The effect of a single, large bolus of vitamin D in healthy adults over the winter and following year: a randomized, double-blind, placebo-controlled trial.

BACKGROUND/OBJECTIVES: Although single, high doses of vitamin D effectively maintain vitamin D sufficiency in several populations, no studies have evaluated healthy adults over winter, during which vitamin D status declines. This study investigated whether high-dose vitamin D3 given once to healthy adults before winter will (1) prevent the wintertime decline in vitamin D status, (2) promote vitamin D sufficiency 1 year following the dose and (3) prevent the rise of parathyroid hormone (PTH) concentrations. SUBJECTS/METHODS: In this double-blind, placebo-controlled trial, we assessed plasma 25(OH)D and PTH concentrations at baseline, 5, 90 and 365 days after drug administration in 28 healthy adults. In all, >80% of subjects returned at each time point. RESULTS: At baseline, the young, healthy participants had a mean plasma 25(OH)D concentration of 17.5±6.1 ng/ml. Only two subjects exhibited plasma 25(OH)D concentrations >30 ng/ml. At 5 days, subjects randomized to vitamin D3 had a higher mean plasma 25(OH)D concentration compared with the placebo group (39.1 vs 19.1 ng/ml, P<0.001). Plasma 25(OH)D concentrations returned to baseline at 90 and 365 days in the vitamin D3 group and remained unchanged in the placebo group. PTH and calcium concentrations were unrelated to changes in 25(OH)D levels and similar between groups over time. CONCLUSIONS: A dose of 250,000 IU of vitamin D3 given once in November resulted in a robust increase in plasma 25(OH)D after 5 days, but it was unable to sustain this increase after 90 days. A larger or more frequent dosing regimen may be needed for long-term vitamin D sufficiency.

Alloy powders for medical applications.

The use of gas atomised alloy powders for the fabrication of medical devices offers a number of cost saving benefits and is therefore gaining in popularity. Their use in established and emerging manufacturing processes for products including hip and knee implants is described.

Rotational moulding.

Rotational moulding promises designers attractive economics and a low-pressure process. The benefits of rotational moulding are compared here with other manufacturing methods such as injection and blow moulding.

Spontaneous germline amplification and translocation of a transgene array.

The majority of the mammalian genome is thought to be relatively stable throughout and between generations. There are no developmentally programmed gene amplifications as seen in lower eukaryotes and prokaryotes, however a number of unscheduled gene amplifications have been documented. Apart from expansion of trinucleotide repeats and minisatellite DNA, which involve small DNA elements, other cases of gene or DNA amplifications in mammalian systems have been reported in tumor samples or permanent cell lines. The mechanisms underlying these amplifications remain unknown. Here, we report a spontaneous transgene amplification through the male germline which resulted in silencing of transgene expression. During routine screening one mouse, phenotypically negative for transgene expression, was found to have a transgene copy number much greater than that of the transgenic parent. Analysis of the transgene expansion revealed that the amplification in the new high copy transgenic line resulted in a copy number approximately 40-60 times the primary transgenic line copy number of 5-8 copies per haploid genome. Genetic breeding analysis suggested that this amplification was the result of insertion at only one integration site, that it was stable for at least two generations and that the site of insertion was different from the site at which the original 5-8 copy array had integrated. FISH analysis revealed that the new high copy array was on chromosome 7 F3/4 whereas the original low copy transgene array had been localised to chromosome 3E3. DNA methylation analysis revealed that the high copy transgene array was heavily methylated. The amplification of transgenes, although a rare event, may give insight into amplification of endogenous genes which can be associated with human disease.

Protein kinases C translocation responses to low concentrations of arachidonic acid.

Arachidonic acid (AA) directly activates protein kinases C (PKC) and may thereby serve as a regulatory signal during cell stimulation. The effect, however, requires a > or =20 microm concentration of the fatty acid. We find that human polymorphonuclear neutrophils (PMN) equilibrated with a ligand for the diacylglycerol receptor on PKC, [(3)H]phorbol dibutyrate (PDB), increased binding of [(3)H]PDB within 15 s of exposure to > or =10-30 nm AA. Other unsaturated fatty acids, but not a saturated fatty acid, likewise stimulated PDB binding. These responses, similar to those caused by chemotactic factors, resulted from a rise in the number of diacylglycerol receptors that were plasma membrane-associated and therefore accessible to PDB. Unlike chemotactic factors, however, AA was fully active on cells overloaded with Ca(2+) chelators. The major metabolites of AA made by PMN, leukotriene B(4) and 5-hydroxyicosatetraenoate, did not mimic AA, and an AA antimetabolite did not block responses to AA. AA also induced PMN to translocate cytosolic PKCalpha, beta(II), and delta to membranes. This response paralleled PDB binding with respect to dose requirements, time, Ca(2+)-independence, resistance to an AA antimetabolite, and induction by another unsaturated fatty acid but not by a saturated fatty acid. Finally, HEK 293 cells transfected with vectors encoding PKCbeta(I) or PKCdelta fused to the reporter enhanced green fluorescent protein (EGFP) were studied. AA caused EGFP-PKCbeta translocation from cytosol to plasma membrane at > or =0.5 microm, and EGFP-PKCdelta translocation from cytosol to nuclear and, to a lesser extent, plasma membrane at as little as 30 nm. We conclude that AA induces PKC translocations to specific membrane targets at concentrations 2-4 orders of magnitude below those activating the enzymes. These responses, at least as they occur in PMN, do not require changes in cell Ca(2+) or oxygenation of the fatty acid. AA seems more suited for signaling the movement than activation of PKC.

