Differential induction of type I interferon responses in myeloid dendritic cells by mosquito and mammalian-cell-derived alphaviruses

Dendritic cells (DCs) are an important early target cell for many mosquito-borne viruses, and in many cases mosquito-cell-derived arboviruses more efficiently infect DCs than viruses derived from mammalian cells. However, whether mosquito-cell-derived viruses differ from mammalian-cell-derived viruses in their ability to induce antiviral responses in the infected dendritic cell has not been evaluated. In this report, alphaviruses, which are mosquito-borne viruses that cause diseases ranging from encephalitis to arthritis, were used to determine whether viruses grown in mosquito cells differed from mammalian-cell-derived viruses in their ability to induce type I interferon (IFN) responses in infected primary dendritic cells. Consistent with previous results, mosquito-cell-derived Ross River virus (mos-RRV) and Venezuelan equine encephalitis virus (mos-VEE) exhibited enhanced infection of primary myeloid dendritic cells (mDCs) compared to mammalian-cell-derived virus preparations. However, unlike the mammalian-cell-derived viruses, which induced high levels of type I IFN in the infected mDC cultures, mos-RRV and mos-VEE were poor IFN inducers. Furthermore, the poor IFN induction by mos-RRV contributed to the enhanced infection of mDCs by mos-RRV. These results suggest that the viruses initially delivered by the mosquito vector differ from those generated in subsequent rounds of replication in the host, not just with respect to their ability to infect dendritic cells but also in their ability to induce or inhibit antiviral type I IFN responses. This difference may have an important impact on the mosquito-borne virus's ability to successfully make the transition from the arthropod vector to the vertebrate host.     

Food quantity and quality regulation of trophic transfer between primary producers and a keystone grazer (Daphnia) in pelagic freshwater food webs

The transfer of energy and nutrients from plants to animals is a key process in all ecosystems. In lakes, inefficient transfer of primary producer derived energy can result in low animal growth rates, accumulation of nuisance phytoplankton blooms and dissipation of energy from the ecosystem. Most research on carbon transfer efficiency in pelagic food webs has focused on either food quantity or food quality, with the latter considered separately as either elemental stoichiometry or biochemical composition. The natural occurrence and magnitude of these types of growth limitations and their combined effects on Daphnia , a keystone grazer in pelagic freshwater ecosystems, are largely unknown. Our empirical models predict that the strength and nature of food quantity and quality limitation varies greatly with lake trophic state (total phosphorus, TP) and that Daphnia growth rates and thus energy and nutrient transfer efficiency are highest in lakes with intermediate trophic status (TP 10–25 μg l⁻¹). We predict that food availability place the greatest constraint on Daphnia growth in nutrient poor lakes (TP≤4 μg l⁻¹). Phosphorus limitation of Daphnia growth increased with decreasing TP, but the overall effect was never predicted to be the dominant constraining factor. Eicosapentaenoic acid (EPA, 20:5ω3) limitation was predicted to occur in both nutrient poor and nutrient rich lakes and placed the primary constraint on food quality in the most productive lakes. Two contrasting EPA-models gave different results on the magnitude of EPA-limitation, implying that additional food quality factors decrease Daphnia growth at high TP. In conclusion, the model predicts that Daphnia growth should peak in mesotrophic lakes, food quantity will place the greatest constraint on growth in oligotrophic lakes and EPA will primarily limit growth in eutrophic lakes.     

