A cassette ligation strategy with thioether replacement of three Gly-Gly peptide bonds: Total chemical synthesis of the 101 residue protein early pregnancy factor [psi(CH2S)(28-29,56-57,76-77)]

The 101 residue protein early pregnancy factor (EPF), also known as human chaperonin 10, was synthesized from four functionalized, but unprotected, peptide segments by a sequential thioether ligation strategy. The approach exploits the differential reactivity of a peptide-NHCH2CH2SH thiolate with XCH2CO-peptides, where X = Cl or I/Br. Initial model studies with short functionalized (but unprotected) peptides showed a significantly faster reaction of a peptide-NHCH2CH2SH thiolate with a BrCH2CO-peptide than with a CICH2CO-peptide, where thiolate displacement of the halide leads to chemoselective formation of a thioether surrogate for the Gly-Gly peptide bond. This rate difference was used as the basis of a novel sequential ligation approach to the synthesis of large polypeptide chains. Thus, ligation of a model bifunctional N-alpha-chloroacetyl, C-terminal thiolated peptide with a second N-alpha-bromoacetyl peptide demonstrated chemoselective bromide displacement by the thiol group. Further investigations showed that the relatively unreactive N-alpha-chloroacetyl peptides could be activated by halide exchange using saturated KI solutions to yield the highly reactive No-iodoacetyl peptides. These findings were used to formulate a sequential thioether ligation strategy for the synthesis of EPF, a 101 amino acid protein containing three Gly-Gly sites approximately equidistantly spaced within the peptide chain. Four peptide segments or cassettes comprising the EPF protein sequence (BrAc-[EPF 78-101] 12, ClAc-[EPF 58-75]-[NHCH2CH2SH] 13, ClAc-[EPF 30-55]-[NHCH2CH2SH] 14, and Ac-[EPF 1-27]-[NHCH2CH2SH] 15) of EPF were synthesized in high yield and purity using Boc SPPS chemistry. In the stepwise sequential ligation strategy, reaction of peptides 12 and 13 was followed by conversion of the N-terminal chloroacetyl functional group to an iodoacetyl, thus activating the product peptide for further ligation with peptide 14. The process of ligation followed by iodoacetyl activation was repeated to yield an analogue of EPF (EPF psi(CH2S)(28-29,56-57,76-77)) 19 in 19% overall yield.

A lipid core peptide construct containing a conserved region determinant of the group a streptococcal M protein elicits heterologous opsonic antibodies

The study reported here investigated the immunogenicity and protective potential of a lipid core peptide (LCP) construct containing a conserved region determinant of M protein, defined as peptide J8. Parenteral immunization of mice with LCP-J8 led to the induction of high-titer serum immunoglobulin G J18-specific antibodies when the construct was coadministered with complete Freund's adjuvant (CFA) or administered alone. LCP-J8 in CFA had significantly enhanced immunogenicity compared with the monomeric peptide J8 given in CFA. Moreover, LCP-J8/CFA and LCP-J8 antisera opsonized four different group A streptococcal (GAS) strains, and the antisera did not cross-react with human heart tissue proteins. These data indicate the potential of an LCP-based M protein conserved region GAS vaccine in the induction of broadly protective immune responses in the absence of a conventional adjuvant.

Communicating vegetation management science to land managers and other stakeholders

Science communication. including extension services. plays a key role in achieving sustainable native vegetation management. One of the pivotal aspects of the debate on sustainable vegetation management is the scientific information underpinning policy-making. In recent years. extension services have Shifted their focus from top-down technology transfer to bottom-up participation and empowerment. I here has also been a broadening of communication strategies to recognise the range of stakeholders involved in native vegetation management and to encompass environmental concerns. This paper examines the differences between government approaches to extension services to deliver policy and the need for effective communication to address broader science issues that underpin native vegetation management. The importance of knowing the learning styles of the stakeholders involved in native vegetation management is discussed at a time of increasing reliance on mass communication for information exchange and the importance of personal communication to achieve on-ground sustainable management. Critical factors for effective science-management communication are identified Such as: (i) undertaking scientific studies (research) with community involvement, acceptance and agreed understanding of project objectives (ii) realistic community consultation periods: (iii) matching communication channels with stakeholder needs; (iv) combining scientific with local knowledge in in holistic (biophysical and social) approach to understanding in issued and (v) regional partnerships. These communication factors are considered to be essential to implementing on-ground natural resource management strategics and actions, including those concerned with native vegetation management.