Complex patterns of inheritance of an imprinted murine transgene suggest incomplete germline erasure.

Here we report a transgenic mouse line that exhibits significant deviations from a classic pattern of parental imprinting. When the transgene is passed through the female germline, it is completely silenced in some offspring while in others expression is reduced. This variable expressivity does not appear to be the result of differences in the presence of unlinked modifiers. Female transmission of the transgene is associated with hypermethylation. The transgene is generally reactivated on passage through the male germline. Extended pedigrees reveal complex patterns of inheritance of the phenotype. The most likely explanation for this result is that the imprint is not completely erased and reset when passed through the germline of either sex. FISH analysis reveals that the transgene has integrated into chromosome 3 band E3, a region not known to carry imprinted genes, and the integration site shows no sign of allele-specific differential methylation. These findings, in conjunction with other recent work, raise the possibility that the introduction of foreign DNA into the mammalian genome, either through retrotransposition or transgenesis, may be associated with parental imprinting that is not always erased and reset during meiosis.

Identification of a novel family of nonclassic yeast phosphatidylinositol transfer proteins whose function modulates phospholipase D activity and Sec14p-independent cell growth.

Yeast phosphatidylinositol transfer protein (Sec14p) is essential for Golgi function and cell viability. We now report a characterization of five yeast SFH (Sec Fourteen Homologue) proteins that share 24-65% primary sequence identity with Sec14p. We show that Sfh1p, which shares 64% primary sequence identity with Sec14p, is nonfunctional as a Sec14p in vivo or in vitro. Yet, SFH proteins sharing low primary sequence similarity with Sec14p (i.e., Sfh2p, Sfh3p, Sfh4p, and Sfh5p) represent novel phosphatidylinositol transfer proteins (PITPs) that exhibit phosphatidylinositol- but not phosphatidylcholine-transfer activity in vitro. Moreover, increased expression of Sfh2p, Sfh4p, or Sfh5p rescues sec14-associated growth and secretory defects in a phospholipase D (PLD)-sensitive manner. Several independent lines of evidence further demonstrate that SFH PITPs are collectively required for efficient activation of PLD in vegetative cells. These include a collective requirement for SFH proteins in Sec14p-independent cell growth and in optimal activation of PLD in Sec14p-deficient cells. Consistent with these findings, Sfh2p colocalizes with PLD in endosomal compartments. The data indicate that SFH gene products cooperate with "bypass-Sec14p" mutations and PLD in a complex interaction through which yeast can adapt to loss of the essential function of Sec14p. These findings expand the physiological repertoire of PITP function in yeast and provide the first in vivo demonstration of a role for specific PITPs in stimulating activation of PLD.

Evaluation of the developmental toxicity of 60 Hz magnetic fields and harmonic frequencies in Sprague-Dawley rats.

Experimental data suggest that exposure to the 50 and 60 Hz sinusoidal components of power-frequency magnetic fields (MFs) does not have an adverse impact on fetal development. However, the possible developmental toxicity of MF harmonics has not been investigated. This study was designed to determine whether exposure to 180 Hz MFs (third harmonic), alone or in combination with 60 Hz MFs, induces birth defects in Sprague-Dawley rats. Groups of sperm-positive dams (> or =20/group) were exposed for 18.5 h per day from gestation days 6 through 19 to (1) ambient MFs only (<0.0001 mT; sham controls); (2) 60 Hz MFs at 0.2 mT; (3) 180 Hz MFs at 0.2 mT; or (4) 60 Hz + 180 Hz MFs (10% third harmonic; total field strength = 0.2 mT). Litter size, litter weight, percentage live births, sex ratio, and number of resorption sites were determined for each dam, and gross external, visceral, cephalic and skeletal examinations were performed on all fetuses. MF exposure had no significant effects on litter size, litter weight, or fetal development. With the exception of common rib variants, the incidence of fetal anomalies was comparable in all groups. A small increase in the incidence of rib variants was seen in the group exposed to 60 Hz + 180 Hz MFs; however, the incidence of rib variants in this group was similar to that in historical controls from our laboratory. These data extend the existing database on developmental toxicity of MFs by demonstrating that exposure to 180 Hz MFs, either alone or superimposed on an underlying 60 Hz signal, does not induce biologically significant developmental toxicity. These data do not support the hypothesis that exposure to power-frequency MFs is an important risk factor for fetal development.

