Results

In the MOF group serum total cholesterol and LDL decreased significantly (P≤0.05) by 5% (n = 11) and 7% (n = 9), respectively in volunteers with elevated baseline levels. Additionally, after 8 weeks the ratio of glutathione to glutathione disulphide in erythrocytes rose from baseline by 22% (n = 15, P<0.05) in MOF supplemented subjects. We also observed that MOF supplementation exerts anti-inflammatory effects in blood towards ex vivo added bacterial endotoxin and significantly reduces expression of inflammatory genes in leukocytes. Conversely, alterations in macro- and microvascular function, platelet aggregation, plasma levels of nitric oxide surrogates, endothelin-1, C-reactive protein, fibrinogen, prostaglandin F2alpha, plasma antioxidant capacity and gene expression levels of antioxidant defense enzymes did not reach statistical significance after 8 weeks MOF supplementation. However, integrating all measured effects into a global, so-called vascular health index revealed a significant improvement of overall vascular health by MOF compared to placebo (P≤0.05).

Conclusion

Our integrative multi-biomarker approach unveiled the pleiotropic vascular health benefit of an 8 weeks supplementation with 200 mg/d MOF in humans.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00742287","term_id":"NCT00742287"}}NCT00742287     

Compressed vertebrae in Atlantic salmon Salmo salar: evidence for metaplastic chondrogenesis as a skeletogenic response late in ontogeny

Anterior/posterior (a/p) compression of the vertebral column, referred to as 'short tails', is a recurring event in farmed Atlantic salmon. Like other skeletal deformities, the problem usually becomes evident in a late life phase, too late for preventive measures, making it difficult to understand the aetiology of the disease. We use structural, radiological, histological, and mineral analyses to study 'short tail' adult salmon and to demonstrate that the study of adult fish can provide important insights into earlier developmental processes. 'Short tails' display a/p compressed vertebrae throughout the spine, except for the first post-cranial vertebrae. The vertebral number is unaltered, but the intervertebral space is reduced and the vertebrae are shorter. Compressed vertebrae are characterized by an unchanged central part, altered vertebral end plates (straight instead of funnel-shaped), an atypical inward bending of the vertebral edges, and structural alterations in the intervertebral tissue. The spongiosa is unaffected. The growth zones of adjacent vertebrae fuse and blend towards the intervertebral space into chondrogenic tissue. This tissue produces different types of cartilage, replacing the notochord. The correspondence in location of intervertebral cartilage and deformed vertebral end plates, and the clearly delimited, unaltered, central vertebral parts suggest that the a/p compression of vertebral bodies is a late developmental disorder that may be related to a metaplastic shift of osteogenic tissue into chondrogenic tissue in the vertebral growth zone. Given the lack of evidence for infections, metabolic disorders and/or genetic disorders, we propose that an altered mechanical load could have caused the transformation of the bone growth zones and the concomitant replacement of the intervertebral (notochord) tissue by cartilaginous tissues in the 'short tails' studied here. This hypothesis is supported by the role that notochord cells are known to play in spine development and in maintaining the structure of the intervertebral disk.     

PAK5 kinase is an inhibitor of MARK/Par-1, which leads to stable microtubules and dynamic actin

MARK/Par-1 is a kinase involved in development of embryonic polarity. In neurons, MARK phosphorylates tau protein and causes its detachment from microtubules, the tracks of axonal transport. Because the target sites of MARK on tau occur at an early stage of Alzheimer neurodegeneration, we searched for interaction partners of MARK. Here we report that MARK2 is negatively regulated by PAK5, a neuronal member of the p21-activated kinase family. PAK5 suppresses the activity of MARK2 toward its target, tau protein. The inhibition requires the binding between the PAK5 and MARK2 catalytic domains, but does not require phosphorylation. In transfected Chinese hamster ovary (CHO) cells both kinases show a vesicular distribution with partial colocalization on endosomes containing AP-1/2. Although MARK2 transfected alone destabilizes microtubules and stabilizes actin stress fibers, PAK5 keeps microtubules stable through the down-regulation of MARK2 but destabilizes the F-actin network so that stress fibers and focal adhesions disappear and cells develop filopodia. The results point to an inverse relationship between actin- and microtubule-related signaling by the PAK5 and MARK2 pathways that affect both cytoskeletal networks.     

Myosin-V, Kinesin-1, and Kinesin-3 cooperate in hyphal growth of the fungus Ustilago maydis

Long-distance transport is crucial for polar-growing cells, such as neurons and fungal hyphae. Kinesins and myosins participate in this process, but their functional interplay is poorly understood. Here, we investigate the role of kinesin motors in hyphal growth of the plant pathogen Ustilago maydis. Although the microtubule plus-ends are directed to the hyphal tip, of all 10 kinesins analyzed, only conventional kinesin (Kinesin-1) and Unc104/Kif1A-like kinesin (Kinesin-3) were up-regulated in hyphae and they are essential for extended hyphal growth. deltakin1 and deltakin3 mutant hyphae grew irregular and remained short, but they were still able to grow polarized. No additional phenotype was detected in deltakin1rkin3 double mutants, but polarity was lost in deltamyo5rkin1 and deltamyo5rkin3 mutant cells, suggesting that kinesins and class V myosin cooperate in hyphal growth. Consistent with such a role in secretion, fusion proteins of green fluorescent protein and Kinesin-1, Myosin-V, and Kinesin-3 accumulate in the apex of hyphae, a region where secretory vesicles cluster to form the fungal Spitzenk?rper. Quantitative assays revealed a role of Kin3 in secretion of acid phosphatase, whereas Kin1 was not involved. Our data demonstrate that just two kinesins and at least one myosin support hyphal growth.     

