Phosphorylation-dependent binding of 14-3-3 terminates signalling by the Gab2 docking protein

Grb2-associated binder (Gab)2 functions downstream of a variety of receptor and cytoplasmic tyrosine kinases as a docking platform for specific signal transducers and performs important functions in both normal physiology and oncogenesis. Gab2 signalling is promoted by its association with specific receptors through the adaptor Grb2. However, the molecular mechanisms that attenuate Gab2 signals have remained unclear. We now demonstrate that growth factor-induced phosphorylation of Gab2 on two residues, S210 and T391, leads to recruitment of 14-3-3 proteins. Together, these events mediate negative-feedback regulation, as Gab2^S210A/T391A exhibits sustained receptor association and signalling and promotes cell proliferation and transformation. Importantly, introduction of constitutive 14-3-3-binding sites into Gab2 renders it refractory to receptor activation, demonstrating that site-selective binding of 14-3-3 proteins is sufficient to terminate Gab2 signalling. Furthermore, this is associated with reduced binding of Grb2. This leads to a model where signal attenuation occurs because 14-3-3 promotes dissociation of Gab2 from Grb2, and thereby uncouples Gab2 from the receptor complex. This represents a novel regulatory mechanism with implications for diverse tyrosine kinase signalling systems.     

Core alpha1,3-fucose is a key part of the epitope recognized by antibodies reacting against plant N-linked oligosaccharides and is present in a wide variety of plant extracts

Carbohydrates have been suggested to account for some IgE cross- reactions between various plant, insect, and mollusk extracts, while some IgG antibodies have been successfully raised against plant glycoproteins. A rat monoclonal antibody raised against elderberry abscission tissue (YZ1/2.23) and rabbit polyclonal antiserum against horseradish peroxidase were screened for reactivity in enzyme-linked immunosorbent assay against a range of plant glycoproteins and extracts as well as neoglycoproteins, bee venom phospholipase, and several animal glycoproteins. Of the oligosaccharides tested, Man3XylFucGlcNAc2(MMXF3) derived from horseradish peroxidase was the most potent inhibitor of the reactivity of both YZ1/2.23 and anti- horseradish peroxidase to native horseradish peroxidase glycoprotein. The reactivity of YZ1/2. 23 and anti-horseradish peroxidase against Sophora japonica lectin was most inhibited by a neoglycoconjugate of bromelain glycopeptide cross-linked to bovine serum albumin, while the defucosylated form of this conjugate was inactive as an inhibitor. A wide range of plant extracts was found to react against YZ1/2.23 and anti-horseradish peroxidase, with particularly high reactivities recorded for grass pollen and nut extracts. All these reactivities were inhibitable with the bromelain glycopeptide/bovine serum albumin conjugate. Bee venom phospholipase and whole bee venom reacted weakly with YZ1/2.23 but more strongly with anti-horseradish peroxidase in a manner inhibitable with the bromelain glycopeptide/bovine serum albumin conjugate, while hemocyanin from Helix pomatia reacted poorly with YZ1/2.23 but did react with anti-horseradish peroxidase. It is concluded that the alpha1, 3-fucose residue linked to the chitobiose core of plant glycoproteins is the most important residue in the epitope recognized by the two antibodies studied, but that the polyclonal anti-horseradish peroxidase antiserum also contains antibody populations that recognize the xylose linked to the core mannose of many plant and gastropod N-linked oligosaccharides.      

Endothelial progenitor cells: Characterization, <Emphasis Type="Italic">in vitro</Emphasis> expansion,and prospects for autologous cell therapy

Injection of hematopoietic stem cells or endothelial progenitor cells (EPCs) expanded ex vivo has been shown to augment neovascularization in adult patients, but the precise origin and identity of the cell population responsible for these clinical benefits are controversial. The limited quantity of EPCs in the circulation has been the main obstacle to clinical trials. Several authors have therefore attempted to expand these cells ex vivo in order to obtain a homogeneous cell therapy product. One possible means of expanding EPCs ex vivo is to activate the thrombin receptor PAR-1 with the specific peptide SFLLRN. Indeed, PAR-1 activation promotes cell proliferation and C-X-C chemokine receptor type 4 (CXCR4) dependent migration and differentiation, with an overall angiogenic effect. This review summarizes the results and rationale of clinical trials of angiogenic therapy, the nature of EPCs, the different methods of ex vivo expansion, and current methods of quantification.     