Reactivation of heritably silenced gene expression in mice.

Epigenetic modifications that suppress gene activity in mammals are generally considered to be cleared in the germline, restoring totipotency of the genome. Here we report the germline inheritance of transcriptional silencing in mice, and reversion to activity after as many as three generations in the silent state. In a series of lines made with a LacZ transgene, one line exhibits variable expressivity: genotypically identical littermates have proportions of beta-Gal-positive erythrocytes that vary over at least four orders of magnitude, and in some offspring expression is completely silenced. The silent state of the transgene is inherited for multiple generations in the founder strain irrespective of the sex of the parent, implying maintenance of the epigenetic state through meiosis. Crosses of silenced mice with C57BL/6 mice result in reactivation of the transgene in approximately a third of F(1) littermates. The silencing involves a stochastic, all-or-none mechanism. Furthermore, silencing is transcriptional and correlates with methylation of the transgene as well as an inaccessible chromatin structure; these changes are reversed when expression is reactivated. This work supports the notion that silent genetic information in mammals can be inherited and later reactivated, and implies a mode of phenotypic inheritance that is less stable than Mendelian inheritance.

Algorithmic stability and sanity-check bounds for leave-one-out cross-validation.

In this article we prove sanity-check bounds for the error of the leave-one-out cross-validation estimate of the generalization error: that is, bounds showing that the worst-case error of this estimate is not much worse than that of the training error estimate. The name sanity check refers to the fact that although we often expect the leave-one-out estimate to perform considerably better than the training error estimate, we are here only seeking assurance that its performance will not be considerably worse. Perhaps surprisingly, such assurance has been given only for limited cases in the prior literature on cross-validation. Any nontrivial bound on the error of leave-one-out must rely on some notion of algorithmic stability. Previous bounds relied on the rather strong notion of hypothesis stability, whose application was primarily limited to nearest-neighbor and other local algorithms. Here we introduce the new and weaker notion of error stability and apply it to obtain sanity-check bounds for leave-one-out for other classes of learning algorithms, including training error minimization procedures and Bayesian algorithms. We also provide lower bounds demonstrating the necessity of some form of error stability for proving bounds on the error of the leave-one-out estimate, and the fact that for training error minimization algorithms, in the worst case such bounds must still depend on the Vapnik-Chervonenkis dimension of the hypothesis class.

Novel developmentally regulated phosphoinositide binding proteins from soybean whose expression bypasses the requirement for an essential phosphatidylinositol transfer protein in yeast.

Phosphatidylinositol transfer proteins (PITPs) have been shown to play important roles in regulating a number of signal transduction pathways that couple to vesicle trafficking reactions, phosphoinositide-driven receptor-mediated signaling cascades, and development. While yeast and metazoan PITPs have been analyzed in some detail, plant PITPs remain entirely uncharacterized. We report the identification and characterization of two soybean proteins, Ssh1p and Ssh2p, whose structural genes were recovered on the basis of their abilities to rescue the viability of PITP-deficient Saccharomyces cerevisiae strains. We demonstrate that, while both Ssh1p and Ssh2p share approximately 25% primary sequence identity with yeast PITP, these proteins exhibit biochemical properties that diverge from those of the known PITPs. Ssh1p and Ssh2p represent high-affinity phosphoinositide binding proteins that are distinguished from each other both on the basis of their phospholipid binding specificities and by their substantially non-overlapping patterns of expression in the soybean plant. Finally, we show that Ssh1p is phosphorylated in response to various environmental stress conditions, including hyperosmotic stress. We suggest that Ssh1p may function as one component of a stress response pathway that serves to protect the adult plant from osmotic insult.

The hydrophobic face orientation of apolipoprotein A-I amphipathic helix domain 143-164 regulates lecithin:cholesterol acyltransferase activation.