Infusing the use of seasonal climate forecasting into crop management practice in North East Australia using discussion support software

Seasonal climate forecasting offers potential for improving management of crop production risks in the cropping systems of NE Australia. But how is this capability best connected to management practice? Over the past decade, we have pursued participative systems approaches involving simulation-aided discussion with advisers and decision-makers. This has led to the development of discussion support software as a key vehicle for facilitating infusion of forecasting capability into practice. In this paper, we set out the basis of our approach, its implementation and preliminary evaluation. We outline the development of the discussion support software Whopper Cropper, which was designed for, and in close consultation with, public and private advisers. Whopper Cropper consists of a database of simulation output and a graphical user interface to generate analyses of risks associated with crop management options. The charts produced provide conversation pieces for advisers to use with their farmer clients in relation to the significant decisions they face. An example application, detail of the software development process and an initial survey of user needs are presented. We suggest that discussion support software is about moving beyond traditional notions of supply-driven decision support systems. Discussion support software is largely demand-driven and can compliment participatory action research programs by providing cost-effective general delivery of simulation-aided discussions about relevant management actions. The critical role of farm management advisers and dialogue among key players is highlighted. We argue that the discussion support concept, as exemplified by the software tool Whopper Cropper and the group processes surrounding it, provides an effective means to infuse innovations, like seasonal climate forecasting, into farming practice. Crown Copyright (C) 2002 Published by Elsevier Science Ltd. All rights reserved.

MR image-based measurement of rates of change in volumes of brain structures. Part 1: Method and validation

A detailed analysis procedure is described for evaluating rates of volumetric change in brain structures based on structural magnetic resonance (MR) images. In this procedure, a series of image processing tools have been employed to address the problems encountered in measuring rates of change based on structural MR images. These tools include an algorithm for intensity non-uniforniity correction, a robust algorithm for three-dimensional image registration with sub-voxel precision and an algorithm for brain tissue segmentation. However, a unique feature in the procedure is the use of a fractional volume model that has been developed to provide a quantitative measure for the partial volume effect. With this model, the fractional constituent tissue volumes are evaluated for voxels at the tissue boundary that manifest partial volume effect, thus allowing tissue boundaries be defined at a sub-voxel level and in an automated fashion. Validation studies are presented on key algorithms including segmentation and registration. An overall assessment of the method is provided through the evaluation of the rates of brain atrophy in a group of normal elderly subjects for which the rate of brain atrophy due to normal aging is predictably small. An application of the method is given in Part 11 where the rates of brain atrophy in various brain regions are studied in relation to normal aging and Alzheimer's disease. (C) 2002 Elsevier Science Inc. All rights reserved.

Roof gutters: A key container for Aedes aegypti and Ochlerotatus notoscriptus (Diptera : Culicidae) in Australia

The contribution of roof gutters to Aedes aegypti (L.) and Ochlerotatus notoscriptus (Skuse) pupal populations was quantified for the first time in Cairns, Australia. Concurrent yard and roof surveys yielded ill estimated 6,934 mosquito pupae, comprising four species. Roof gutters were all uncommon but productive source of Ae. aegypti in both wet season (n = 11) and dry season (n = 2) surveys, producing 52.6% and 39.5% of the respective populations. First story gutters accounted for 92.3% of the positive gutters. Therefore, treatment of roof gutters is a critical element in Ae. aegypti control campaigns during dengue outbreaks. In wet season yards, the largest standing, crops of Ae. aegypti occurred in garden accoutrements, discarded household items, and rubbish (36.4%, 28.0%, and 20.6%, respectively). In dry season yards, rubbish produced 79.6% of the Ae. aegypti pupae. The number of Ae. aegypti pupae/person was 2.36 and 0.59 for the wet and dry season surveys, respectively.

