Factors associated with bicycle helmet use among young adolescents in a multinational sample.

OBJECTIVE: To determine factors associated with variation in bicycle helmet use by youth of different industrialized countries. DESIGN: A multinational cross sectional nationally representative survey of health behaviors including symptoms, risk taking, school setting, and family context. SETTING: School based survey of 26 countries. SUBJECTS: School students, ages 11, 13, and 15 years totaling 112,843. OUTCOME MEASURES: Reported frequency of bicycle helmet use among bicycle riders. RESULTS: Reported helmet use varied greatly by country from 39.2% to 1.9%, with 12 countries reporting less than 10% of the bicycle riders as frequent helmet users and 14 countries more than 10%. Reported helmet use was highest at 11 years and decreased as children's age increased. Use was positively associated with other healthy behaviors, with parental involvement, and with per capita gross domestic product of the country. It is negatively associated with risk taking behaviors. Countries reported to have interventions promoting helmet use, exemplified by helmet giveaway programmes, had greater frequency of reported helmet use than those without programmes. CONCLUSIONS: Bicycle helmet use among young adolescents varies greatly between countries; however, helmet use does not reach 50% in any country. Age is the most significant individual factor associated with helmet for helmet using countries. The observation that some helmet promotion programmes are reported for countries with relatively higher student helmet use and no programmes reported for the lowest helmet use countries, suggests the possibility of a relation and the need for objective evaluation of programme effectiveness.

Commitment to folded and aggregated states occurs late in interleukin-1 beta folding.

A point mutation, lysine 97 to isoleucine, in the all-beta cytokine interleukin-1 beta (IL-1 beta) exhibits an increased propensity to form inclusion bodies in vivo and aggregates in vitro. In an effort to better understand the aggregation reaction and determine when intervention may allow rescue of protein from aggregation during renaturation, we developed a novel application of mass spectrometry using isotopic labeling to determine the step(s) at which K97I commits to either the native or aggregated state. Interestingly, despite the early formation of a folding intermediate ensemble at an observed rate lambda(2) of 4.0 s(-1), K97I commits to folding at a significantly slower rate lambda(CF) of 0.021 s(-1). This rate of commitment to folding is in excellent agreement with the observed rate of K97I native state formation (lambda(1) = 0.018 s(-1)). K97I also commits slowly to aggregation at an observed rate lambda(CA) of 0.023 s(-1). Earlier folding species and aggregates present prior to these commitment steps are likely to be in a reversible equilibrium between monomeric folding intermediates and higher-order oligomers. Kinetic and equilibrium experimental measurements of folding and aggregation processes are consistent with a nucleation-dependent model of aggregation.

The structure of the chromophore within DsRed, a red fluorescent protein from coral.

DsRed, a brilliantly red fluorescent protein, was recently cloned from Discosoma coral by homology to the green fluorescent protein (GFP) from the jellyfish Aequorea. A core question in the biochemistry of DsRed is the mechanism by which the GFP-like 475-nm excitation and 500-nm emission maxima of immature DsRed are red-shifted to the 558-nm excitation and 583-nm emission maxima of mature DsRed. After digestion of mature DsRed with lysyl endopeptidase, high-resolution mass spectra of the purified chromophore-bearing peptide reveal that some of the molecules have lost 2 Da relative to the peptide analogously prepared from a mutant, K83R, that stays green. Tandem mass spectrometry indicates that the bond between the alpha-carbon and nitrogen of Gln-66 has been dehydrogenated in DsRed, extending the GFP chromophore by forming C==N==C==O at the 2-position of the imidazolidinone. This acylimine substituent quantitatively accounts for the red shift according to quantum mechanical calculations. Reversible hydration of the C==N bond in the acylimine would explain why denaturation shifts mature DsRed back to a GFP-like absorbance. The C==N bond hydrolyses upon boiling, explaining why DsRed shows two fragment bands on SDS/PAGE. This assay suggests that conversion from green to red chromophores remains incomplete even after prolonged aging.

Evidence for an obligatory intermediate in the folding of interleukin-1 beta.

The folding of the beta-sheet protein, interleukin-1 beta, was examined at pH 5.0 and 25 degrees C using pulse-labelling hydrogen exchange and electrospray ionization mass spectrometric analysis, as well as stopped-flow circular dichroism and fluorescence spectroscopies. The first detectable event is the formation of a partially folded intermediate in a kinetic step with a relaxation time of 126 +/- 26 ms. There is a lag in native protein production of at least 400 ms. Optical studies indicate that the intermediate is converted to the native species in a reaction with a relaxation time of 43 +/- 5 s. The kinetic rates determined from stopped-flow fluorescence, circular dichroism and pulse-labelling experiments are similar and consistent with a simple sequential model for the folding pathway of interleukin-1 beta at pH 5.0 and 25 degrees C. Taken together, our data provide kinetic evidence that formation of the native state of interleukin-1 beta proceeds through an obligatory intermediate. We explain our results in terms of the classical and new views of protein folding.