Characterisation and biological activity of Glu3 amino acid substituted GIP receptor antagonists

Glucose-dependent insulinotropic polypeptide (GIP) is an important gastrointestinal hormone, which regulates insulin release and glucose homeostasis, but is rapidly inactivated by enzymatic N-terminal truncation. Here we report the enzyme resistance and biological activity of several Glu(3)-substituted analogues of GIP namely; (Ala(3))GIP, (Lys(3))GIP, (Phe(3))GIP, (Trp(3))GIP and (Tyr(3))GIP. Only (Lys(3))GIP demonstrated moderately enhanced resistance to DPP-IV (p<0.05 to p<0.01) compared to native GIP. All analogues demonstrated a decreased potency in cAMP production (EC(50) 1.47 to 11.02 nM; p<0.01 to p<0.001) with (Lys(3))GIP and (Phe(3))GIP significantly inhibiting GIP-stimulated cAMP production (p<0.05). In BRIN-BD11 cells, (Lys(3))GIP, (Phe(3))GIP, (Trp(3))GIP and (Tyr(3))GIP did not stimulate insulin secretion with both (Lys(3))GIP and (Phe(3))GIP significantly inhibiting GIP-stimulated insulin secretion (p<0.05). Injection of each GIP analogue together with glucose in ob/ob mice significantly increased the glycaemic excursion compared to control (p<0.05 to p<0.001). This was associated with lack of significant insulin responses. (Ala(3))GIP, (Phe(3))GIP and (Tyr(3))GIP, when administered together with GIP, significantly reduced plasma insulin (p<0.05 to p<0.01) and impaired the glucose-lowering ability (p<0.05 to p<0.01) of the native peptide. The DPP-IV resistance and GIP antagonism observed were similar but less pronounced than (Pro(3))GIP. These data demonstrate that position 3 amino acid substitution of GIP with (Ala(3)), (Phe(3)), (Tyr(3)) or (Pro(3)) provides a new class of functional GIP receptor antagonists.     

Hierarchical delivery of an essential host colonization factor in enteropathogenic Escherichia coli

Many significant bacterial pathogens use a type III secretion system to inject effector proteins into host cells to disrupt specific cellular functions, enabling disease progression. The injection of these effectors into host cells is often dependent on dedicated chaperones within the bacterial cell. In this report, we demonstrate that the enteropathogenic Escherichia coli (EPEC) chaperone CesT interacts with a variety of known and putative type III effector proteins. Using pull-down and secretion assays, a degenerate CesT binding domain was identified within multiple type III effectors. Domain exchange experiments between selected type III effector proteins revealed a modular nature for the CesT binding domain, as demonstrated by secretion, chaperone binding, and infection assays. The CesT-interacting type III effector Tir, which is crucial for in vivo intestinal colonization, had to be expressed and secreted for efficient secretion of other type III effectors. In contrast, the absence of other CesT-interacting type III effectors did not abrogate effector secretion, indicating an unexpected hierarchy with respect to Tir for type III effector delivery. Coordinating the expression of other type III effectors with cesT in the absence of tir partially restored total type III effector secretion, thereby implicating CesT in secretion events. Collectively, the results suggest a coordinated mechanism involving both Tir and CesT for type III effector injection into host cells.     

Mycobacterium avium subspecies paratuberculosis in Bison (Bison bison) from Northern Canada

Nested polymerase chain reaction (PCR) using the Mycobacterium avium subspecies paratuberculosis (Map)-specific region, locus 251, was used as a screening tool for the detection of Map DNA in fecal samples from northern Canadian bison herds. Further characterization of positive samples (26/835) was performed because Map DNA was found without signs of disease. Strain typing, using PCR-Restriction endonucleas assay (REA), was limited to two samples but revealed that the samples corresponded to a cattle-related strain and a sheep-related strain. Sequencing of part of the IS1311 region from the two samples revealed a unique three base-pair region, which is only found within the northern Canadian bison isolates.     

Cdk-inhibitory activity and stability of p27Kip1 are directly regulated by oncogenic tyrosine kinases

p27Kip1 controls cell proliferation by binding to and regulating the activity of cyclin-dependent kinases (Cdks). Here we show that Cdk inhibition and p27 stability are regulated through direct phosphorylation by tyrosine kinases. A conserved tyrosine residue (Y88) in the Cdk-binding domain of p27 can be phosphorylated by the Src-family kinase Lyn and the oncogene product BCR-ABL. Y88 phosphorylation does not prevent p27 binding to cyclin A/Cdk2. Instead, it causes phosphorylated Y88 and the entire inhibitory 3(10)-helix of p27 to be ejected from the Cdk2 active site, thus restoring partial Cdk activity. Importantly, this allows Y88-phosphorylated p27 to be efficiently phosphorylated on threonine 187 by Cdk2 which in turn promotes its SCF-Skp2-dependent degradation. This direct link between transforming tyrosine kinases and p27 may provide an explanation for Cdk kinase activities observed in p27 complexes and for premature p27 elimination in cells that have been transformed by activated tyrosine kinases.     

