Antibiotic activity and characterization of BB-3497, a novel peptide deformylase inhibitor.

Peptide deformylase (PDF) is an essential bacterial metalloenzyme which deformylates the N-formylmethionine of newly synthesized polypeptides and as such represents a novel target for antibacterial chemotherapy. To identify novel PDF inhibitors, we screened a metalloenzyme inhibitor library and identified an N-formyl-hydroxylamine derivative, BB-3497, and a related natural hydroxamic acid antibiotic, actinonin, as potent and selective inhibitors of PDF. To elucidate the interactions that contribute to the binding affinity of these inhibitors, we determined the crystal structures of BB-3497 and actinonin bound to Escherichia coli PDF at resolutions of 2.1 and 1.75 A, respectively. In both complexes, the active-site metal atom was pentacoordinated by the side chains of Cys 90, His 132, and His 136 and the two oxygen atoms of N-formyl-hydroxylamine or hydroxamate. BB-3497 had activity against gram-positive bacteria, including methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecalis, and activity against some gram-negative bacteria. Time-kill analysis showed that the mode of action of BB-3497 was primarily bacteriostatic. The mechanism of resistance was via mutations within the formyltransferase gene, as previously described for actinonin. While actinonin and its derivatives have not been used clinically because of their poor pharmacokinetic properties, BB-3497 was shown to be orally bioavailable. A single oral dose of BB-3497 given 1 h after intraperitoneal injection of S. aureus Smith or methicillin-resistant S. aureus protected mice from infection with median effective doses of 8 and 14 mg/kg of body weight, respectively. These data validate PDF as a novel target for the design of a new generation of antibacterial agents.

The National Population Health Survey--its longitudinal nature.

OBJECTIVES: This article discusses some of the benefits and challenges of data from a longitudinal panel as exemplified by the National Population Health Survey (NPHS). DATA SOURCE: The NPHS collects both cross-sectional and longitudinal data from a sample of randomly selected individuals. The longitudinal sample will be reinterviewed every 2 years for up to 20 years. Two NPHS cycles have been completed: cycle 1 in 1994/95 and cycle 2 in 1996/97. SUMMARY: Selected findings from the NPHS are presented to illustrate the benefits of longitudinal data. An overview of questionnaire content, collection methods follows, and sample design is provided. A summary of response rates is followed by a discussion of the methods used to maintain response and to adjust the survey weights in order to reduce nonresponse bias. Confidentiality, dissemination, inconsistencies in reporting, proxy reporting and changes in coding conventions are also discussed.

Quantitation of matrix metalloproteinases in cultured rat astrocytes using the polymerase chain reaction with a multi-competitor cDNA standard.

Matrix metalloproteinases (MMPs) are a family of Zn2+ endopeptidases that are expressed in many inflammatory conditions and that contribute to connective tissue breakdown and the release of the pro-inflammatory cytokine tumour necrosis factor-alpha (TNF-alpha). There is emerging evidence that MMPs have a role in inflammatory disorders of the central nervous system (CNS) such as multiple sclerosis. However, little is known about the expression of MMPs by inflamed tissue within the CNS or by the glia, neurones, and leucocytes which participate in the inflammatory response. To address this issue we have developed a polymerase chain reaction (PCR)-based method for the quantitation of rat MMP mRNA levels, which we have applied to astrocyte cultures with and without inflammatory stimulation. The technique relies on a competition reaction in which a synthetic standard cDNA is co-amplified with the target cDNA in the same PCR reaction. Standard multi-competitor cDNAs, containing priming sites for nine MMPs, and two housekeeping genes were constructed. We have shown that MMP activity is increased over three-fold in neonatal rat astrocyte cultures following stimulation with lipopolysaccharide (LPS). At the mRNA level, MT-MMP-1, 72 kDa gelatinase, and stromelysin-3 were constitutively expressed and unaffected by LPS treatment, whereas 92 kDa gelatinase, and stromelysin-1 were strongly induced (1,000-fold). Stromelysin-2, rat collagenase, and macrophage metalloelastase were modestly upregulated by LPS treatment. Matrilysin was not expressed. This technique is suitable for quantifying MMP expression in the cells which contribute to inflammation in the CNS and could also be applied directly to tissue samples from animal models of disease.

Mental health supplement to the Ontario Health Survey: methodology.

