Structure of the PII signal transduction protein of Neisseria meningitidis at 1.85 Ȃ resolution

Copyright @ 2006 International Union of CrystallographyThe PII signal transduction proteins GlnB and GlnK are implicated in the regulation of nitrogen assimilation in Escherichia coli and other enteric bacteria. PII-like proteins are widely distributed in bacteria, archaea and plants. In contrast to other bacteria, Neisseria are limited to a single PII protein (NMB 1995), which shows a high level of sequence identity to GlnB and GlnK from Escherichia coli (73 and 62%, respectively). The structure of the PII protein from N. meningitidis (serotype B) has been solved by molecular replacement to a resolution of 1.85 Ȃ. Comparison of the structure with those of other PII proteins shows that the overall fold is tightly conserved across the whole population of related proteins, in particular the positions of the residues implicated in ATP binding. It is proposed that the Neisseria PII protein shares functions with GlnB/GlnK of enteric bacteria.This study is funded by the Medical Research Council UK and Europe (SPINE) consortium (European Commission Grant No. QLG2-CT-2002-00988)

Proceedings of the 2nd IWDG International Whale Conference. Muc Mhara Ireland's Smallest Whale

Muc Mhara – Ireland’s smallest whale. Proceedings of the 2nd Irish Whale and Dolphin Group International Whale Conference. Papers presented include, “Introduction: The harbour porpoise or Muc Mhara”, “An Irish name for the humble harbour porpoise”, “Life in the Fast Lane: Ecology and Behaviour of harbour porpoises in the Gulf of Maine”, “The ecology of harbour porpoise (Phocoena phocoena) in Irish waters: what strandings programmes tell us.”, “Passive acoustic monitoring of the harbour porpoise (Phocoena phocoena) in Irish waters”, “Abundance estimates of harbour porpoises in Irish waters”, “Satellite tracking of harbour porpoises in European Waters”, “Satellite tracking of harbour porpoises in European Waters”, “A cost of green energy: Are offshore renewables: a threat to porpoises?”, “Harbour porpoise populations and protection in an EU context”, “Assessment of Acoustic Deterrent Devices ‘Pingers’ and porpoise by catch rates in Irish Gillnet Fisheries in the Celtic Sea” and “Harbour porpoise Conservation in the Republic of Ireland”

Polychlorinated Biphenyls and Organochlorines in By-Caught Harbour Porpoises Phocoena phocoena and Common Dolphins Delphinus delphis from Irish Coastal Waters

Peer-reviewedConcentrations of 11 organochlorine (OC) pesticides and 10 individual polychlorinated biphenyls (PCB) in blubber and liver from 12 harbour porpoise Phocoena phocoena and eight common dolphins Delphinus delphis incidentally caught in fishing nets in Irish waters are presented. Female harbour porpoises had highest concentrations of OC in blubber and male common dolphins in liver. Harbour porpoises had higher mean concentrations of lindane (121-154 ng/g extractable lipid), dieldrin (116-121 ng/g) and BHC (54-128 ng/g) but common dolphins had greater overall concentrations of DDT (9444-3998 ng/g). The predominant DDT metabolite was pp-DDE and for the chlordanes was t-nonachlor. Concentrations of ICES 7 PCB (liver-blubber) were similar in both species (4075-7999 ng/g in harbour porpoise and 4076-8945 in common dolphins). The sum of ICES 7 PCB in porpoises ranged from 3041-12270 ng/g extractable lipid in the blubber of females and from 2911-10429 ng/g in males and 798-11074 ng/g in the blubber of female common dolphins and 1555-15883 ng/g in males. Contaminant levels were generally similar to those reported from Scotland but lower than reported from Scandinavia. Ratios of DDT to DDE suggests that there are limited new sources of DDT into the Irish marine environment. These results provide a baseline for monitoring of persistent pollutants in the Irish marine environment

Crystal structure of nitrogen regulatory protein IIA(Ntr )from Neisseria meningitidis

BACKGROUND: The NMB0736 gene of Neisseria meningitidis serogroup B strain MC58 encodes the putative nitrogen regulatory protein, IIA(Ntr )(abbreviated to NM-IIA(Ntr)). The homologous protein present in Escherichia coli is implicated in the control of nitrogen assimilation. As part of a structural proteomics approach to the study of pathogenic Neisseria spp., we have selected this protein for structure determination by X-ray crystallography. RESULTS: The NM-IIA(Ntr )was over-expressed in E. coli and was shown to be partially mono-phosphorylated, as assessed by mass spectrometry of the purified protein. Crystals of un-phosphorylated protein were obtained and diffraction data collected to 2.5 Å resolution. The structure of NM-IIA(Ntr )was solved by molecular replacement using the coordinates of the E. coli nitrogen regulatory protein IIA(ntr )[PDB: 1A6J] as the starting model. The overall fold of the Neisseria enzyme shows a high degree of similarity to the IIA(Ntr )from E. coli, and the position of the phosphoryl acceptor histidine residue (H67) is conserved. The orientation of an adjacent arginine residue (R69) suggests that it may also be involved in coordinating the phosphate group. Comparison of the structure with that of E. coli IIA(mtl )complexed with HPr [PDB: 1J6T] indicates that NM-IIA(Ntr )binds in a similar way to the HPr-like enzyme in Neisseria. CONCLUSION: The structure of NM-IIA(Ntr )confirms its assignment as a homologue of the IIA(Ntr )proteins found in a range of other Gram-negative bacteria. We conclude that the NM- IIA(Ntr )protein functions as part of a phosphorylation cascade which, in contrast to E. coli, shares the upstream phosphotransfer protein with the sugar uptake phosphoenolpyruvate:sugar phosphotransferase system (PTS), but in common with E. coli has a distinct downstream effector mechanism

