A system for the heterologous expression of complex redox proteins in Rhodobacter capsulatus: characterisation of recombinant sulphite : cytochrome c oxidoreductase from Starkeya novella

The phototrophic purple non-sulfur bacterium Rhodobacter capsulatus expresses a wide variety of complex redox proteins in response to changing environmental conditions. Here we report the construction and evaluation of an expression system for recombinant proteins in that organism which makes use of the dor promoter from the same organism. A generic expression vector, pDorEX, was constructed and used to express sulphite:cytochrome c oxidoreductase from Starkeya novella, a heterodimeric protein containing both molybdenum and haem c. The recombinant protein was secreted to the periplasm and its biochemical properties were very similar to those of the native enzyme. The pDorEX system therefore seems to be potentially useful for heterologous expression of multi-subunit proteins containing complex redox cofactors. (C) 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

Coordinated expression of scleraxis and Sox9 genes during embryonic development of tendons and cartilage

Embryonic development of tendons is in close association with that of cartilage and bone. Although these tissues are derived from mesenchymal progenitor cells which also give rise to muscle and fat, their fates clearly diverse in early embryonic stages, Transcription factors may play pivotal roles in the process of determination and differentiation of tendon cells as well as other cells in the skeletal system. Scleraxis, a basic helix-loop-helix (bHLH) type transcription factor. is expressed in mesenchymal progenitors that later form connective tissues including tendons. Sox9 is an HMG-box containing transcription factor, which is expressed at high levels in chondrocytes. We hypothesized that the two transcription factors regulate the fate of cells that interact with each other at the interface between the two tissues during divergence of their differentiation pathways, To address this point, we investigated scleraxis and Sox9 rnRNA expression during mouse embyogenesis focusing on the coordinated development of tendons and skeletons, In the early stage of mesenchymal tissue development at 10.5 d.p.c., scleraxis and Sox9 transcripts were expressed in the mesenchymal progenitor cells in the appendicular and axial mesenchyme. At 11.5 d.p.c.. scleraxis transcripts were observed in the mesenchymal tissue surrounding skeletal primordia which express Sox9. From this stage, scleraxis expression was closely associated with, but distinct from, formation of skeletal primordia, At 13.5 d.p.c., scleraxis was expressed broadly in the interface between muscle and skeletal primordia while Sox9 expression is confined within the early skeletal primordia. Then. at 15.5 d.p.c., scleraxis transcripts were more restricted to tendons. These observations revealed the presence of temporal and spatial association of scleraxis expression during embryonic development of tendon precursor cells in close association with that of So,0 expression in chondrogenic cells in skeletal tissues. (C) 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.

Extra-pair paternity and mate-guarding behaviour in the brown thornbill

We used multilocus DNA fingerprinting to assess parentage in the brown thornbill, Acanthiza pusilla, a socially monogamous Australian passerine. Extra-pair paternity was uncommon (6.2% of 178 offspring; 11.9% of 67 broods) and there was no evidence of intra-specific brood parasitism. Extra-pair paternity was limited because pairs spent more time together when females were fertile and males were able to evict intruding males before they could approach the female. Males were responsible for the close proximity of partners during the fertile period. Mate guarding therefore appears to be a male tactic aimed at preventing female infidelity rather than a cooperative behaviour of the pair aimed at preventing extra-pair copulations and/or female harassment. Females did not attempt to escape male guarding and were rarely observed to solicit copulations from intruding males. Nevertheless, females paired to smaller and younger males were more likely to cuckold their mates than females paired to larger and older males. This suggests that females may be more likely to seek or accept extra-pair matings when paired to small, young males or that old, large males are better at preventing their mates from engaging in extra-pair copulations. We found that male age but not male size influences mate-guarding behaviour. Older males tended to respond more aggressively to intruders. We therefore speculate that the relationship between male size/age and extra-pair paternity in brown thornbills may arise because female thornbills prefer large males as mates but are unable to express this preference as easily when paired to older males.

Should we debrief and counsel people who have had psychological shock?

