High level expression, purification, and characterization of the shrimp antimicrobial peptide, Ch-penaeidin, in Pichia pastoris

Penaeidins, members of a new family of antimicrobial peptides constitutively produced and stored in the haemocytes of penaeid shrimp, display antimicrobial activity against bacteria, and fungi. Here, a DNA sequence encoding the mature Ch-penaeidin peptide was cloned into the pPIC9K vector and transformed into Pichia pastoris. The transformed cells were screened for multi-copy plasmids using increasing concentrations of G418. Positive colonies carrying chromosomal integrations of the Chp gene were identified by phenotype and PCR. When transformed cells were induced with methanol, SDS-PAGE and Western blotting revealed the production of a similar to6100 Da recombinant CHP (rCHP) expression product. Large scale expression revealed that rCHP was produced at 108 mg/L under optimal conditions in the highest Chp-producing P. pastoris clone. The antimicrobial activities of rCHP were studied by liquid phase analysis, which revealed that rCHP exhibited activities against some Gram-negative and Gram-positive bacteria, but had a relatively low activity against some fungi. Purification of rCHP by cation exchange chromatography and subsequent automated amino acid sequencing revealed the presence of four additional amino acids (YVEF) at the N-terminus that belonged to the cleaved fusion signal peptide; these residues may account for the observed decrease in antifungal activity. Together, these observations indicate that rCHP is an effective antimicrobial peptide that can be successfully produced at high levels in the yeast, and therefore may be a potential antimicrobial candidate for practical use. (C) 2004 Elsevier Inc. All rights reserved.

Delegation, Arbitration and High-Level Service Discovery as Key Elements of a Software Infrastructure for Pervasive Computing

The dream of pervasive computing is slowly becoming a reality. A number of projects around the world are constantly contributing ideas and solutions that are bound to change the way we interact with our environments and with one another. An essential component of the future is a software infrastructure that is capable of supporting interactions on scales ranging from a single physical space to intercontinental collaborations. Such infrastructure must help applications adapt to very diverse environments and must protect people's privacy and respect their personal preferences. In this paper we indicate a number of limitations present in the software infrastructures proposed so far (including our previous work). We then describe the framework for building an infrastructure that satisfies the abovementioned criteria. This framework hinges on the concepts of delegation, arbitration and high-level service discovery. Components of our own implementation of such an infrastructure are presented.

Chronic active hepatitis with high level of anti-ds-DNA detected by a solid phase radioimmunoassay

SCOPUS: ar.j

High Level Environmental Screening Study for Offshore Wind Farm Developments – Marine Habitats and Species Project

High level environmental screening study for offshore wind farm developments – marine habitats and species This report provides an awareness of the environmental issues related to marine habitats and species for developers and regulators of offshore wind farms. The information is also relevant to other offshore renewable energy developments. The marine habitats and species considered are those associated with the seabed, seabirds, and sea mammals. The report concludes that the following key ecological issues should be considered in the environmental assessment of offshore wind farms developments: • likely changes in benthic communities within the affected area and resultant indirect impacts on fish, populations and their predators such as seabirds and sea mammals; • potential changes to the hydrography and wave climate over a wide area, and potential changes to coastal processes and the ecology of the region; • likely effects on spawning or nursery areas of commercially important fish and shellfish species; • likely effects on mating and social behaviour in sea mammals, including migration routes; • likely effects on feeding water birds, seal pupping sites and damage of sensitive or important intertidal sites where cables come onshore; • potential displacement of fish, seabird and sea mammals from preferred habitats; • potential effects on species and habitats of marine natural heritage importance; • potential cumulative effects on seabirds, due to displacement of flight paths, and any mortality from bird strike, especially in sensitive rare or scarce species; • possible effects of electromagnetic fields on feeding behaviour and migration, especially in sharks and rays, and • potential marine conservation and biodiversity benefits of offshore wind farm developments as artificial reefs and 'no-take' zones. The report provides an especially detailed assessment of likely sensitivity of seabed species and habitats in the proposed development areas. Although sensitive to some of the factors created by wind farm developments, they mainly have a high recovery potential. The way in which survey data can be linked to Marine Life Information Network (MarLIN) sensitivity assessments to produce maps of sensitivity to factors is demonstrated. Assessing change to marine habitats and species as a result of wind farm developments has to take account of the natural variability of marine habitats, which might be high especially in shallow sediment biotopes. There are several reasons for such changes but physical disturbance of habitats and short-term climatic variability are likely to be especially important. Wind farm structures themselves will attract marine species including those that are attached to the towers and scour protection, fish that associate with offshore structures, and sea birds (especially sea duck) that may find food and shelter there. Nature conservation designations especially relevant to areas where wind farm might be developed are described and the larger areas are mapped. There are few designated sites that extend offshore to where wind farms are likely to be developed. However, cable routes and landfalls may especially impinge on designated sites. The criteria that have been developed to assess the likely marine natural heritage importance of a location or of the habitats and species that occur there can be applied to survey information to assess whether or not there is anything of particular marine natural heritage importance in a development area. A decision tree is presented that can be used to apply ‘duty of care’ principles to any proposed development. The potential ‘gains’ for the local environment are explored. Wind farms will enhance the biodiversity of areas, could act as refugia for fish, and could be developed in a way that encourages enhancement of fish stocks including shellfish.