Alkyne substrate interaction within the nitrogenase MoFe protein

Nitrogenase catalyzes the biological reduction of N(2) to ammonia (nitrogen fixation), as well as the two-electron reduction of the non-physiological alkyne substrate acetylene (HC triple bond CH). A complex metallo-organic species called FeMo-cofactor provides the site of substrate reduction within the MoFe protein, but exactly where and how substrates interact with FeMo-cofactor remains unknown. Recent results have shown that the MoFe protein alpha-70(Val) residue, whose side chain approaches one Fe-S face of FeMo-cofactor, plays a significant role in defining substrate access to the active site. For example, substitution of alpha-70(Val) by alanine results in an increased capacity for the reduction of the larger alkyne propyne (HC triple bond C-CH(3)), whereas, substitution by isoleucine at this position nearly eliminates the capacity for the reduction of acetylene. These and complementary spectroscopic studies led us to propose that binding of short chain alkynes occurs with side-on binding to Fe atom 6 within FeMo-cofactor. In the present work, the alpha-70(Val) residue was substituted by glycine and this MoFe protein variant shows an increased capacity for reduction of the terminal alkyne, 1-butyne (HC triple bond C-CH(2)-CH(3)). This protein shows no detectable reduction of the internal alkyne 2-butyne (H(3)C-C triple bond C-CH(3)). In contrast, substitution of the nearby alpha-191(Gln) residue by alanine, in combination with the alpha-70(Ala) substitution, does result in significant reduction of 2-butyne, with the exclusive product being 2-cis-butene. These results indicate that the reduction of alkynes by nitrogenases involves side-on binding of the alkyne to Fe6 within FeMo-cofactor, and that a terminal acidic proton is not required for reduction. The successful design of amino acid substitutions that permit the targeted accommodation of an alkyne that otherwise is not a nitrogenase substrate provides evidence to support the current model for alkyne interaction within the nitrogenase MoFe protein.     

Association of ALOX5AP with ischemic stroke: a population-based case-control study

Arachidonate 5-lipoxygenase activating protein (ALOX5AP) has been reported to demonstrate linkage and association with ischemic stroke and myocardial infarction. However, replication studies have been conflicting and to date, a significant proportion of blacks have not been studied. We prospectively recruited cases of ischemic stroke from all 16 hospitals in the Greater Cincinnati/Northern Kentucky region and demographically matched them to stroke-free population-based controls. Single nucleotide polymorphisms (SNPs) were selected based on association with ischemic stroke in prior studies. Allelic, genotypic and haplotypic association testing was performed using HAPLOVIEW. Multiple logistic regression was used to control for the presence of traditional risk factors including hypertension, diabetes, hypercholesterolemia and smoking. A total of 357 cases and 482 controls were genotyped. The SNPs, rs9579646 and rs4769874 were found to be significantly associated at both allelic (P = 0.019 and P < 10⁻⁴, respectively) and genotypic level with ischemic stroke among whites after correction for multiple testing. Haplotype association was identified with ischemic stroke as well as ischemic stroke subtypes among whites. Although an overall haplotype association with ischemic stroke was identified among blacks no evidence of association among individual haplotypes, alleles or genotypes were observed. Allele frequencies for the SNPs examined were markedly different among whites and blacks. In conclusion, we report significant association of variants of ALOX5AP with ischemic stroke and ischemic stroke subtypes among whites. No significant association was identified among blacks.