Protection of mice from group A streptococcal infection by intranasal immunisation with a peptide vaccine that contains a conserved M protein B cell epitope and lacks a T cell autoepitope

Infection with group A streptococci (GAS) can lead to rheumatic fever (RF) and rheumatic heart disease (RHD) which are a major health concern particularly in indigenous populations worldwide, and especially in Australian Aboriginals. A primary route of GAS infection is via the upper respiratory tract, and therefore, a major goal of research is the development of a mucosal-based GAS vaccine, The majority of the research to date has focused on the GAS M protein since immunity to GAS is mediated by M protein type-specific opsonic antibodies. There are two major impediments to the development of a vaccine-the variability in M proteins and the potential for the induction of an autoimmune response. To develop a safe and broad-based vaccine, we have therefore focused on the GAS M protein conserved C-region, and have identified peptides, J8 and the closely related J8 peptide (J14), which may be important in protective immunity to GAS infection. Using a mucosal animal model system, our data have shown a high degree of throat GAS colonisation in B10.BR mice 24 h following intranasal immunisation with the mucosal adjuvant, cholera toxin B subunit (CTB), and/or diptheria toxoid (dT) carrier, or PBS alone, and challenge with the M1 GAS strain. However, GAS colonisation of the throat was significantly reduced following intranasal immunisation of mice with the vaccine candidate J8 conjugated to dT or J14-dT when administered with CTB. Moreover, J8-dT/CTB and J14-dT/CTB-immunised mice had a significantly higher survival when compared to CTB and PBS-immunised control mice. These data indicate that immunity to GAS infection can be evoked by intranasal immunisation with a GAS M protein C-region peptide vaccine that contains a protective B cell epitope and lacks a T cell autoepitope. (C) 2002 Published by Elsevier Science Ltd.

Lipoamino acid-based adjuvant carrier system: Enhanced immunogenicity of group a streptococcal peptide epitopes

Lipoamino acid-based synthetic peptides (lipid core peptides, LCP) derived from the type-specific and conserved region determinants of group A streptococci (GAS) were evaluated as potential candidate sequences in a vaccine to prevent GAS-associated diseases, including rheumatic heart, disease and poststreptococcal acute glomerulonephritis. The LCP peptides had significantly enhanced immunogenicity as compared with the monomeric peptide epitopes. Furthermore, the peptides incorporated into the LCP system generated epitope-specific antibodies without the use of any conventional adjuvant.

Effect of manual hyperinflation on hemodynamics, gas exchange, and respiratory mechanics in ventilated patients

The authors investigated the effect of manual hyperinflation (MHI) with set parameters applied to patients on mechanical ventilation on hemodynamics, respiratory mechanics, and gas exchange. Sixteen critically ill patients post-septic shock, with acute lung injury, were studied. Heart rate, arterial pressure, and mean pulmonary artery pressure were recorded every minute. pulmonary artery occlusion pressure, cardiac output, arterial blood gases, and dynamic compliance (C-dyn) were recorded pre- and post-MHI. From this, systemic vascular resistance index (SVRI), cardiac index, oxygen delivery, and partial pressure of oxygen:fraction of inspired oxygen (PaO2:FiO(2)) ratio were calculated. There were significant increases in SVRI (P < 0.05) post-MHI and diastolic arterial pressure (P < 0.01)during MHI. C-dyn increased post-MHI (P < 0.01) and was sustained at 20 minutes post-MHI (P < 0.01). Subjects with an intrapulmonary cause of lung disease had a significant decrease (P = 0.02) in PaO2:FiO(2), and those with extrapulmonary causes of lung disease had a significant increase (P < 0.001) in PaO2:FiO(2) post-MHI. In critically ill patients, MHI resulted in an improvement in lung mechanics and an improvement in gas exchange in patients with lung disease due to extrapulmonary events and did not result in impairment of the cardiovascular system.