Antagonist and agonist binding models of the human gonadotropin-releasing hormone receptor

G-protein-coupled receptors (GPCRs) constitute one of the most important classes of drug targets. Since the first high-resolution structure of a GPCR was determined by Palczewski and co-workers [K. Palczewski, T. Kumasaka, T. Hori, C.A. Behnke, H. Motoshima, B.A. Fox, I. Le Trong, D.C. Teller, T. Okada, R.E. Stenkamp, M. Yamamoto, M. Miyano, Crystal structure of rhodopsin: a G-protein-coupled receptor, Science 289 (2000) 739-745], development of in silico models of rhodopsin-like GPCRs could be rationally founded. In this work, we present a model of the human gonadotropin-releasing hormone receptor based on the rhodopsin structure. The transmembrane helices are modeled by homology, while the extra- and intra-cellular loops are modeled in such a way that experimentally determined interactions and microdomains (e.g., hydrophobic cores) are retained. We conclude that specifically tailored models, compared to more automatic approaches, have the benefit that known interactions are easily introduced early in the homology modeling. Furthermore, tailored models, although more tedious to construct, are better suited for drug lead finding and for compound optimization. To test the stability of the receptor, we performed a 1 ns molecular dynamics simulation. Moreover, we docked two agonists (native GnRH and Triptorelin, [dTrp(6)]-GnRH) and two antagonists (Cetrorelix, dNal(1)-dCpa(2)-dPal(3)-Ser(4)-Tyr(5)-dCit(6)-Leu(7)-Arg(8)-Pro(9)-dAla(10)), and the covalently constrained dicyclic decapeptide dicyclo(1,1'-5/4-10)[Ac-Glu(1)(Gly(1)')-dCpa(2)-dTrp(3)-Asp(4)-dbu(5)-dNal(6)-Leu(7)-Arg(8)-Pro(9)-dpr(10)-NH(2)] into the putative receptor binding site. The docked ligand conformations result in ligand-receptor interactions that are generally in good agreement with site-directed mutagenesis and ligand-binding studies presented in the literature. Our results indicate that the binding conformation of the antagonists differs from that of the agonists. This difference can be linked to the activation or inhibition of the receptor.     

A novel role of CD30/CD30 ligand signaling in the generation of long-lived memory CD8+ T cells

Memory CD8+ T cells can be divided into two subsets, central memory (T(CM)) and effector memory (T(EM)) CD8+ T cells. We found that CD30, a member of the TNFR-associated factor (TRAF)-linked TNFR superfamily, signaling is involved in differentiation of long-lived CD8+ T(CM) cells following Listeria monocytogenes infection. Although CD8+ T(EM) cells transiently accumulated in the nonlymphoid tissues of CD30 ligand (CD153-/-) mice after infection, long-lived memory CD8+ T(CM) cells were poorly generated in these mice. CCR7 mRNA expression was down-regulated in CD8+ T cells of the spleen of CD153-/- mice in vivo and the expression was up-regulated in CD8+ T(EM) cells by anti-CD30 mAb cross-linking in vitro. These results suggest that CD30/CD30 ligand signaling plays an important role in the generation of long-lived memory CD8+ T cells at least partly by triggering homing receptors for T(CM) cells.     

Fermentation performance of engineered and evolved xylose-fermenting Saccharomyces cerevisiae strains

Lignocellulose hydrolysate is an abundant substrate for bioethanol production. The ideal microorganism for such a fermentation process should combine rapid and efficient conversion of the available carbon sources to ethanol with high tolerance to ethanol and to inhibitory components in the hydrolysate. A particular biological problem are the pentoses, which are not naturally metabolized by the main industrial ethanol producer Saccharomyces cerevisiae. Several recombinant, mutated, and evolved xylose fermenting S. cerevisiae strains have been developed recently. We compare here the fermentation performance and robustness of eight recombinant strains and two evolved populations on glucose/xylose mixtures in defined and lignocellulose hydrolysate-containing medium. Generally, the polyploid industrial strains depleted xylose faster and were more resistant to the hydrolysate than the laboratory strains. The industrial strains accumulated, however, up to 30% more xylitol and therefore produced less ethanol than the haploid strains. The three most attractive strains were the mutated and selected, extremely rapid xylose consumer TMB3400, the evolved C5 strain with the highest achieved ethanol titer, and the engineered industrial F12 strain with by far the highest robustness to the lignocellulosic hydrolysate.