Methylotrophy in Freshwater Beggiatoa alba Strains

Two freshwater strains of the gammaproteobacterium Beggiatoa alba, B18LD and OH75-2a, are able to use methanol as a sole carbon and energy source under microoxic conditions. Genes encoding a methanol dehydrogenase large-subunit homolog and four enzymes of the tetrahydromethanopterin-dependent C₁ oxidation pathway were identified in B18LD. No evidence of methanotrophy was detected.     

The tyrosine phosphorylation of Munc18c induces a switch in binding specificity from syntaxin 4 to Doc2beta

Glucose-stimulated insulin secretion is mediated by syntaxin 4-based SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein complexes and the Sec1/Munc18 protein Munc18c. Our laboratory recently reported that Munc18c-syntaxin 4 complexes are further regulated by the competitive binding of the double C2 domain protein Doc2beta to Munc18c, although the underlying mechanism for this is unknown. Because the Doc2beta binding region of Munc18c contained residue Tyr-219 and this residue becomes phosphorylated in response to glucose stimulation, we hypothesized that the mechanism would involve Munc18c phosphorylation. Coimmunoprecipitation analyses using detergent lysates prepared from pervanadate-treated MIN6 beta cells revealed that the tyrosine phosphorylation of Munc18c corresponded to a 60% decrease in Munc18c-syntaxin 4 association with a coordinate 2-fold increase in Munc18c-Doc2beta binding. In vitro binding assays identified syntaxin 4 residues 118-194 as sufficient to confer its interaction with Munc18c; residues 118-194 contain the Hc alpha-helix and flexible linker region controlling transition of syntaxins between closed and open conformations. When overexpressed in MIN6 cells, this Hc-linker region functioned as a competitive inhibitor of endogenous syntaxin 4-Munc18c binding, increased syntaxin 4 binding to VAMP2, and significantly enhanced glucose-stimulated secretion. Molecular modeling of these new interactions yielded the predictions 1) that Tyr-219 of Munc18c remains buried under basal conditions in a conformation that is favorable for interaction with "closed" syntaxin 4 and 2) that stimulation leads to changes in syntaxin 4 contacts to facilitate exposure of Munc18c Tyr-219 for phosphorylation and Doc2beta binding.     

Protection against respiratory syncytial virus by a recombinant Newcastle disease virus vector

Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract disease in infants and the elderly, but no safe and effective RSV vaccine is yet available. For reasons that are not well understood, RSV is only weakly immunogenic, and reinfection occurs throughout life. This has complicated the search for an effective live attenuated viral vaccine, and past trials with inactivated virus preparations have led to enhanced immunopathology following natural infection. We have tested the hypothesis that weak stimulation of innate immunity by RSV correlates with ineffective adaptive responses by asking whether expression of the fusion glycoprotein of RSV by Newcastle disease virus (NDV) would stimulate a more robust immune response to RSV than primary RSV infection. NDV is a potent inducer of both alpha/beta interferon (IFN-alpha/beta) production and dendritic cell maturation, while RSV is not. When a recombinant NDV expressing the RSV fusion glycoprotein was administered to BALB/c mice, they were protected from RSV challenge, and this protection correlated with a robust anti-F CD8+ T-cell response. The effectiveness of this vaccine construct reflects the differential abilities of NDV and RSV to promote dendritic cell maturation and is retained even in the absence of a functional IFN-alpha/beta receptor.     

Impact of dose,route, and composition on the immunogenicity of immune polyelectrolyte multilayers delivered on gold templates

Biomaterial vaccines offer new capabilities that can be exploited for both infectious disease and cancer. We recently developed a novel vaccine platform based on self‐assembly of immune signals into immune polyelectrolyte multilayers (iPEMs). These iPEM vaccines are electrostatically assembled from peptide antigens and nucleic acid‐based toll‐like receptor agonists (TLRas) that serve as molecular adjuvants. Gold nanoparticles (AuNPs) coated with iPEMs stimulate effector cytokine secretion in vitro and expand antigen‐specific T cells in mice. Here we investigated how the dose, injection route, and choice of molecular adjuvant impacts the ability of iPEMs to generate T cell immunity and anti‐tumor response in mice. Three injection routes—intradermal, subcutaneous, and intramuscular—and three iPEM dosing levels were employed. Intradermal injection induced the most potent antigen‐specific T cell responses and, for all routes, the level of response was dose‐dependent. We further discovered that these vaccines generate durable memory, indicated by potent, antigen‐specific CD8⁺ T cell recall responses in mice challenged with vaccine 49 days after a prime‐boost immunization regimen. In a common exogenous antigen melanoma model, iPEM vaccines slowed or stopped tumor growth more effectively than equivalent ad‐mixed formulations. Further, iPEMs containing CpG—a TLR9a—were more potent compared with iPEMs containing polyIC, a TLR3a. These findings demonstrate the ability of iPEMs to enhance response to several different classes of vaccine cargos, supporting iPEMs as a simple vaccine platform that mimics attractive features of other nanoparticles using immune signals that can be self‐assembled or coated on substrates. Biotechnol. Bioeng. 2017;114: 423–431. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

