Use of proline mutants to help solve the NMR solution structure of type III antifreeze protein

To help understand the structure/function relationships in antifreeze proteins (AFP), and to define the motifs required for ice binding, a Type III AFP suitable for two-dimensional (2D) NMR studies was produced in Escherichia coli. A synthetic gene for one of the Type III AFP isoforms was assembled in a T7 polymerase-directed expression vector. The 67-amino acid-long gene product differed from the natural AFP by inclusion of an N-terminal methionine but was indistinguishable in activity. The NMR spectra of this AFP were complicated by cis-trans proline isomerization from the C-terminal sequence YPPA. Substitution of this sequence by YAA eliminated isomer signals without altering the activity or structure of the mutant AFP. This variant (rQAE m1.1) was selected for sequential assignment and the secondary structure determination using 2D 1H NMR spectroscopy. Nine beta-strands are paired to form two triple-stranded antiparallel sheets and one double-stranded antiparallel sheet. Two further proline replacements, P29A and P33A, were made to delineate the role of conserved prolines in Type III AFP. These mutants were valuable in clarifying ambiguous NMR spectral assignments amongst the remaining six prolines of rQAE m1.1. In contrast to the replacement of the C-terminal prolyl residues, the exchange of P29 and P33 caused some structural changes and significantly decreased protein solubility and antifreeze activity.     

The reproducibility of submaximal work heart rates-an interim report

The present article describes the background and principles of a programme for community control of hypertension, which is part of the North Karelia project--a comprehensive community programme for the control of cardiovascular diseases. The hypertension programme also forms part of an international co-operative study by WHO. The programme consists of a community intervention integrated with the service structure. The methods used include health education of the public, education of health service personnel, organization of a service for the spreading of information and also for screening, treatment and follow-up. Every hypertensive person in the community is registered in the hypertension register and checked on annually. The evaluation is made mainly on baseline and terminal survey data from the intervention area and a reference area, the hypertension register, a myocardial infarction register and a stroke register in the intervention area.     

The effect of substrate preparation on the adhesion of polyethylene to aluminium

Polyethylene coatings were applied to aluminium by melting, and adhesion measured by a peel test. The formation of hydrated oxide films on the metal is shown to greatly increase adhesion for coatings applied in nitrogen, through formation of needle-shaped oxide crystals. Particles of oxide deposited on the metal are found to have a similar effect. These treatments improve the durability of the bond when exposed to water, especially when coatings have been applied in air.     

Publication Sponsored by the Society's Scarce Resources Committee — IV

This paper attempts to summarize the cost areas related to the operation of a modern stenter and suggests ways of improving efficiency that can be introduced by the practical finisher. For them to be implemented it is essential that adequate instrumentation is available.     

Recommendations for the presentation of NMR structures of proteins and nucleic acids

The recommendations presented here are designed to support easier communication of NMR data and NMR structures of proteins and nucleic acids through unified nomenclature and reporting standards. Much of this document pertains to the reporting of data in journal articles; however, in the interest of the future development of structural biology, it is desirable that the bulk of the reported information be stored in computer-accessible form and be freely accessible to the scientific community in standardized formats for data exchange. These recommendations stem from an IUPAC-IUBMB-IUPAB inter-union venture with the direct involvement of ICSU and CODATA. The Task Group has reviewed previous formal recommendations and has extended them in the light of more recent developments in the field of biomolecular NMR spectroscopy. Drafts of the recommendations presented here have been examined critically by more than 50 specialists in the field and have gone through two rounds of extensive modification to incorporate suggestions and criticisms.     

Single nucleotide polymorphisms (SNPs) in key cytokines may modulate food allergy phenotypes

Single nucleotide polymorphisms (SNPs) can play a direct or indirect role in phenotypic expression in food allergy pathogenesis. Our goal was to quantitate the expression of SNPs in relevant cytokines that were expressed in food allergic patients. SNPs in cytokine genes IL-4 and IL-10 are known to be important in IgE generation and regulation. We examined IL-4 (C-590T), IL-4Rα (1652A/G) and IL-10 (C-627A) SNPs using real-time PCR followed by restriction fragment length polymorphism (RFLP) analysis. Our results show that the AA, AG and GG genotypes for IL-4Rα (1652A/G) polymorphisms were statistically different in radioallergosorbent test (RAST) positive versus negative patients, and although no statistically significant differences were observed between genotypes in the IL-4 (C-590T) and IL-10 (C-627A) SNPs, we observed a significant decrease in IL-4 (C-590T) gene expression and increase in IL-4Rα (1652A/G) and IL-10 (C-627A) gene expression between RAST⁺ versus RAST^? patients, respectively. We also observed significant modulation in the protein expression of IL-4 and IL-10 in the serum samples of the RAST⁺ patients as compared to the RAST^? patients indicating that changes in SNP expression resulted in altered phenotypic response in these patients.     

Investigations of the effects of gender, diurnal variation, and age in human urinary metabolomic profiles

Metabolomics may have the capacity to revolutionize disease diagnosis through the identification of scores of metabolites that vary during environmental, pathogenic, or toxicological insult. NMR spectroscopy has become one of the main tools for measuring these changes since an NMR spectrum can accurately identify metabolites and their concentrations. The predominant approach in analyzing NMR data has been through the technique of spectral binning. However, identification of spectral areas in an NMR spectrum is insufficient for diagnostic evaluation, since it is unknown whether areas of interest are strictly caused by metabolic changes or are simply artifacts. In this paper, we explore differences in gender, diurnal variation, and age in a human population. We use the example of gender differences to compare traditional spectral binning techniques (NMR spectral areas) to novel targeted profiling techniques (metabolites and their concentrations). We show that targeted profiling produces robust models, generates accurate metabolite concentration data, and provides data that can be used to help understand metabolic differences in a healthy population. Metabolites relating to mitochondrial energy metabolism were found to differentiate gender and age. Dietary components and some metabolites related to circadian rhythms were found to differentiate time of day urine collection. The mechanisms by which these differences arise will be key to the discovery of new diagnostic tests and new understandings of the mechanism of disease.