Developmental studies on two ecdysone deficient mutants ofDrosophila melanogaster

Summary This paper describes two ecdysone-deficient, recessive-lethal mutants,lethal(1)giant ring gland (grg) andlethal(1)suppressor of forked ^mad-ts (mad-ts: Jürgens and Gateff 1979) and compares their ecdysteroid titers with that of the wild-type. Mutant larvae show a much reduced ecdysteroid content, amounting to 1/10 to 1/30 of the wild-type values, but never a true titer peak. They fail to pupate and die after 1–3 weeks. Ecdysteroid feeding elicits different responses in the larvae of the two mutants.mad-ts larvae pupate within 24 h, thus showing that their low ecdysteroid titer is directly connected to their inability to pupate.mad-ts resembles the mutantlethal (3)ecdysone-1 ^ts (Garen et al. 1977). Thegrg mutant larvae, on the other hand, fail to pupate after 20-hydroxyecdysone feeding as well as injection. The primary defect of thegrg mutant is not entirely clear. Thegrg larval salivary gland cells appear to possess normal ecdysteroid receptors. Furthermore, the low ecdysteroid titer ingrg is not the result of an increased ecdysteroid catabolism. The primary defect in the mutant may lie in the malfunctioning neurosecretory cells which do not show neurosecretion in histological preparations. Further support for this notion comes from electronmicrographs of the enlargedgrg ring glands which, in contrast to the wild-type, do not possess nerve endings.In the wild-type three ecdysteroid peaks were found: one shortly before puparium formation, the second at approximately 12 h and the third at about 30 h after pupation. The ecdysteroid titer peak in late third instar, wild-type larvae is mainly due to the presence of 20-dydroxyecdysone as shown by radioimmunoassays after thin layer chromatography and derivatization followed by gas liquid chromatography and mass spectroscopy. In addition, a number of unidentified polar and apolar metabolites were also present.     

A topologically distinct class of photolyases specific for UV lesions within single-stranded DNA

Photolyases are ubiquitously occurring flavoproteins for catalyzing photo repair of UV-induced DNA damages. All photolyases described so far have a bilobal architecture with a C-terminal domain comprising flavin adenine dinucleotide (FAD) as catalytic cofactor and an N-terminal domain capable of harboring an additional antenna chromophore. Using sequence-similarity network analysis we discovered a novel subgroup of the photolyase/cryptochrome superfamily (PCSf), the NewPHLs. NewPHL occur in bacteria and have an inverted topology with an N-terminal catalytic domain and a C-terminal domain for sealing the FAD binding site from solvent access. By characterizing two NewPHL we show a photochemistry characteristic of other PCSf members as well as light-dependent repair of CPD lesions. Given their common specificity towards single-stranded DNA many bacterial species use NewPHL as a substitute for DASH-type photolyases. Given their simplified architecture and function we suggest that NewPHL are close to the evolutionary origin of the PCSf.     

Low temperature delays timing and enhances the cost of nitrogen fixation in the unicellular cyanobacterium Cyanothece

Marine nitrogen-fixing cyanobacteria are largely confined to the tropical and subtropical ocean. It has been argued that their global biogeographical distribution reflects the physiologically feasible temperature range at which they can perform nitrogen fixation. In this study we refine this line of argumentation for the globally important group of unicellular diazotrophic cyanobacteria, and pose the following two hypotheses: (i) nitrogen fixation is limited by nitrogenase activity at low temperature and by oxygen diffusion at high temperature, which is manifested by a shift from strong to weak temperature dependence of nitrogenase activity, and (ii) high respiration rates are required to maintain very low levels of oxygen for nitrogenase, which results in enhanced respiratory cost per molecule of fixed nitrogen at low temperature. We tested these hypotheses in laboratory experiments with the unicellular cyanobacterium Cyanothece sp. {"type":"entrez-nucleotide","attrs":{"text":"BG043511","term_id":"12489990","term_text":"BG043511"}}BG043511. In line with the first hypothesis, the specific growth rate increased strongly with temperature from 18 to 30 °C, but leveled off at higher temperature under nitrogen-fixing conditions. As predicted by the second hypothesis, the respiratory cost of nitrogen fixation and also the cellular C:N ratio rose sharply at temperatures below 21 °C. In addition, we found that low temperature caused a strong delay in the onset of the nocturnal nitrogenase activity, which shortened the remaining nighttime available for nitrogen fixation. Together, these results point at a lower temperature limit for unicellular nitrogen-fixing cyanobacteria, which offers an explanation for their (sub)tropical distribution and suggests expansion of their biogeographical range by global warming.     

