Proceedings of the Rank Forum on Vitamin D.

The Rank Forum on Vitamin D was held on 2nd and 3rd July 2009 at the University of Surrey, Guildford, UK. The workshop consisted of a series of scene-setting presentations to address the current issues and challenges concerning vitamin D and health, and included an open discussion focusing on the identification of the concentrations of serum 25-hydroxyvitamin D (25(OH)D) (a marker of vitamin D status) that may be regarded as optimal, and the implications this process may have in the setting of future dietary reference values for vitamin D in the UK. The Forum was in agreement with the fact that it is desirable for all of the population to have a serum 25(OH)D concentration above 25 nmol/l, but it discussed some uncertainty about the strength of evidence for the need to aim for substantially higher concentrations (25(OH)D concentrations>75 nmol/l). Any discussion of 'optimal' concentration of serum 25(OH)D needs to define 'optimal' with care since it is important to consider the normal distribution of requirements and the vitamin D needs for a wide range of outcomes. Current UK reference values concentrate on the requirements of particular subgroups of the population; this differs from the approaches used in other European countries where a wider range of age groups tend to be covered. With the re-emergence of rickets and the public health burden of low vitamin D status being already apparent, there is a need for urgent action from policy makers and risk managers. The Forum highlighted concerns regarding the failure of implementation of existing strategies in the UK for achieving current vitamin D recommendations.

Impact of baseline vitamin B12 status on the effect of vitamin B12 supplementation on neurologic function in older people: secondary analysis of data from the OPEN randomised controlled trial.

BACKGROUND/OBJECTIVES: The available evidence from randomised controlled trials suggests that vitamin B12 supplementation does not improve neurologic function in older people with marginal but not deficient Vitamin B12 status. This secondary analysis used data from the Older People and Enhanced Neurological function (OPEN) randomised controlled trial to assess whether baseline vitamin B12 status or change in vitamin B12 status over 12 months altered the effectiveness of dietary vitamin B12 supplementation on neurologic function in asymptomatic older people with depleted vitamin B12 status at study entry. SUBJECTS/METHODS: Vitamin B12 status was measured as serum concentrations of vitamin B12, holotranscobalamin, homocysteine and via a composite indicator (cB12). Neurological function outcomes included eleven electrophysiological measures of sensory and motor components of peripheral and central nerve function. Linear regression analyses were restricted to participants randomised into the intervention arm of the OPEN trial (n=91). RESULTS: Analyses revealed an inconsistent pattern of moderate associations between some measures of baseline vitamin B12 status and some neurological responses to supplementation. The directions of effect varied and heterogeneity in effect across outcomes could not be explained according to type of neurological outcome. There was no evidence of differences in the neurological response to vitamin B12 supplementation according to change from baseline over 12 months in any indicator of B12 status. CONCLUSIONS: This secondary analysis of high-quality data from the OPEN trial provides no evidence that baseline (or change from baseline) vitamin B12 status modifies the effect of vitamin B12 supplementation on peripheral or central nerve conduction among older people with marginal vitamin B12 status. There is currently insufficient evidence of efficacy for neurological function to support population-wide recommendations for vitamin B12 supplementation in healthy asymptomatic older people with marginal vitamin B12 status.

The yeast cyclophilin multigene family: purification, cloning and characterization of a new isoform.

Cyclophilins (Cyps) constitute a highly conserved family of proteins present in a wide variety of organisms. Historically, Cyps were first identified by their ability to bind the immunosuppressive agent cyclosporin A (CsA) with high affinity; they later were found to have peptidyl-prolyl cis-trans isomerase (PPIase) activity, which catalyzes the folding of oligopeptides at proline-peptide bonds in vitro and may be important for protein folding in vivo. Cells of Saccharomyces cerevisiae contain at least two distinct Cyp-related PPIases encoded by the genes CYP1 and CYP2. A yeast strain (GL81) containing genomic disruptions of three known yeast PPIase-encoding genes [CYP1, CYP2 and RBP1 (for rapamycin-binding protein); Koltin et al., Mol. Cell. Biol. 11 (1991) 1718-1723] was previously constructed and found to be viable. Soluble fractions of these cells possess residual CsA-sensitive PPIase activity (2-5% of that present in wild-type cells as assayed in vitro). We have purified an approx. 18-kDa protein exhibiting PPIase activity from a soluble fraction of GL81 cells and determined that its N-terminal amino acid (aa) sequence exhibits significant homology (but nonidentity) to the Cyp1 and Cyp2 proteins. We designate the gene for this new protein, CYP3. Using a degenerate oligodeoxyribonucleotide (oligo) based on the N-terminal aa sequence, plus an internal oligo homologous to a conserved region within the portion of CYP1 and CYP2 that had been deleted in the genome, a CYP3-specific DNA fragment was generated by the polymerase chain reaction (PCR) using GL81 genomic DNA as a substrate. This PCR fragment was used as a probe to isolate CYP3 genomic and cDNA clones.(ABSTRACT TRUNCATED AT 250 WORDS)

Recombinant HIV-1 reverse transcriptase: purification, primary structure, and polymerase/ribonuclease H activities.