OBJECTIVE: To describe the methodology of a province-wide, cross-sectional, epidemiologic study of psychiatric disorder among those aged 15 years and over living in household dwellings in Ontario. METHOD: Respondents for the survey were drawn from households (N = 13002) participating in a province-wide health survey. One person per household was selected, and 9953 (76.5%) participated. RESULTS: Participants and nonparticipants were similar to each other. An extensive array of data, including measures of psychiatric disorder classified using a revised version of the Composite International Diagnostic Interview (CIDI), are available for all respondents. CONCLUSIONS: The Ontario Health Supplement is contained in a public-use data file at the Ontario Ministry of Health and is available to investigators for study. A strong survey design, careful measurement, and acceptable levels of response provide the rationale for our inviting researchers to access and use the Ontario Health Supplement data base.

Identification of MMP-18, a putative novel human matrix metalloproteinase.

A partial cDNA encoding the 3' end of a putative novel human matrix metalloproteinase (MMP) was identified by sequence similarity searching of databases containing expressed sequence tags. The remaining 5' end of the MMP cDNA was amplified by PCR from human mammary gland cDNA. The predicted protein product displays all the structural features characteristic of the MMP family and has closest identity with MMP-1, -3, -10, and 11. We have provisionally designated this novel MMP as MMP-18. MMP-18 mRNA is expressed in a wide variety of normal human tissues, including mammary gland, placenta, lung, pancreas, ovary, small intestine, spleen, thymus, prostate, testis, colon, and heart, but is not detected in brain, skeletal muscle, kidney, liver, or peripheral blood leucocytes.

A healthy outlook.

The sense of coherence-a healthy outlook-can be thought of as a measure of positive health, that is, a factor promoting resilience which enables an individual to remain healthy. Based on National Population Health Survey (NPHS) data, three health measures were analyzed in relation to sense of coherence. The sense of coherence accounted for a substantial proportion of the total variance for two of the three measures. Theoretically, people with a healthy outlook are more able to cope successfully with trauma and stress. According to NPHS data, on average, those who reported at least one traumatic event had a lower sense of coherence than those who did not. For people who experienced trauma during childhood and young adulthood, yet had a strong sense of coherence, the impact of that trauma on their health was diminished.

Sample design of the national population health survey.

In 1994, Statistics Canada began data collection for the National Population Health Survey (NPHS), a household survey designed to measure the health status of Canadians and to expand knowledge of health determinants. The survey is longitudinal, with data being collected on selected panel members every second year. This article focuses on the NPHS sample design and its rationale. Topics include sample allocation, representativeness, and selection; modifications in Quebec and the territories; and integration of the NPHS with the National Longitudinal Survey of Children. The final section considers some methodological issues to be addressed in future waves of the survey.

The role of culture in grief.

Reports of the effects of the death of a loved one were collected from subjects at the University of Massachusetts in the United States and the University of Madrid in Spain. Although reports of the overall severity of the experience were similar, differences between the samples were apparent in the specific nature of the experience. Americans indicated that, following the death of a loved one, their self-esteem was diminished, as was their liking and trust of others. In contrast, Spaniards reported a greater negative effect on self-esteem and a positive effect on liking and trust of others. The role of culture in the experience of bereavement is discussed.

The National Population Health Survey: highlights of initial developments.

A National Population Health Survey is being developed for implementation across Canada in 1994. A description is given of the objectives, plans and current status of the survey. The current assumptions concerning the survey methodology are also outlined along with an early indication of the content.

Two PDGF-B chain residues, arginine 27 and isoleucine 30, mediate receptor binding and activation.

PDGF may be involved in the pathogenesis of a variety of disorders including atherosclerosis and certain types of cancer. There is currently little understanding of the molecular structure of PDGF and of the critical amino acid residues involved in receptor binding and cell activation. Two such PDGF-B chain residues, arginine 27 and isoleucine 30, have been identified by a site-directed mutagenesis programme. Substitutions in these positions can lead to PDGF mutants defective in both receptor affinity and cell activation as judged by displacement of [125I]PDGF-BB, mitogenic assay and inositol lipid turnover. Circular dichroism and fluorescence spectroscopy show that such mutations do not disrupt the structure of PDGF.

Secretion of human epidermal growth factor from Saccharomyces cerevisiae using synthetic leader sequences.

We have investigated different leader sequences for their ability to direct the efficient secretion of human epidermal growth factor (hEGF) from Saccharomyces cerevisiae. We designed a consensus signal sequence which directs secretion of hEGF from yeast as efficiently as the yeast invertase signal sequence. However, secretion is increased over fivefold by the introduction, after the signal sequence, of a synthetic 19-amino acid (aa) pro-sequence containing a cleavage recognition site for the KEX2 protease. Even in the absence of an Asn-linked glycosylation site, secretion of hEGF using the synthetic prepro-leader was as efficient as that directed by the alpha-factor leader. The role of the KEX2 protease cleavage site was investigated by mutation of the yeast alpha-factor KEX2 site (cleavage after Lys-Arg). Cleavage was obtained with the following order of efficiency, Lys-Arg greater than Pro-Arg greater than Asp-Arg, although the sequence context was also found to affect efficiency.