Design: Randomised controlled trial of psychological debriefing. Setting: A British teaching hospital (the Radcliffe Hospital, Oxford). Patients: 66 men and 40 women, aged 17–69 years, admitted to hospital after a motor vehicle accident. Most had been the driver of a car. Median admission duration was four days for the 52 control patients and eight days for the 54 who underwent the intervention. Interventions: A debriefing of about one hour on Day 2 of admission, encouraging patients to describe the accident and express their emotions, followed by a cognitive appraisal which included describing common reactions to traumatic experiences and suggesting a range of people who might be able to assist in the future, including the patient's general practitioner. 91 patients were assessed at four months and 61 were assessed at three years. Control patients had no debriefing or counselling. Main outcome measures: Impact of Event Scale (IES, which focuses on intrusive thoughts and avoidance of similar situations to the event); Brief Symptom Inventory (BSI, a measure of 53 symptoms); and other questions related to physical pain and functional activities. Main results: At four months there was still considerable psychological morbidity among the patients who were followed up. There was a significant difference (P < 0.05) in changes of IES between the 42 who received the intervention, in whom it increased from 15 (standard deviation [SD], 15) to 16 (SD, 15), and the 49 controls, in whom it fell from 15 (SD, 12) to 13 (SD, 14). Similarly, two subscales of the BSI score changed significantly between the intervention group, among whom it deteriorated from 0.5 (SD, 0.5) to 0.6 (SD, 0.8), and the control s, in whom it hardly changed from 0.4 (SD, 0.3) to 0.4 (SD, 0.4). Among the 61 patients followed for three years, the 30 randomised to receive the intervention were significantly worse, by self-report, both psychologically and physically. Their mean IES score deteriorated from a baseline of 15 (SD, 14) to 16 (SD, 18). In comparison, scores for the 31 control patients improved from 16 (SD, 12) to 13 (SD, 17). The difference in change was significant (P < 0.05). Among all patients with high initial scores, these decreased among the controls but not among those receiving the intervention. Conclusion: Psychological counselling should only be used in the context of trials rather than routine care.

How well do general practitioners deliver palliative care? A systematic review

General practitioners (GPs) deliver the majority of palliative care to patients in the last year of life. This article seeks to examine the nature of GP care, perceptions of the GPs themselves and others of that care, the adequacy of palliative care training, issues relating to accessibility of GPs to palliative care patients, and strategies that may be of use in encouraging more effective delivery of palliative care by GPs. Medline and PubMed databases from 1966 to 2000 were searched, and 135 references identified. Sixty-six of these described studies relevant to GP palliative care. GPs value this part of their work. Most of the time, patients appreciate the contribution the GP makes to palliative care particularly if the GP is accessible, takes time to listen, allows patient and carer to ventilate their feelings, and is seen to be making efforts made regarding symptom relief. However, reports from bereaved relatives suggest that palliative care is performed less well in the community than in other settings. GPs express discomfort about their competence to perform palliative care adequately. They tend to miss symptoms which are not treatable by them, or which are less common. However, with appropriate specialist support and facilities, GPs have been shown to deliver sound and effective care. GP comfort working with specialist teams increases with exposure to this form of patient management, as does the understanding of the potential other team members have in contributing to the care of the patient. Formal arrangements engaging GPs to work with specialist teams have been shown to improve functional outcomes, patient satisfaction, improve effective use of resources and improve effective physician behaviour in other areas of medicine. Efforts by specialist services to develop formal involvement of GPs in the care of individual patients, may be an effective method of improving GP palliative care skills and appreciation of the roles specialist services can play.

Dinoflagellate symbiosis: strategies and adaptations for the aquisition and fixation of inorganic carbon

Dinoflagellates exist in symbiosis with a number of marine invertebrates including giant clams, which are the largest of these symbiotic organisms. The dinoflagellates (Symbiodinium sp.) live intercellularly within tubules in the mantle of the host clam. The transport of inorganic carbon (Ci) from seawater to Symbiodinium (=zooxanthellae) is an essential function of hosts that derive the majority of their respiratory energy from the photosynthate exported by the zooxanthellae. Immunolocalisation studies show that the host has adapted its physiology to acquire, rather than remove CO2, from the haemolymph and clam tissues. Two carbonic anhydrase (CA) isoforms (32 and 70 kDa) play an essential part in this process. These have been localised to the mantle and gill tissues where they catalyse the interconversion of HCO3- to CO2, which then diffuses into the host tissues. The zooxanthellae exhibit a number of strategies to maximise Ci acquisition and utilisation. This is necessary as they express a form II Rubisco that has poor discrimination between CO2 and O-2. Evidence is presented for a carbon concentrating mechanism (CCM) to overcome. this disadvantage. The CCM incorporates the presence of a light-activated CA activity, a capacity to take up both HCO3- and CO2, an ability to accumulate an elevated concentration of Ci within the algal cell, and localisation of Rubisco to the pyrenoid. These algae also express both external and intracellular CAs, with the intracellular isoforms being localised to the thylakoid lumen and pyrenoid. These results have been incorporated into a model that explains the transport of Ci from seawater through the clam to the zooxanthellae.