Recombinant HIV-1 reverse transcriptase (RT) was stably overproduced as a soluble protein in Escherichia coli using a double-plasmid expression system in which an RT precursor protein was expressed and processed in vivo by HIV-1 protease produced in trans. The RT thus produced consisted of an equimolar mixture of two polypeptides, p66 and p51, which were copurified to greater than 90% homogeneity and were found to share a common NH2 terminus as judged by sequence analysis of the polypeptide mixture. The observed sequence confirmed correct in vivo cleavage by protease at the protease-RT polyprotein junction to yield an NH2 terminus identical to that of genuine viral RT (M. M. Lightfoote et al. (1986) J. Virol. 60, 771-775; F. diMarzo Veronese et al. (1986) Science 231, 1289-1291). The bacterially expressed RT had a specific activity similar to that of viral RT and inhibition studies with phosphonoformate confirmed that it was indistinguishable from the viral enzyme with respect to sensitivity to this inhibitor. Polymerase activated gel analysis of the mixture indicated that p66 was associated with a higher level of RT activity than p51. RNase H activated gel analysis suggested that the purified preparation of recombinant RT was free of endogenous E. coli RNase H, and that the RNase H activity of RT was exclusively associated with the p66 polypeptide, supporting the hypothesis that the RNase H domain is located in the COOH-terminal region of the molecule.

Secretion of interleukin-1 beta and Escherichia coli galactokinase by Streptomyces lividans.

The functionality of the Streptomyces lividans beta-galactosidase signal peptide to direct heterologous protein export was examined. The signal peptide plus eight amino acids of mature protein were sufficient to export not only a naturally exported protein, interleukin-1 beta, but also a naturally occurring cytoplasmic protein, Escherichia coli galactokinase. Interestingly, cells which expressed yet exported galactokinase were phenotypically Gal-. The potential use of the exported galactokinase system for the isolation and characterization of mutations within signal peptides and the export machinery of the host is discussed.

Secretion of N-glycosylated interleukin-1 beta in Saccharomyces cerevisiae using a leader peptide from Candida albicans. Effect of N-linked glycosylation on biological activity.

Human interleukin-1 beta (IL-1 beta) is expressed in activated monocytes as a 31-kDa precursor protein which is processed and secreted as a mature, unglycosylated 17-kDa carboxyl-terminal fragment, despite the fact that it contains a potential N-linked glycosylation site near the NH2 terminus (-Asn7-Cys8-Thr9-). cDNA coding for authentic mature IL-1 beta was fused to the signal sequence from the Candida albicans glucoamylase gene, two amino acids downstream from the signal processing site. Upon expression in Saccharomyces cerevisiae, approximately equimolar amounts of N-glycosylated (22 kDa) and unglycosylated (17 kDa) IL-1 beta protein were secreted. The N-glycosylated yeast recombinant IL-1 beta exhibited a 5-7-fold lower specific activity compared to the unglycosylated species. The mechanism responsible for inefficient glycosylation was also studied. We found no differences in secretion kinetics or processing between the two extracellular forms of IL-1 beta. The 17-kDa protein, which was found to lack core sugars, does not result from deglycosylation of the 22-kDa protein in vivo and does not result from saturation of the glycosylation enzymatic machinery through overexpression. Alteration of the uncommon Cys8 residue in the -Asn-X-Ser/Thr-glycosylation site to Ser also had no effect. However, increasing the distance between Asn7 and the signal processing site increased the extent of core N-linked glycosylation, suggesting a reduction in glycosylation efficiency near the NH2 terminus.

Purification and characterization of human recombinant interleukin-1 beta.

A human interleukin-1 (IL-1) beta cDNA was cloned, and the region coding for the mature protein was expressed in Escherichia coli. The 17-kDa biologically active product was purified in 40% yield to apparent homogeneity, without chaotropes, from the soluble fraction of sonicated cell lysates. The recombinant IL-1 beta was characterized by amino acid analysis, NH2- and COOH-terminal sequence analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, spectroscopy, and biological assay. Specific biological activity was 4.6 X 10(8) units/mg in a co-mitogenic IL-2 induction assay using cultured EL-4 T-lymphocytes. The molar extinction coefficient was determined to be 10,300 cm-1 M-1 at 280 nm. NH2-terminal sequence analysis revealed that 70% of the product begins with the Ala corresponding to the NH2 terminus of the natural protein, while 30% begins with the following Pro. No initiator Met was observed. Both of the sulfhydryl groups are reactive to Ellman's reagent and to iodoacetamide under nonreducing conditions, indicating that the Cys residues do not form disulfide bonds. S-Carboxamidomethyl-Cys-rIL-1 beta retained biological activity in the IL-2 induction assay. Circular dichroism suggested an extensive beta sheet structure for rIL-1 beta.