Apolipoprotein A-I (apoA-I) activates the plasma enzyme lecithin:cholesterol acyltransferase (LCAT), catalyzing the rapid conversion of lipoprotein cholesterol to cholesterol ester. Structural mutants of apoA-I have been used to study the details of apoA-I-LCAT-catalyzed cholesterol ester formation. Several studies have shown that the alpha-helical segments corresponding to amino acids 143-164 and 165-186 (repeats 6 and 7) are essential for LCAT activation. In the present studies, we examined how the orientation of the hydrophobic face, independent of an increase in overall hydrophobicity, affects LCAT activation. We designed, expressed, and characterized a mutant, reverse of 6 apoA-I (RO6 apoA-I), in which the primary amino acid sequence of repeat 6 (amino acids 143-164) was reversed from its normal orientation. This mutation rotates the hydrophobic face of repeat 6 approximately 80 degrees. Lipid-free RO6 apoA-I showed a marked stabilization when denatured by guanidine hydrochloride, but showed significant destabilization to guanidine hydrochloride denaturation in the lipid-bound state compared with wild-type apoA-I. Recombinant high density lipoprotein discs (rHDL) formed from RO6 apoA-I, sn-1-palmitoyl-sn-2-oleoyl phosphati-dylcholine, and cholesterol were approximately 12 A smaller than wild-type apoA-I rHDL. The reduced size suggests that one of the repeats did not effectively participate in phospholipid binding and organization. The sn-1-palmitoyl-sn-2-oleoyl phosphatidylcholine RO6 rHDL were a less effective substrate for LCAT. Mapping the entire lipid-free and lipid-bound RO6 apoA-I with a series of monoclonal antibodies revealed that both the lipid-free and lipid-bound RO6 apoA-I displayed altered or absent epitopes in domains within and adjacent to repeat 6. Together, these results suggest that the proper alignment and orientation of the hydrophobic face of repeat 6 is an important determinant for maintaining and stabilizing helix-bilayer and helix-helix interactions.

Evaluation of a 20 minute 14C urea breath test for the diagnosis of Helicobacter pylori infection.

The use of 14C-urea breath testing for diagnosis of Helicobacter pylori infection in gastric mucosa has gained widespread acceptance and utilisation. We evaluated a 14C urea breath test (UBT) in 116 patients undergoing endoscopy. Seventy four patients were administered 185 kBq (5 mCi-conventional dose), and 42 patients reduced dose (92.5 IBq, 2.5 mCi) of 14C-urea. All were tested for H. pylori using culture, direct microscopy of gastric biopsies and histological evaluation of paraffin stained sections. Using the mean + three standard deviations as the cut-off value, a sensitivity of 96% and specificity of 100% was found for the conventional dose test. At reduced dose, sensitivity was 100% and specificity 96%. Positive and negative predictive values were 100% and 93% for the conventional dose test, and 96% and 100% for testing at reduced dose. We conclude that the UBT is a simple, non-invasive and useful diagnostic alternative for detection of H. pylori in infected patients. We advocate its use in patients less than 45 years of age without alarm symptoms, and also in cases where the need for endoscopic evaluation is not vital, such as after eradication therapy.

Alteration in apolipoprotein A-I 22-mer repeat order results in a decrease in lecithin:cholesterol acyltransferase reactivity.

Apolipoprotein A-I contains eight 22-amino acid and two 11-amino acid tandem repeats that comprise 80% of the mature protein. These repeating units are believed to be the basic motif responsible for lipid binding and lecithin:cholesterol acyltransferase (LCAT) activation. Computer analysis indicates that despite a fairly high degree of compositional similarity among the tandem repeats, significant differences in hydrophobic and amphipathic character exist. Our previous studies demonstrated that deletion of repeat 6 (143-164) or repeat 7 (165-186) resulted in a 98-99% reduction of LCAT activation as compared with wild-type apoA-I. To determine the effects of substituting one of these repeats with a more hydrophobic repeat we constructed a mutant apoA-I protein in which residues 143-164 (repeat 6) were replaced with repeat 10 (residues 220-241). The cloned mutant protein, 10F6 apoA-I, was expressed and purified from an Sf-9 cell baculoviral system and then analyzed using a number of biophysical and biochemical techniques. Recombinant complexes prepared at a 100:5:1 molar ratio of L-alpha-dimyristoylphosphatidylcholine:cholesterol:wild-type or 10F6 apoA-I showed a doublet corresponding to Stokes diameters of 114 and 108 A on nondenaturing 4-30% polyacrylamide gel electrophoresis. L-alpha-Dimyristoylphosphatidylcholine 10F6 apoA-I complexes had a 5-6-fold lower apparent Vmax/apparent Km as compared with wild-type apoA-I containing particles. As expected, monoclonal antibody epitope mapping of the lipid-free and lipid-bound 10F6 apoA-I confirmed that a domain expressed between residues 143 and 165 normally found in wild-type apoA-I was absent. The region between residues 119 and 144 in 10F6 apoA-I showed a marked reduction in monoclonal antibody binding capacity. Therefore, we speculate that the 5-6-fold lower LCAT reactivity in 10F6 compared with wild-type apoA-I recombinant particles results from increased stabilization within the 121-165 amino acid domain due to more stable apoprotein helix phospholipid interactions as well as from conformational alterations among adjacent amphipathic helix repeats.