Streptococcus pyogenes prtFII, but not sfbI, sfbII or fbp54, is represented more frequently among invasive-disease isolates of tropical Australia

Streptococcus pyogenes (group A streptococcus) strains may express several distinct fibronectin-binding proteins (FBPs) which are considered as major streptococcal adhesins. Of the FBPs, SfbI was shown in vitro to promote internalization of the bacterium into host cells and has been implicated in persistence. In the tropical Northern Territory, where group A streptococcal infection is common, multiple genotypes of the organism were found among isolates from invasive disease cases and no dominant strains were observed. To determine whether any FBPs is associated with invasive disease propensity of S. pyogenes, we have screened streptococcal isolates from bacteraemic and necrotizing fasciitis patients and isolates from uncomplicated infections for genetic endowment of 4 FBPs. No difference was observed in the distribution of sfbII, fbp54 and sfbI between the blood isolates' and isolates from uncomplicated infection. We conclude that the presence of sfbI does not appear to promote invasive diseases, despite its association with persistence. We also show a higher proportion of group A streptococcus strains isolated from invasive disease cases possess prtFII when compared to strains isolated from non-invasive disease cases. We suggest that S. pyogenes may recruit different FBPs for different purposes.

The embryonic development of the bodywall and nervous system of the cestode flatworm Hymenolepis diminuta

Cestodes (tapeworms) are a derived, parasitic clade of the phylum Platyhelminthes (flatworms). The cestode body wall represents an adaptation to its endoparasitic lifestyle. The epidermis forms a nonciliated syncytium, and both muscular and nervous system are reduced. Morphological differences between cestodes and free-living flatworms become apparent already during early embryogenesis. Cestodes have a complex life cycle that begins with an infectious larva, called the oncosphere. In regard to cell number, cestode oncospheres are among the simplest multicellular organisms, containing in the order of 50-100 cells. As part of our continuing effort to analyze embryonic development in flatworms, we describe here the staining pattern obtained with acTub in embryos and larvae of the cestode Hymenolepis diminuta and, briefly, the monogenean Neoheterocotyle rhinobatidis. In addition, we labeled the embryonic musculature of Hymenolepis with phalloidin. In Hymenolepis embryos, two different cell types that we interpret as neurons and epidermal gland cells express acTub. There exist only two neurons that develop close to the midline at the anterior pole of the embryo. The axons of these two neurons project posteriorly into the center of the oncosphere, where they innervate the complex of muscles that is attached to the booklets. In addition to neurons, acTub labels a small and invariant set of epidermal gland cells that develop at superficial positions, anteriorly adjacent to the neurons, in the dorsal midline, and around the posteriorly located hooklets. During late stages of embryogenesis they spread and form a complete covering of the embryo. We discuss these data in the broader context of platyhelminth embryology.

Generation of CTL responses using Kunjin replicon RNA

The Kunjin replicon was used to express a polytope that consisted of seven hepatitis C virus cytotoxic T lymphocyte epitopes and one influenza cytotoxic T lymphocyte epitope for vaccination studies. The self-replicating nature of, and expression from, the ribonucleic acid was confirmed in vitro . Initial vaccinations with one dose of Kun-Poly ribonucleic acid showed that an influenza-specific cytotoxic T lymphocyte response was elicited more efficiently by intradermal inoculation compared with intramuscular delivery. Two micrograms of ribonucleic acid delivered in the ear pinnae of mice was sufficient to elicit a detectable cytotoxic T lymphocyte response 10 days post-vaccination. Further vaccination studies showed that four of the seven hepatitis C virus cytotoxic T lymphocyte epitopes were able to elicit weak cytotoxic T lymphocyte responses whereas the influenza epitope was able to elicit strong, specific cytotoxic T lymphocyte responses following three doses of Kun-Poly ribonucleic acid. These studies vindicate the use of the Kunjin replicon as a vector to deliver encoded proteins for the development of cell-mediated immune responses.