Crystal structure of the Aequorea victoria green fluorescent protein.

The green fluorescent protein (GFP) from the Pacific Northwest jellyfish Aequorea victoria has generated intense interest as a marker for gene expression and localization of gene products. The chromophore, resulting from the spontaneous cyclization and oxidation of the sequence -Ser65 (or Thr65)-Tyr66-Gly67-, requires the native protein fold for both formation and fluorescence emission. The structure of Thr65 GFP has been determined at 1.9 angstrom resolution. The protein fold consists of an 11-stranded beta barrel with a coaxial helix, with the chromophore forming from the central helix. Directed mutagenesis of one residue adjacent to the chromophore, Thr203, to Tyr or His results in significantly red-shifted excitation and emission maxima.

Understanding, improving and using green fluorescent proteins.

Green fluorescent proteins (GFPs) are presently attracting tremendous interest as the first general method to create strong visible fluorescence by purely molecular biological means. So far, they have been used as reporters of gene expression, tracers of cell lineage, and as fusion tags to monitor protein localization within living cells. However, the GFP originally cloned from the jellyfish Aequorea victoria has several nonoptimal properties including low brightness, a significant delay between protein synthesis and fluorescence development, and complex photoisomerization. Fortunately, the protein can be re-engineered by mutagenesis to ameliorate these deficiencies and shift the excitation and emission wavelengths, creating different colors and new applications.

The human NME2 gene lies within 18kb of NME1 in chromosome 17.

The NME1 gene, localized to human chromosome 17 at q22, shows reduced expression in tumors of high metastatic potential. A homologous gene, NME2, with similar reduced expression in breast carcinoma, has recently been reported. We have isolated and characterized five yeast artificial chromosome (YAC) clones and three cosmid clones that contain both genes, demonstrating that the NME2 gene is also located on chromosome 17 and is separated by not more than 18 kb from the NME1 gene. The two putative tumor suppressor genes, encoding the two polypeptide chains of nucleoside diphosphate (NDP) kinase, are thus quite close to each other on chromosome 17, indicating that they may well have arisen by a tandem duplication. Both genes now appear to be excluded as candidates for the early-onset breast cancer (BRCA1) locus.

Phenotypic and functional similarities between 5-azacytidine-treated T cells and a T cell subset in patients with active systemic lupus erythematosus.

OBJECTIVE: Antigen-specific CD4+ T cells treated with DNA methylation inhibitors become autoreactive, suggesting a novel mechanism for autoimmunity. To test whether this mechanism might be involved in systemic lupus erythematosus (SLE), phenotypic markers for the autoreactive cells were sought. METHODS: Cloned normal T cells were treated with the DNA methylation inhibitor 5-azacytidine (5-azaC) and studied for altered gene expression. T cells from patients with active SLE were then studied for a similar change in gene expression, and cells expressing the marker were tested for autoreactivity. RESULTS: 5-azaC-treated normal T cells had increased CD11a (leukocyte function-associated antigen 1 alpha) expression relative to other membrane molecules. A T cell subset with similar CD11a expression was found in patients with active SLE. This subset contained cells that spontaneously lysed autologous macrophages, with a specificity similar to that of 5-azaC-treated cells.

Procainamide inhibits DNA methyltransferase in a human T cell line.

Procainamide, a widely used antiarrythmic, causes DNA hypomethylation in the human T cell line Jurkat, but the mechanism is unknown. We report that procainamide inhibits the DNA methyltransferase catalyzed transfer of methyl groups from S-adenosylmethionine to DNA, but has no effect on other known regulators of DNA methylation. Our results suggest that procainamide could inhibit cellular DNA methylation by inhibiting DNA methyltransferase activity.

New technique for reapproximation of the calcaneofibular ligament after double ligament rupture.

Ankle sprains are often inadequately treated in the emergency room, because of the feeling that, "It is only a sprain." A thorough examination is performed at St. John Hospital Macomb Center to determine the extent of ligamentous damage. If a double ligament rupture presents, and surgical intervention is necessary, a procedure similar to the one described by Bröstrom is performed. The calcaneofibular ligament's frayed ends, as well as its location, make suturing the ligament difficult. Utilizing medium-sized Ligaclips on either side of the rupture provides an excellent scaffold for reapproximation of the calcaneofibular ligament. The Ligaclips could also be used to determine if a re-rupture has occurred.

Pressure and temperature dependence of the self-broadened linewidths of the carbon dioxide laser bands.

The results of laboratory measurements of high-resolution infrared spectra of carbon dioxide in the region near 10 microm at several temperatures and pressures are described. Spectra of the CO(2) laser bands centered at 961 and 1064 cm(-1) were collected at a resolution ofO005 cm(-1) with the gas at temperatures between 50 and 150 degrees C. The self-broadened rotation-vibration line shapes were modeled using a nonlinear least-squares regression technique. Careful attention was given to the problem of base line correction. The temperature exponent of the pressure-broadening coefficient was estimated for thirty lines. The linewidths obtained previously using indirect methods compare well to data obtained in the present investigation.