The Sec1/Munc18 protein Vps45 regulates cellular levels of its SNARE binding partners Tlg2 and Snc2 in Saccharomyces cerevisiae

Intracellular membrane trafficking pathways must be tightly regulated to ensure proper functioning of all eukaryotic cells. Central to membrane trafficking is the formation of specific SNARE (soluble N-ethylmeleimide-sensitive factor attachment protein receptor) complexes between proteins on opposing lipid bilayers. The Sec1/Munc18 (SM) family of proteins play an essential role in SNARE-mediated membrane fusion, and like the SNAREs are conserved through evolution from yeast to humans. The SM protein Vps45 is required for the formation of yeast endosomal SNARE complexes and is thus essential for traffic through the endosomal system. Here we report that, in addition to its role in regulating SNARE complex assembly, Vps45 regulates cellular levels of its SNARE binding partners: the syntaxin Tlg2 and the v-SNARE Snc2: Cells lacking Vps45 have reduced cellular levels of Tlg2 and Snc2; and elevation of Vps45 levels results in concomitant increases in the levels of both Tlg2 and Snc2. As well as regulating traffic through the endosomal system, the Snc v-SNAREs are also required for exocytosis. Unlike most vps mutants, cells lacking Vps45 display multiple growth phenotypes. Here we report that these can be reversed by selectively restoring Snc2 levels in vps45 mutant cells. Our data indicate that as well as functioning as part of the machinery that controls SNARE complex assembly, Vps45 also plays a key role in determining the levels of its cognate SNARE proteins; another key factor in regulation of membrane traffic

Nematode Symbiont for Photorhabdus asymbiotica

Photorhabdus asymbiotica is an emerging bacterial pathogen that causes locally invasive soft tissue and disseminated bacteremic infections in the United States and Australia. Although the source of infection was previously unknown, we report that the bacterium is found in a symbiotic association with an insect-pathogenic soil nematode of the genus Heterorhabditis

From knock-out phenotype to three-dimensional structure of a promising antibiotic target from streptococcus pneumoniae

Given the rise in drug-resistant Streptococcus pneumoniae, there is an urgent need to discover new antimicrobials targeting this pathogen and an equally urgent need to characterize new drug targets. A promising antibiotic target is dihydrodipicolinate synthase (DHDPS), which catalyzes the rate-limiting step in lysine biosynthesis. In this study, we firstly show by gene knock out studies that S. pneumoniae (sp) lacking the DHDPS gene is unable to grow unless supplemented with lysine-rich media. We subsequently set out to characterize the structure, function and stability of the enzyme drug target. Our studies show that sp-DHDPS is folded and active with a kcat = 22 s-1 , KM PYR = 2.55 ± 0.05 mM and KM ASA = 0.044 ± 0.003 mM. Thermal denaturation experiments demonstrate sp-DHDPS exhibits an apparent melting temperature (TM app) of 72 °C, which is significantly greater than Escherichia coli DHDPS (Ec-DHDPS) (TM app = 59 °C). Sedimentation studies show that sp-DHDPS exists in a dimer-tetramer equilibrium with a KD 4→2 = 1.7 nM, which is considerably tighter than its E. coli ortholog (KD 4→2 = 76 nM). To further characterize the structure of the enzyme and probe its enhanced stability, we solved the high resolution (1.9 Å) crystal structure of sp-DHDPS (PDB ID 3VFL). The enzyme is tetrameric in the crystal state, consistent with biophysical measurements in solution. Although the sp-DHDPS and Ec-DHDPS active sites are almost identical, the tetramerization interface of the s. pneumoniae enzyme is significantly different in composition and has greater buried surface area (800 Å2 ) compared to its E. coli counterpart (500 Å2 ). This larger interface area is consistent with our solution studies demonstrating that sp-DHDPS is considerably more thermally and thermodynamically stable than Ec-DHDPS