Comparison of diagnostic performances of ten different immunoassays detecting anti-CCHFV IgM and IgG antibodies from acute to subsided phases of Crimean-Congo hemorrhagic fever

Crimean-Congo Hemorrhagic Fever Virus (CCHFV) is a geographically widespread tick-borne arbovirus that has been recognized by the WHO as an emerging pathogen needing urgent attention to ensure preparedness for potential outbreaks. Therefore, availability of accurate diagnostic tools for identification of acute cases is necessary.A panel comprising 121 sequential serum samples collected during acute, convalescent and subsided phase of PCR-proven CCHFV infection from 16 Kosovar patients was used to assess sensitivity. Serum samples from 60 healthy Kosovar blood donors were used to assess specificity. All samples were tested with two IgM/IgG immunofluorescence assays (IFA) from BNITM, the CCHFV Mosaic 2 IgG and IgM indirect immunofluorescence tests (IIFT) from EUROIMMUN, two BlackBox ELISAs for the detection of CCHFV-specific IgM and IgG antibodies (BNITM), two Anti-CCHFV ELISAs IgM and IgG from EUROIMMUN using recombinant structural proteins of CCHFV antigens, and two ELISAs from Vector-Best (IgM: μ-capture ELISA, IgG: indirect ELISA using immobilized CCHFV antigen). Diagnostic performances were compared between methods using sensitivity, specificity, concordance and degree of agreement with particular focus on the phase of the infection.In early and convalescent phases of infection, the sensitivities for detecting specific IgG antibodies differed for the ELISA test. The BlackBox IgG ELISA yielded the highest, followed by the EUROIMMUN IgG ELISA and finally the VectorBest IgG ELISA with the lowest sensitivities. In the subsided phase, the VectorBest IgM ELISA detected a high rate of samples that were positive for anti-CCHFV IgM antibodies. Both test systems based on immunofluorescence showed an identical sensitivity for detection of anti-CCHFV IgM antibodies in acute and convalescent phases of infection.Available serological test systems detect anti-CCHFV IgM and IgG antibodies accurately, but their diagnostic performances vary with respect to the phase of the infection.     

Long-Term Survival of an Urban Fruit Bat Seropositive for Ebola and Lagos Bat Viruses

Ebolaviruses (EBOV) (family Filoviridae) cause viral hemorrhagic fevers in humans and non-human primates when they spill over from their wildlife reservoir hosts with case fatality rates of up to 90%. Fruit bats may act as reservoirs of the Filoviridae. The migratory fruit bat, Eidolon helvum, is common across sub-Saharan Africa and lives in large colonies, often situated in cities. We screened sera from 262 E. helvum using indirect fluorescent tests for antibodies against EBOV subtype Zaire. We detected a seropositive bat from Accra, Ghana, and confirmed this using western blot analysis. The bat was also seropositive for Lagos bat virus, a Lyssavirus, by virus neutralization test. The bat was fitted with a radio transmitter and was last detected in Accra 13 months after release post-sampling, demonstrating long-term survival. Antibodies to filoviruses have not been previously demonstrated in E. helvum. Radio-telemetry data demonstrates long-term survival of an individual bat following exposure to viruses of families that can be highly pathogenic to other mammal species. Because E. helvum typically lives in large urban colonies and is a source of bushmeat in some regions, further studies should determine if this species forms a reservoir for EBOV from which spillover infections into the human population may occur.