Engineered assembly of intertwined oligomers of an immunoglobulin chain.

Domain 1 of CD2 (CD2.D1) forms a conventional Ig fold stabilised by non-covalent antiparallel contacts between beta-strands. Removing two residues from the middle of the protein sequence, where the polypeptide chain normally folds back upon itself, stabilises an open conformation. In this modified molecule, the optimum evolved contacts between side-chains can only be satisfied through the antiparallel association of two chains to create a symmetrical pair of pseudo-domains. Here, we describe the dynamics of the switch between monomeric and dimeric states and demonstrate the extension of this novel underlying principle to trimer and tetramer formation. The ability of a protein molecule to form higher-order antiparallel structures is reminiscent of the behaviour of hairpins, duplexes, three-way and Holliday junctions in DNA.

The Risk Approach: unassailable logic?

The Risk Approach (RA) is a framework for selective provision of health care services advocated by the World Health Organization (WHO). Although the potential utility of RA is stressed in the WHO literature, the framework remains largely untested as a basis for resource deployment. Despite this, advocates claim the logic of RA is 'theoretically unassailable'. The purpose of this paper is to challenge this claim by critically examining some of the assumptions and proposed methods contained in the WHO literature concerning RA. Two areas of concern are discussed, relating to (i) prediction and the nature of relations between markers and outcomes, and (ii) marker measurement and scoring.

On the epistemology of risk: language, logic and social science.

'Risk' is a widely used concept in literatures related to health, health care and medicine. In recent decades, three bodies of literature have emerged in which 'risk' is the primary focus of concern: Health Risk Appraisal, the Risk Approach and Risk Analysis/Assessment/Management. These literatures overlook important concepts and theoretical developments in contemporary social science. They also lack conceptual coherence. Reduction of incoherence will require re-examination of the epistemology of risk in relation to both its language and its logic in light of developments in social science.

Social inequality, population health, and housing: a study of two Vancouver neighborhoods.

An emerging 'population health' framework for understanding inequalities in health identifies the structure of social relations as a crucial factor in shaping human health and well-being. However, there remain many unanswered questions about the mechanisms through which social relations might shape the health status of individuals and populations. Housing plays a central role in routinized, everyday life and is fundamentally bound up in one's sense of control over life circumstances. Housing and property markets are significant in the distribution of wealth and are an important arena for the exercise of power relations. Housing circumstance is crucial in the production and reproduction of social identity and social status. Yet little has been written on the influence of inequalities generated by housing and housing markets on the differential distribution of health status. This paper reports the findings of an empirical study of relationships between socioeconomic status, material and meaningful dimensions of housing and home, and health status. Our objective is to investigate ways in which material and meaningful factors related to housing, in conjunction with other dimensions of the social environment, could operate to produce systematic inequalities in health status across social strata. The data for this study were obtained through a mailed survey of residents in the Mount Pleasant (n = 322) and Sunset (n = 206) neighborhoods of Vancouver, Canada. They suggest that, in concert with commonly used measures of socioeconomic status, both material and meaningful dimensions of housing and home are associated with health status in a direction consistent with expectations following from our analytical model.

The ecology of health services utilization in Grenada, West Indies.

Few empirical investigations into the nature of health services utilization fully acknowledge that the home environment may act as a socio-geographic focus of both disease transmission and of learned health behavior. This paper examines the role of the home environment, as well as of personal characteristics and accessibility, in the utilization of health services in Grenada, West Indies. Bivariate and logit analyses of household survey data are employed to identify markers of high user individuals and households. Aspects of each domain of the home environment (physical and behavioral environment, demographics, and residential mobility) emerge as contributors to the utilization phenomenon in the study communities. Both etiological and socio-economic linkages are postulated to underlie the observed relationships. The implications of this work for health planning in developing countries is discussed.

Reported versus recorded health service utilization in Grenada, West Indies.