An international perspective on sunken state vessels as underwater cultural heritage

On 2 November 2001, the General Assembly of the United Nations Scientific, Economic and Cultural Organisation (UNESCO) adopted the convention on the Protection of the Underwater Cultural Heritage. Among the many complex issues addressed in the convention is the legal status of sunken state-owned vessels, including warships. Prior to the adoption of this convention, no conventional or customary international law existed with regards to the question of abandonment of state-owned vessels or the application of the principle of sovereign immunity to sunken state vessels. While difficulties between coastal states and maritime and former colonial powers resulted in a regime that does not comprehensively address the issues, the convention does provide some guidance in this regard and may provide a basis for further development.

Tyrosinase-Cre mice for tissue-specific gene ablation in neural crest and neuroepithelial-derived tissues

This study describes the derivation of two new lines of transgenic mice that express Cre recombinase under the control of tyrosinase transcriptional elements. To determine the suitability of the Tyrosinase-Cre transgene for tissue-specific gene ablation studies, a fate map of Cre expression domains was determined using the Z/AP reporter strain. It was shown that Cre-expressing cells contribute to a wide array of neural crest and neuroepithelial-derived lineages. The melanocytes of the harderian gland and eye choroid, sympathetic cephalic ganglia, leptomeninges of the telencephalon, as well as cranial nerves (V), (VII), and (IX) are derived either fully or partly from Cre-expressing cephalic crest. The cells contributing to the cranial nerves were the first to exhibit Cre expression at E10.5 as they were migrating into the branchial arches. The melanocytes, chromaffin cells of the adrenal medulla, and dorsal root ganglia are derived from trunk neural crest that either express Cre or were derived from Cre-expressing precursors. An array of brain tissue including the basal forebrain, hippocampus, olfactory bulb, and the granule cell layer of the lateral cerebellum, as well as the retinal pigmented epithelium and glia of the optic nerve originate from Cre-expressing neuroepithelial cells. (C) 2003 Wiley-Liss, Inc.

Carboxy-fluorescein diacetate, succinimidyl ester labeled papillomavirus virus-like particles fluoresce after internalization and interact with heparan sulfate for binding and entry

Human papillomaviruses (HPVs) infect epithelial cells and are associated with genital carcinoma. Most epithelial cell lines express cell-surface glycosaminoglycans (GAGs) usually found attached to the protein core of proteoglycans. Our aim was to study how GAGs influenced HPV entry. Using a human keratinocyte cell line (HaCaT), preincubation of HPV virus-like particles (VLPs) with GAGs showed a dose-dependent inhibition of binding. The IC50 (50% inhibition) was only 0.5 mug/ml for heparin, 1 mug/ml for dextran sulfate, and 5-10 mug/ml for heparan sulfate from mucosal origin. Mutated chinese hamster ovary (CHO) cell lines lacking heparan sulfate or all GAGs were unable to bind HPV VLPs. Here we also report a method to study internalization by using VLPs labeled with carboxy-fluorescein diacetate, succinimidyl ester, a fluorochrome that is only activated after cell entry. Pretreatment of labeled HPV VLPs with heparin inhibited uptake, suggesting a primary interaction between HPV and cell-surface heparan sulfate. (C) 2003 Elsevier Science (USA). All rights reserved.

Enhanced effector and memory CTL responses generated by incorporation of receptor activator of NF-kappa B(RANK)/RANK ligand costimulatory molecules into dendritic cell immunogens expressing a human tumor-specific antigen