Fully differential cross sections for transfer ionization - a sensitive probe of high level correlation effects in atoms

The transfer ionization process offers a unique opportunity to study radial and angular electron correlations in the helium atom. We report calculations for the multiple differential cross sections of the transfer ionization process p + He --> H + He++ + e(-). The results of these calculations demonstrate the strong sensitivity of the fully differential cross sections to fine details of electron correlation in the target atom. Specifically, angular electron correlation in the ground state of helium results in a broad peak in the electron emission spectra in the backward direction, relative to the incoming beam. Our model explains some of the key effects observed in measurements of multiple differential cross sections using the COLTRIMS technique.

Multiple Regulatory Pathways Associated with High-Level Ciprofloxacin and Multidrug Resistance in Salmonella enterica Serovar Enteritidis: Involvement of ramA and Other Global Regulators

Mechanisms of antibiotic resistance were examined in nalidixic acid-resistant Salmonella enterica serovar Enteritidis field isolates displaying decreased susceptibility to ciprofloxacin and in in vitro-derived ciprofloxacin-resistant mutants (104-cip and 5408-cip). All field isolates harbored a single gyrA mutation (D87Y). Deletion of acrB and complementation with wild-type gyrA increased quinolone susceptibility. Selection for ciprofloxacin resistance was associated with the development of an additional gyrA (S83F) mutation in 104-cip, novel gyrB (E466D) and parE (V461G) mutations in 5408-cip, overexpression of acrB and decreased susceptibility to nonquinolone antibiotics in both mutants, and decreased OmpF production and altered lipopoly- saccharide in 104-cip. Complementation of mutated gyrA and gyrB with wild-type alleles restored susceptibility to quinolones in 104-cip and significantly decreased the ciprofloxacin MIC in 5408-cip. Complementation of parE had no effect on quinolone MICs. Deletion of acrB restored susceptibility to ciprofloxacin and other antibiotics tested. Both soxS and marA were overexpressed in 104-cip, and ramA was overexpressed in 5408-cip. Inactivation of each of these global regulators lowered ciprofloxacin MICs, decreased expression of acrB, and restored susceptibility to other antibiotics. Mutations were found in soxR (R20H) and in soxS (E52K) in 104-cip and in ramR (G25A) in 5408-cip. In conclusion, both efflux activity and a single gyrA mutation contribute to nalidixic acid resistance and reduced ciprofloxacin sensitivity. Ciprofloxacin resistance and decreased susceptibility to multiple antibiotics can result from different genetic events leading to development of target gene mutations, increased efflux activity resulting from differential expression of global regulators associated with mutations in their regulatory genes, and possible altered membrane permeability.