Calls for household surveys to provide information on service utilization in less developed countries raise questions regarding the accuracy and reliability of reporting. This paper compares reported to recorded health service utilization for diarrhoea and any other morbidity over a 2-week and 3-month period for information obtained from a household survey in Grenada, West Indies. A sensitivity analysis is used to derive minimum and maximum estimates of the accuracy of reported utilization. Over-reporting utilization was found to be between 33 and 62% for diarrhoea and 49 and 81% for any other morbidity. Under-reporting of all utilization was estimated to be between 47 and 65%. These results cast doubt on the utility of household surveys as a reliable source of information regarding service utilization.

Isolation and characterisation of an antifungal antibiotic (GR135402) with protein synthesis inhibition.

A novel antifungal antibiotic GR135402 has been isolated from a fermentation broth of Graphium putredinis which inhibited protein synthesis in Candida albicans but not rabbit reticulocytes. The spectrum of activity included C. albicans and Cryptococcus neoformans but not some other Candida species or Aspergillus species. Therapeutic efficacy in a mouse model of systemic candidosis was attained following parenteral dosing.

Population health promotion: responsible sharing of future directions.

Population health promotion illustrates most robustly that health is a shared responsibility. Improving our understanding of the social production of health and the purchase population health promotion has on shaping social welfare policy presents a number of challenges to the future development of this discourse. Three are briefly discussed in this paper. First is the matter of language we use to describe our understanding of processes and influences. Second is the conceptualization of the pathways that shape population health status. Finally, cultural practices both extant and required to improve health status and reduce inequalities are addressed.

Toward a lexicon of population health.

Despite its undeniable currency in research and policy circles, there remains considerable confusion about what 'population health' is. We propose a lexicon for population health in the hope of clarifying issues and advancing this important research emphasis and policy agenda. It distinguishes population health in its literal meaning from a population health perspective, population health research, a population health framework, and a population health approach to policy. Population health is more than just thinking in aggregate terms or about identifying vulnerable or at-risk subpopulations. A population health perspective is fundamentally concerned with the social nature of health influences. The social structures that shape health experiences transcend the characteristics or actions of any one individual, providing population health with analytic advantages over individualistic-oriented approaches to health and to health policy.

Developmental marketing strategies for community mental health.

This article contains a marketing plan that was specifically designed for The Greater Lawrence Mental Health Center in Lawrence, Massachusetts. It is a full service, non-profit mental health facility that employs 85 allied health professionals and serves Lawrence and its neighboring communities. The plan was developed by a marketing consultant, in conjunction with the agency's Executive and Administrative Directors. This article illustrates a format for developing a marketing plan, and includes specific approaches that must be considered. The concepts are generic, and can be applied to similar endeavors undertaken by a comparable agency.

Mode of action of ceftazidime: affinity for the penicillin-binding proteins of Escherichia coli K12, Pseudomonas aeruginosa and Staphylococcus aureus.

The competition of a new aminothiazolyl cephalosporin, ceftazidime, for the penicillin-binding proteins (PBP's) of Escherichia coli K12, Pseudomonas aeruginosa and Staphylococcus aureus has been studied. Ceftazidime caused filamentation and eventually cell lysis of both E. coli and Ps. aeruginosa at its minimum inhibitory concentration, due to its primary activity against PBP-3. The antibiotic also inhibited PBP's 1 a and 1 bs, the 'essential' cell elongation proteins at higher, therapeutically achievable concentrations and consequently induced rapid lysis of both E. coli and Ps. aeruginosa. In Staph. aureus ceftazidime showed high affinity for PBP-1, -2 and less affinity for PBP-3. The results indicate that in E. coli K12 and Ps. aeruginosa, ceftazidime owes its good antibacterial activity to high affinity for PBP-3, the 'essential' binding protein involved in cell division combined with favourable outer membrane penetration.

Three beta-lactamases isolated from Aeromonas salmonicida, including a carbapenemase not detectable by conventional methods.

The beta-lactamases of seven strains of Aeromonas salmonicida subsp. achromogenes resistant to amoxicillin (MIC > 1024 mg/l) and responsible for furunculosis in farmed Atlantic salmon in Scotland were examined to establish the mechanisms of beta-lactam resistance. Separation of a cell-free extract on an isoelectric focusing gel stained with the chromogenic cephalosporin nitrocefin showed the presence of two beta-lactamases, one with a pI of 7.9 and the other with a pI of 6.0. Hydrolysis assays of cell-free extracts of these strains demonstrated carbapenemase, penicillinase and cephalosporinase activity. However, when the beta-lactamases were separated by anion exchange chromatography, the carbapenemase activity could not be retrieved in either of the peak fractions containing the separated enzymes that had been visualised by nitrocefin. Consequently, a novel carbapenemase was discovered which cannot be detected with nitrocefin.