Ontario Child Health Study follow-up: evaluation of sample loss.

This article presents an analysis of sample loss in a 4-year follow-up of children aged 4 to 12 who participated in the Ontario Child Health Study in 1983. Of the 1,617 children participating in the original Ontario Child Health Study, 1,172 (72.5%) were located and enlisted at follow-up in 1987. Based on wave-one assessments, nonparticipants at follow-up tended to have higher levels of psychopathology and family risk variables. Respondents were matched with nonparticipants and differentially weighted to compensate for selective loss. In comparing estimates based on actual (observed) and weighted responses in the follow-up sample, it was found that the effects of sample loss depended on the analytical focus. Evaluations of outcome of disorder and risk for disorder were not affected by sample loss. Evaluation of variables that predict persistence of disorder (prognosis) was affected by a bias toward the null.

Ontario Child Health Study. I. Methodology.

We developed the methodology for a community survey to determine the prevalence of emotional and behavioral disorders among children 4 to 16 years of age in Ontario, Canada. Our discussion includes the objectives of the survey, the measurement of disorder, sampling methods and survey design, and a description of the data collected and instrumentation. Among 2052 households with eligible children, 1869 (91%) participated in the survey. The results can be used to help plan the future allocation of mental health resources in Ontario.

Chemical mutagenesis of human interferon-beta: construction, expression in E. coli, and biological activity of sodium bisulfite-induced mutations.

A series of modified human interferon-beta (IFN-beta) genes was produced by sodium bisulfite treatment of the IFN-beta gene cloned in M13. A library of mutated sequences was generated from which subgenomic fragments containing one or a small number of coding alterations were isolated and substituted into the IFN-beta gene in an E. coli expression vector. A number of modified genes and their expression products were evaluated. In several instances levels of expression and biological activity profiles are altered compared to the parental gene product. A number of key amino acids can be identified, whose substitutions have marked effects on biological activity of IFN-beta.

Novel modified beta-interferons: gene cloning, expression, and biological activity in bacterial extracts.

A series of novel, modified interferons based on the structure of human beta-interferon have been expressed in Escherichia coli. Modified interferon genes were constructed from sequences derived from the natural beta-interferon gene, a synthetic beta-interferon gene, or a specific combination of the two. A total of 23 out of the 25 novel interferons exhibited antiviral (AV) and antiproliferative (AP) activity which varied from 3 to 230% and 8 to 490% of the values for beta-interferon, respectively. None of the novel interferons had only AV or AP activity, although one had a much reduced ratio of AV/AP activity compared with beta-interferon. Substitution of beta-interferon amino acids 2-7 or 28-46 resulted in interferons with significantly increased AP activity on Daudi lymphoblastoid cells (four- to fivefold). All the novel interferons except two with modifications in the 82-105 region reacted with a neutralizing beta-interferon monoclonal antibody.

Effective participation in cooperative clinical trials by an independent community organization. An NCOG Model.

The NCCP has established a model for community involvement in study group clinical trials, based on the use of independent community groups as participating entities. The structure and operation of the Sacramento, California demonstration project are presented. Evaluation of the NCOG small cell lung cancer study 2061 reveals that the data submitted by the community group equalled or exceeded university-generated and group-wide data for evaluability, response rate, survival, and quality control.

Biological properties of human interferon beta 1 synthesized in recombinant bacteria.

Human fibroblast interferon, designated IFN-beta 1, has been produced in E. coli by direct expression of the cloned cDNA coding for the mature polypeptide. Bacterial lysates from recombinant cultures contain a polypeptide with an apparent molecular weight of 17,500 that corresponds in size to the unglycosylated IFN-beta 1 molecule. The latter could be specifically immunoprecipitated by antibodies to purified natural IFN-beta and could inhibit the replication of Herpes simplex virus types 1 and 2 in many different cell lines. Like the natural fibroblast IFN-beta, the bacterial IFN-beta 1 was active in many human cell lines, less active in a monkey cell line and inactive in rabbit and mouse fibroblasts. The antibody titre required to neutralise the anti-herpes activity of both IFN preparations was similar suggesting that they have the same specific activities. Similarly, the bacterial IFN-beta 1 was equally active in inhibiting the proliferation of Daudi cells grown in culture. Bacterial IFN-beta 1 was also capable of enhancing natural killer cell activity and antibody-dependent cellular cytotoxicity in vitro. Thus, IFN-beta 1 produced in recombinant bacteria displays a large range of biological properties ascribed to the natural fibroblast IFN-beta molecule.