Structure guided design of a series of selective pyrrolopyrimidinone MARK inhibitors

The initial structure activity relationships around an isoindoline uHTS hit will be described. Information gleaned from ligand co-crystal structures allowed for rapid refinements in both MARK potency and kinase selectivity. These efforts allowed for the identification of a compound with properties suitable for use as an in vitro tool compound for validation studies on MARK as a viable target for Alzheimer’s disease.

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Appropriateness of biotechnology to African agriculture: Striga and maize as paradigms

Plant Cell, Tissue and Organ Culture (PCTOC) -     

Recent advances in bio‐chemical,molecular and physiological aspects of membrane lipid derivatives in plant pathology

Plant pathogens pose a significant threat to the food industry and food security accounting for 10–40% crop losses annually on a global scale. Economic losses from plant diseases are estimated at $300B for major food crops and are associated with reduced food availability and accessibility and also high food costs. Although strategies exist to reduce the impact of diseases in plants, many of these introduce harmful chemicals to our food chain. Therefore, it is important to understand and utilize plants' immune systems to control plant pathogens to enable more sustainable agriculture. Lipids are core components of cell membranes and as such are part of the first line of defense against pathogen attack. Recent developments in omics technologies have advanced our understanding of how plant membrane lipid biosynthesis, remodelling and/or signalling modulate plant responses to infection. Currently, there is limited information available in the scientific literature concerning lipid signalling targets and their biochemical and physiological consequences in response to plant pathogens. This review focusses on the functions of membrane lipid derivatives and their involvement in plant responses to pathogens as biotic stressors. We describe major plant defense systems including systemic‐acquired resistance, basal resistance, hypersensitivity and the gene‐for‐gene concept in this context.     

S(-)-Nornicotine Increases Dopamine Release in a Calcium-Dependent Manner from Superfused Rat Striatal Slices

Abstract: The present study demonstrates that S (-)-nornicotine evoked a concentration-dependent increase in dopamine (DA) release from superfused rat striatal slices. The increase in DA release was indicated by an S (-)-nornicotine-induced overflow of endogenous 3,4-dihydroxyphenyl-acetic acid (DOPAC) in the striatal superfusate and by an S (-)-nornicotine-induced increase in tritium overflow from striatal slices preloaded with [³H]DA. Low concentrations (0.01–1.0 μ M ) of S (-)-nornicotine, which did not evoke endogenous DOPAC overflow, also were unable to modulate electrically evoked DOPAC overflow. The increase in DOPAC overflow induced by S (-)-nornicotine was compared with that produced by S (-)-nicotine. Comparing equimolar concentrations (0.1-100 μ M ) of S (-)-nornicotine and S (-)-nicotine, superfusion with S (-)-nornicotine resulted in a significantly greater DOPAC overflow. In contrast to the effect of S (-)-nicotine, S (-)-nornicotine evoked a sustained increase in DOPAC over-flow for the entire period of S (-)-nornicotine exposure. Furthermore, DOPAC overflow evoked by S (-)-nornicotine in control Krebs buffer was inhibited by superfusion with a low-calcium buffer. Moreover, in the low-calcium buffer, DOPAC overflow induced by 30 and 100 μ M S (-)-nornicotine was not different from that with no S (-)-nornicotine. The results indicate that S (-)-nornicotine, a constituent of tobacco products and a known metabolite of S (-)-nicotine, increases DA release in a calcium-dependent manner in superfused rat striatal slices. It is interesting that unlike S (-)-nicotine, there does not appear to be desensitization to this effect of S (-)-nornicotine.