Synthesis of peptidoglycan in vivo in methicillin-resistant Staphylococcus aureus.

The cell-wall composition and degree of cross-linking of peptidoglycan in a strain of Staphylococcus aureus (strain MR-1) which is highly resistant to methicillin were similar to those of other strains of S. aureus. When the organism was grown in the presence of very low concentrations of methicillin (equivalent to 3 x 10(-4) x minimum growth-inhibitory concentration [MGIC] there was a large decrease in the degree of cross-linking of the peptidoglycan. Increasing concentrations of methicillin (up to 1.25 x 10(-2) x MGIC) caused a further decrease in cross-linkage but thereafter a minimum value was reached. This remained unchanged even after growth of the organisms in much higher concentrations of the antibiotic up to 0.3 x minimum growth-inhibitory concentration. S. aureus MR-1 was able to grow normally for many generations under these conditions and reduction in cross-linkage of peptidoglycan was the only change detected in wall chemistry. Growth in the presence of methicillin (up to 0.3 x MGIC) (or other beta-lactam antibiotics) did not lead to an imbalance in the biosynthesis of peptidoglycan since no soluble polymers were secreted into the growth medium and nucleotide-linked precursors did not accumulate intracellularly. High concentrations of beta-lactam antibiotics (5 x MGIC) were bacteriostatic not bactericidal and this may be related to an apparent deficiency in the endogenous autolytic enzymes of strain MR-1. Studies of the penicillin-binding proteins after growth in the presence of methicillin suggest that one of these proteins remains resistant to very high concentrations of the antibiotic. We propose that this protein acts as the primary transpeptidase responsible for the incorporation of newly synthesised peptidoglycan into the growing wall.

The 'hidden' carbapenemase of Aeromonas hydrophila.

It has been presumed that there are just two beta-lactamases in the motile Aeromonas species, a carbapenemase and a cephalosporinase, based on the premise that all beta-lactamases can be detected by hydrolysis of the chromogenic cephalosporin, nitrocefin. However, when it was recently found that a non-motile species of Aeromonas that causes furunculosis in salmon, contained three beta-lactamases, one of which was a carbapenemase which could not be detected with nitrocefin, it was hypothesised that genetic exchange could occur between fish pathogens and human pathogens resulting in the transfer of the carbapenemase-encoding gene. This could have a potentially serious impact on intensive therapy units where carbapenems are employed. The purpose of this study was to determine whether the human pathogen Aeromonas hydrophila demonstrated the same beta-lactamase profile. After anion and cation exchange chromatography had been employed to separate the beta-lactamases of a clinical strain of A. hydrophila, three different beta-lactamases were found, one of which is a carbapenemase which does not hydrolyse nitrocefin. It is, therefore, probable that many strains of Aeromonas spp. contain a similar array of beta-lactamases which include a carbapenemase that cannot be detected with nitrocefin. Similar carbapenemases may well remain hidden in other species of bacteria unless appropriate techniques to detect the enzymes are employed.

Elongation factor 3 (EF-3) from Candida albicans shows both structural and functional similarity to EF-3 from Saccharomyces cerevisiae.