Multiwavelength Study of M8.9/3B Solar Flare from AR NOAA 10960

We present a multi-wavelength analysis of a long duration white-light solar flare (M8.9/3B) event that occurred on 4 June 2007 from NOAA AR 10960. The flare was observed by several spaceborne instruments, namely SOHO/MDI, Hinode/SOT, TRACE and STEREO/SECCHI. The flare was initiated near a small, positive-polarity, satellite sunspot at the centre of the AR, surrounded by opposite-polarity field regions. MDI images of the AR show considerable amount of changes in a small positive-polarity sunspot of delta configuration during the flare event. SOT/G-band (4305 A) images of the sunspot also suggest the rapid evolution of the positive-polarity sunspot with highly twisted penumbral filaments before the flare event, which were oriented in the counterclockwise direction. It shows the change in orientation and also remarkable disappearance of twisted penumbral filaments (~35-40%) and enhancement in umbral area (~45-50%) during the decay phase of the flare. TRACE and SECCHI observations reveal the successive activations of two helical twisted structures associated with this sunspot, and the corresponding brightening in the chromosphere as observed by the time-sequence images of SOT/Ca II H line (3968 A). The secondary-helical twisted structure is found to be associated with the M8.9 flare event. The brightening starts 6-7 min prior to the flare maximum with the appearance of secondary helical-twisted structure. The flare intensity maximizes as this structure moves away from the AR. This twisted flux-tube associated with the flare triggering, is found to be failed in eruption. The location of the flare is found to coincide with the activation site of the helical twisted structures. We conclude that the activations of successive helical twists in the magnetic flux tubes/ropes plays a crucial role in the energy build-up process and triggering of M-class solar flare without a CME.Comment: 22 pages, 12 figures, Accepted for Publication in Solar Physic

Vaccination with DNA plasmids expressing Gn coupled to C3d or alphavirus replicons expressing Gn protects mice against rift valley fever virus

Background: Rift Valley fever (RVF) is an arthropod-borne viral zoonosis. Rift Valley fever virus (RVFV) is an important biological threat with the potential to spread to new susceptible areas. In addition, it is a potential biowarfare agent. Methodology/Principal Findings: We developed two potential vaccines, DNA plasmids and alphavirus replicons, expressing the Gn glycoprotein of RVFV alone or fused to three copies of complement protein, C3d. Each vaccine was administered to mice in an all DNA, all replicon, or a DNA prime/replicon boost strategy and both the humoral and cellular responses were assessed. DNA plasmids expressing Gn-C3d and alphavirus replicons expressing Gn elicited high titer neutralizing antibodies that were similar to titers elicited by the live-attenuated MP12 virus. Mice vaccinated with an inactivated form of MP12 did elicit high titer antibodies, but these antibodies were unable to neutralize RVFV infection. However, only vaccine strategies incorporating alphavirus replicons elicited cellular responses to Gn. Both vaccines strategies completely prevented weight loss and morbidity and protected against lethal RVFV challenge. Passive transfer of antisera from vaccinated mice into naïve mice showed that both DNA plasmids expressing Gn-C3d and alphavirus replicons expressing Gn elicited antibodies that protected mice as well as sera from mice immunized with MP12. Conclusion/Significance: These results show that both DNA plasmids expressing Gn-C3d and alphavirus replicons expressing Gn administered alone or in a DNA prime/replicon boost strategy are effective RVFV vaccines. These vaccine strategies provide safer alternatives to using live-attenuated RVFV vaccines for human use. © 2010 Bhardwaj et al

Human helminth therapy to treat inflammatory disorders - where do we stand?

Parasitic helminths have evolved together with the mammalian immune system over many millennia and as such they have become remarkably efficient modulators in order to promote their own survival. Their ability to alter and/or suppress immune responses could be beneficial to the host by helping control excessive inflammatory responses and animal models and pre-clinical trials have all suggested a beneficial effect of helminth infections on inflammatory bowel conditions, MS, asthma and atopy. Thus, helminth therapy has been suggested as a possible treatment method for autoimmune and other inflammatory disorders in humans

The impact of transglutaminase on soy protein and tofu texture

The enzyme transglutaminase was investigated for its cross-linking effect on the soy proteins of tofu. In vitro incubations confirmed that soy proteins are excellent substrates for transglutaminase, especially when denatured. The macroscopic effects resulting from the addition of transglutaminase were compared to changes at the microstructural and molecular level. Treatment produced a firmer tofu, with a significantly increased fracture force. Examination by SEM showed a change in the matrix structure, with transglutaminase resulting in a finer-stranded, uniform network that accounted for the increase in fracture force. At the molecular level, little, if any, cross-linking occurred within the tofu matrix in situ. This suggests that the change in functional properties afforded by addition of transglutaminase to tofu is due to a side reaction of the enzyme, for example hydrolysis of glutamine residues, rather than its cross-linking activity. These ideas are further explored in the accompanying paper

D6.7 Report on the experience of conducting the case studies

One of the main aims of the case studies was to publish improved market reports. The data collected as part of the six case studies have been, or will shortly be, published in the five improved national organic market reports and one first regional market report (MOAN case study). This will make a contribution towards filling the many gaps that continue to exist in organic market data collection in Europe