The outcome of dendritic cell (DC) presentation of Ag to T cells via the TCR/MHC synapse is determined by second signaling through CD80/86 and, importantly, by ligation of costimulatory ligands and receptors located at the DC and T cell surfaces. Downstream signaling triggered by costimulatory molecule ligation results in reciprocal DC and T cell activation and survival, which predisposes to enhanced T cell-mediated immune responses. In this study, we used adenoviral vectors to express a model tumor Ag (the E7 oncoprotein of human papillomavirus 16) with or without coexpression of receptor activator of NF-kappaB (RANK)/RANK ligand (RANKL) or CD40/CD40L costimulatory molecules, and used these transgenic DCs to immunize mice for the generation of E7-directed CD8(+) T cell responses. We show that coexpression of RANK/RANKL, but not CD40/CD40L, in E7-expressing DCs augmented E7-specific IFN-gamma-secreting effector and memory T cells and E7-specific CTLs. These responses were also augmented by coexpression of T cell costimulatory molecules (RANKL and CD40L) or DC costimulatory molecules (RANK and CD40) in the E7-expressing DC immunogens. Augmentation of CTL responses correlated with up-regulation of CD80 and CD86 expression in DCs transduced with costimulatory molecules, suggesting a mechanism for enhanced T cell activation/survival. These results have generic implications for improved tumor Ag-expressing DC vaccines, and specific implications for a DC-based vaccine approach for human papillomavirus 16-associated cervical carcinoma.

Genetic characterization of pilin glycosylation and phase variation in Neisseria meningitidis

Pili of Neisseria meningitidis are a key virulence factor, being the major adhesin of this capsulate organism and contributing to specificity for the human host. Pili are post-translationally modified by addition of either an O-linked trisaccharide, Gal (beta1-4) Gal (alpha1-3) 2,4-diacetamido-2,4,6-trideoxyhexose or an O-linked disaccharide Gal (alpha1,3) GlcNAc. The role of these structures in meningococcal pathogenesis has not been resolved. In previous studies we identified two separate genetic loci, pglA and pglBCD, involved in pilin glycosylation. Putative functions have been allocated to these genes; however, there are not enough genes to account for the complete biosynthesis of the described structures, suggesting additional genes remain to be identified. In addition, it is not known why some strains express the trisaccharide structure and some the disaccharide structure. In order to find additional genes involved in the biosynthesis. of these structures, we used the recently published group A strain Z2491 and group B strain MC58 Neisseria meningitidis genomes and the unfinished Neisseria meningitidis group C strain FAM18 and Neisseria gonorrhoeae strain FA1090 genomes to identify novel genes involved in pilin glycosylation, based on homology to known oligosaccharide biosynthetic genes. We identified a new gene involved in pilin glycosylation designated pglE and examined four additional genes pgIB/B2, pglF, pglG and pglH. A strain survey revealed that pglE and pglF were present in each strain examined. The pglG, pglH and pgIB2 polymorphisms were not found in strain C311#3 but were present in a large number of clinical isolates. Insertional mutations were constructed in pglE and pglF in N. meningitidis strain C311#3, a strain with well-defined lipopolysaccharide (LPS) and pilin-linked glycan structures. Increased gel migration of the pilin subunit molecules of pglE and pglF mutants was observed by Western analysis, indicating truncation of the trisaccharide structure. Antisera specific for the C311#3 trisaccharide failed to react with pilin from these pglE and pglF mutants. GC-MS analysis of the sugar composition of the pglE mutant showed a reduction in galactose compared with C311#3 wild type. Analysis of amino acid sequence homologies has suggested specific roles for pglE and pglF in the biosynthesis of the trisaccharide structure. Further, we present evidence that pglE, which contains heptanucleotide repeats, is responsible for the phase variation between trisaccharide and disaccharide structures in strain C311#3 and other strains. We also present evidence that pglG, pglH and pgIB2 are potentially phase variable.

Boletim eletr??nico de bibliografias especializadas: pol??ticas p??blicas em direitos humanos

Os direitos humanos s??o os direitos essenciais a todos os seres humanos, sem que haja discrimina????o por ra??a, cor, g??nero, idioma, nacionalidade ou por qualquer outro motivo. Eles podem ser civis ou pol??ticos, como o direito ?? vida, ?? igualdade perante a lei e ?? liberdade de express??o. Podem tamb??m ser econ??micos, sociais e culturais, como o direito ao trabalho e ?? educa????o e coletivos, como o direito ao desenvolvimento. As Pol??ticas P??blicas em Direitos Humanos ?? tema deste boletim bibliogr??fico, o qual traz publica????es que mostram que estas n??o devem ser apenas programas governamentais, mas um instrumento de participa????o da sociedade civil na delimita????o do espa??o p??blico.