As with many other fungi, including the budding yeast Saccharomyces cerevisiae, the dimorphic fungus Candida albicans encodes the novel translation factor, elongation factor 3 (EF-3). Using a rapid affinity chromatography protocol, EF-3 was purified to homogeneity from C. albicans and shown to have an apparent molecular mass of 128 kDa. A polyclonal antibody raised against C. albicans EF-3 also showed cross-reactivity with EF-3 from S. cerevisiae. Similarly, the S. cerevisiae TEF3 gene (encoding EF-3) showed cross-hybridization with genomic DNA from C. albicans in Southern hybridization analysis, demonstrating the existence of a single gene closely related to TEF3 in the C. albicans genome. This gene was cloned by using a 0.7 kb polymerase chain reaction-amplified DNA fragment to screen to C. albicans gene library. DNA sequence analysis of 200 bp of the cloned fragment demonstrated an open reading frame showing 51% predicted amino acid identity between the putative C. albicans EF-3 gene and its S. cerevisiae counterpart over the encoded 65-amino-acid stretch. That the cloned C. albicans sequence did indeed encode EF-3 was confirmed by demonstrating its ability to rescue an otherwise non-viable S. cerevisiae tef3:HIS3 null mutant. Thus EF-3 from C. albicans shows both structural and functional similarity to EF-3 from S. cerevisiae.

A role in vivo for penicillin-binding protein-4 of Staphylococcus aureus.

The degree of cross-linking of the peptidoglycan of Staphylococcus aureus H and mutants lacking penicillin-binding proteins 1 and 4 was studied. No major changes were observed in organisms lacking protein 1 whereas loss of protein 4 was accompanied by a marked reduction in the degree of cross-linking and the absence of a membrane-bound 'model' transpeptidase activity. A similar effect was achieved when cultures of the staphylococci were treated with the beta-lactam antibiotic cefoxitin. At low concentrations (0.05 microgram ml-1) cefoxitin shows highest affinity for protein 4 to which it appears to bind irreversibly. Treatment of the mutant lacking protein 4 with this concentration of the antibiotic did not affect the degree of cross-linkage. The possibility that the decrease in cross-linkage was a consequence of DD-carboxypeptidase activity on peptidoglycan precursors was investigated. Although both S. aureus H and the mutants possessed such activity it was insensitive to benzylpenicillin and cefoxitin and the role of this enzyme(s) in peptidoglycan biosynthesis remains unknown. We conclude that in vivo protein 4 acts as a transpeptidase involved in the secondary cross-linking of peptidoglycan and this activity is necessary to achieve the high degree of cross-linkage observed in the peptidoglycan of staphylococci.

Cross-linking and O-acetylation of newly synthesized peptidoglycan in Staphylococcus aureus H.

Staphylococcus aureus H growing exponentially was labelled with N-acetyl[14C]glucosamine, which became incorporated into the peptidoglycan. The portion of peptidoglycan not linked to teichoic acid (60-75% of the whole) was degraded with Chalaropsis muramidase to yield disaccharide-peptide monomers and dimers, trimers and oligomers formed by biosynthetic cross-linking of the monomers. The degree of O-acetylation of these fragments was also examined. Pulse-chase experiments showed that the proportion of label initially in the monomer fraction immediately after the 1 min pulse declined rapidly during a 3 min chase, while the oligomer fraction (fragments greater than trimer) gained the radioactivity proportionately. The radioactivity of the dimer and trimer fractions remained virtually unchanged. At 4 min after the commencement of labelling (i.e. approx. one-tenth of a generation time) final values had been reached. The O-acetylation of all fragments had achieved final values even at 1 min, except for the monomer fraction, which showed an increase from 40% to 60% during the first 3 min of chase. Although O-acetylation was clearly a very rapid process, no O-acetylated peptidoglycan lipid-intermediates could be detected.

One sequence, four folds: transitions between an ensemble of metastable folds for the N-terminal domain of CD2.

Recombinant forms of the N-terminal domain of the cell adhesion receptor CD2 adopt a variety of olds by exchange of beta-sheets between adjacent polypeptide chains. Although these interdigitated forms are normally metastable, we have used site-directed mutagenesis to alter the kinetics of formation and relative stabilities of these states, leading to spontaneous formation of monomeric, dimeric, trimeric and tetrameric intertwined folded states. A characteristic feature of these fold-disorder-alternative fold transitions is the independence of each domain folding event, as deduced from kinetic analysis of folding data. Structures for fully interdigitated trimeric and tetrameric forms have been modelled, consistent with both the crystallographic and kinetic data. Although the biological role of these alternative folded states remains unclear, these structures form a remarkable demonstration of the fluidity of structure generated from a single polypeptide chain.