A versatile ligation-independent cloning method suitable for high-throughput expression screening applications

This article describes the construction of a set of versatile expression vectors based on the In-Fusion™ cloning enzyme and their use for high-throughput cloning and expression screening. Modifications to commonly used vectors rendering them compatible with In-Fusion™ has produced a ligation-independent cloning system that is (1) insert sequence independent (2) capable of cloning large PCR fragments (3) efficient over a wide (20-fold) insert concentration range and (4) applicable to expression in multiple hosts. The system enables the precise engineering of (His6-) tagged constructs with no undesirable vector or restriction-site-derived amino acids added to the expressed protein. The use of a multiple host-enabled vector allows rapid screening in both E. coli and eukaryotic hosts (HEK293T cells and insect cell hosts, e.g. Sf9 cells). These high-throughput screening activities have prompted the development and validation of automated protocols for transfection of mammalian cells and Ni-NTA protein purification

Structure of the PII signal transduction protein of Neisseria meningitidis at 1.85 Å resolution

The structure of the PII signal transduction protein of N. meningitidis at 1.85 Å resolution is described

Standing up in Multiple Sclerosis (SUMS): Protocol for a multi-centre randomised controlled trial evaluating the clinical and cost effectiveness of a home-based self-management standing frame programme in people with progressive multiple sclerosis.

This study is funded by the NIHR Health Technology Assessment Programme (14/176/12), United Kingdom.Background:  Impaired mobility is a cardinal feature of multiple sclerosis (MS) and is rated by people with MS as their highest priority. By the secondary progressive phase, balance, mobility and physical activity levels are significantly compromised; an estimated 70% of people with secondary progressive MS fall regularly. Our ongoing research has systematically developed ‘Balance Right in MS’ (BRiMS), an innovative, manualised 13-week guided self-management programme tailored to the needs of people with MS, designed to improve safe mobility and minimise falls. Our eventual aim is to assess the clinical and cost effectiveness of BRiMS in people with secondary progressive MS by undertaking an appropriately statistically powered, multi-centre, assessor-blinded definitive, randomised controlled trial. This feasibility study will assess the acceptability of the intervention and test the achievability of running such a definitive trial. Methods/design:  This is a pragmatic multi-centre feasibility randomised controlled trial with blinded outcome assessment. Sixty ambulant people with secondary progressive MS who self-report two or more falls in the previous 6 months will be randomly allocated (1:1) to either the BRiMS programme plus usual care or to usual care alone. All participants will be assessed at baseline and followed up at 15 weeks and 27 weeks post-randomisation. The outcomes of this feasibility trial include: • Feasibility outcomes, including trial recruitment, retention and completion • Assessment of the proposed outcome measures for the anticipated definitive trial (including measures of walking, quality of life, falls, balance and activity level) • Measures of adherence to the BRiMS programme • Data to inform the economic evaluation in a future trial • Process evaluation (assessment of treatment fidelity and qualitative evaluation of participant and treating therapist experience) Discussion:  The BRiMS intervention aims to address a key concern for MS service users and providers. However, there are several uncertainties which need to be addressed prior to progressing to a full-scale trial, including acceptability of the BRiMS intervention and practicality of the trial procedures. This feasibility trial will provide important insights to resolve these uncertainties and will enable a protocol to be finalised for use in the definitive trial.Publisher PDFPeer reviewe

The optimization of in vitro high-throughput chemical lysis of Escherichia coli. Application to ACP domain of the polyketide synthase ppsC from Mycobacterium tuberculosis

Protein production in Escherichia coli involves high-level expression in a culture, followed by harvesting of the cells and finally their disruption, or lysis, to release the expressed proteins. We compare three high-throughput chemical lysis methods to sonication, using a robotic platform and methodologies developed in our laboratory [1]. Under the same expression conditions, all lysis methods varied in the degree of released soluble proteins. With a set of 96 test proteins, we used our split GFP to quantify the soluble and insoluble protein fractions after lysis. Both the amount of soluble protein and the percentage recovered in the soluble fraction using SoluLyse® were well correlated with sonication. Two other methods, Bugbuster® and lysozyme, did not correlate well with sonication. Considering the effects of lysis methods on protein solubility is especially important when accurate protein solubility measurements are needed, for example, when testing adjuvants, growth media, temperature, or when establishing the effects of truncation or sequence variation on protein stability

Spatio-temporal genetic tagging of a cosmopolitan planktivorous shark provides insight to gene flow, temporal variation and site-specific re-encounters

Migratory movements in response to seasonal resources often influence population structure and dynamics. Yet in mobile marine predators, population genetic consequences of such repetitious behaviour remain inaccessible without comprehensive sampling strategies. Temporal genetic sampling of seasonally recurring aggregations of planktivorous basking sharks, Cetorhinus maximus, in the Northeast Atlantic (NEA) affords an opportunity to resolve individual re-encounters at key sites with population connectivity and patterns of relatedness. Genetic tagging (19 microsatellites) revealed 18% of re-sampled individuals in the NEA demonstrated inter/multi-annual site-specific re-encounters. High genetic connectivity and migration between aggregation sites indicate the Irish Sea as an important movement corridor, with a contemporary effective population estimate (Ne) of 382 (CI = 241–830). We contrast the prevailing view of high gene flow across oceanic regions with evidence of population structure within the NEA, with early-season sharks off southwest Ireland possibly representing genetically distinct migrants. Finally, we found basking sharks surfacing together in the NEA are on average more related than expected by chance, suggesting a genetic consequence of, or a potential mechanism maintaining, site-specific re-encounters. Long-term temporal genetic monitoring is paramount in determining future viability of cosmopolitan marine species, identifying genetic units for conservation management, and for understanding aggregation structure and dynamics