Speciation and distribution of the members of the Anopheles punctulatus (Diptera : Culicidae) group in Papua New Guinea

Mosquito collections were made throughout the mainland of Papua New Guinea to identify the members of the Anopheles punctulatus group present and to determine their distribution. Identification was made using morphology, DNA hybridization, and polymerase chain reaction (PCR)-RFLP analysis. Nine members of the group were identified: An. farauti s.s. Laveran, An.farauti 2, An. koliensis Owen, and An. punctulatus Donitz, were common and widespread; An. farauti 4 was restricted to the north of the central ranges where it was common; An. farauti 6 was found only in the highlands above 1,000 m; and An. farauti 3, An. sp. near punctulatus and An. clowi Rozeboom & Knight were uncommon and had restricted distributions. Identification of An. koliensis and An. punctulatus using proboscis morphology was found to be unreliable wherever An. farauti 4 occurred. The distribution and dispersal of the members of the An. punctulatus group is discussed in regard to climate, larval habitats, distance from the coast, elevation, and proximity to human habitation.

Fast neutron mutagenesis of soybean (Glycine soja L.) produces a supernodulating mutant containing a large deletion in linkage group H

We describe for the first time the application of fast neutron mutagenesis to the genetic dissection of root nodulation in legumes. We demonstrate the utility of chromosomal deletion mutations through production of a soybean supernodulation mutant FN37 that lacks the internal autoregulation of nodulation mechanism. After inoculation with microsymbiont Bradyrhizobium japonicum, FN37 forms at least 10 times more nodules than the wild type G. soja parent and has a phenotype identical to that of chemically induced allelic mutants nts382 and nts1007 (NTS-1 locus). Reciprocal grafting of shoots and roots confirmed systemic shoot control of the FN37 nodulation phenotype. RFLP/PCR marker pUTG132a and AFLP marker UQC-IS1 which are tightly linked to NTS-1 allowed the isolation of BAC contigs delineating both ends of the deletion. The genetic/physical distance ratio in the NTS-1 region is 279 kb/cM. The deletion is estimated to be about 460 kb based on the absence of markers and bacterial artificial chromosomes (BAC) ends as well as genetic and physical mapping. Deletion break points were determined physically and placed within flanking BAC contigs.

Distribution and evolution of the Anopheles punctulatus group (Diptera : Culicidae) in Australia and Papua New Guinea

The members of the Anopheles punctulatus group are major vectors of malaria and Bancroftian filariasis in the southwest Pacific region. The group is comprised of 12 cryptic species that require DNA-based tools for species identification. From 1984 to 1998 surveys were carried out in northern Australia, Papua New Guinea and on islands in the southwest Pacific to determine the distribution of the A. punctulatus group. The results of these surveys have now been completed and have generated distribution data from more than 1500 localities through this region. Within this region several climatic and geographical barriers were identified that restricted species distribution and gene flow between geographic populations. This information was further assessed in light of a molecular phylogeny derived from the ssrDNA (18S). Subsequently, hypotheses have been generated on the evolution and distribution of the group so that future field and laboratory studies may be approached more systematically. This study suggested that the ability for widespread dispersal was found to have appeared independently in species that show niche-specific habitat preference (Anopheles farauti s.s. and A. punctulatus) and conversely in species that showed diversity in their larval habitat (Anopheles farauti 2). Adaptation to the monsoonal climate of northern Australia and southwest Papua New Guinea was found to have appeared independently in A. farauti s.s., A. farauti 2 and Anopheles farauti 3. Shared or synapomorphic characters were identified as saltwater tolerance (A. farauti s.s. and Anopheles farauti 7) and elevational affinities above 1500 m (Anopheles farauti 5, Anopheles farauti 6 and A. farauti 2). (C) 2002 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved.