The x-ray crystallographic structure of the heterodimeric plant toxin ricin has been determined at 2.8-A resolution. The A chain enzyme is a globular protein with extensive secondary structure and a reasonably prominent cleft assumed to be the active site. The B chain lectin folds into two topologically similar domains, each binding lactose in a shallow cleft. In each site a glutamine residue forms a hydrogen bond to the OH-4 of galactose, accounting for the epimerimic specificity of binding. The interface between the A and B chains shows some hydrophobic contacts in which proline and phenylalanine side chains play a prominent role.

It has been shown that proteasome activity is required for cancer cell survival and consumption of fruits and vegetables is associated with decreased cancer risk. Previously, we reported that grape extract could inhibit proteasome activity and induce apoptosis in tumor cells. In this study, we examined the flavonoids apigenin, quercetin, kaempferol and myricetin for their proteasome-inhibitory and apoptosis-inducing abilities in human tumor cells. We report that apigenin and quercetin are much more potent than kaempferol and myricetin at: (i) inhibiting chymotrypsin-like activity of purified 20S proteasome and of 26S proteasome in intact leukemia Jurkat T cells; (ii) accumulating putative ubiquitinated forms of two proteasome target proteins, Bax and Inhibitor of nuclear factor kappabeta-alpha in Jurkat T cells and (iii) inducing activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase in Jurkat T cells. The proteasome-inhibitory abilities of these compounds correlated with their apoptosis-inducing potencies. Results from computational modeling of the potential interactions of these flavonoids to the chymotrypsin site (beta5 subunit) of the proteasome were consistent with the obtained proteasome-inhibitory activities. We found that the C(4) carbon may be a site of nucleophilic attack by the OH group of N-terminal threonine of proteasomal beta5 subunit and that the C(3) hydroxyl may alter the ability of these flavonoids to inhibit the proteasome. Finally, apigenin neither effectively inhibited the proteasome activity nor induced apoptosis in non-transformed human natural killer cells. Our results suggested that the proteasome may be a target of these dietary flavonoids in human tumor cells and that inhibition of the proteasome by flavonoids may be one of the mechanisms responsible for their cancer-preventive effects.

This survey summarizes the findings, accumulated within the last 2 years, concerning melatonin's role in defending against toxic free radicals. Free radicals are chemical constituents that have an unpaired electron in their outer orbital and, because of this feature, are highly reactive. Inspired oxygen, which sustains life, also is harmful because up to 5% of the oxygen (O2) taken in is converted to oxygen-free radicals. The addition of a single electron to O2 produces the superoxide anion radical (O2-.); O2-. is catalytic-reduced by superoxide dismutase, to hydrogen peroxide (H2O2). Although H2O2 is not itself a free radical, it can be toxic at high concentrations and, more importantly, it can be reduced to the hydroxyl radical (.OH). The .OH is the most toxic of the oxygen-based radicals and it wreaks havoc within cells, particularly with macromolecules. In recent in vitro studies, melatonin was shown to be a very efficient neutralizer of the .OH; indeed, in the system used to test its free radical scavenging ability it was found to be significantly more effective than the well known antioxidant, glutathione (GSH), in doing so. Likewise, melatonin has been shown to stimulate glutathione peroxidase (GSH-Px) activity in neural tissue; GSH-PX metabolizes reduced glutathione to its oxidized form and in doing so it converts H2O2 to H2O, thereby reducing generation of the .OH by eliminating its precursor. More recent studies have shown that melatonin is also a more efficient scavenger of the peroxyl radical than is vitamin E. The peroxyl radical is generated during lipid peroxidation and propagates the chain reaction that leads to massive lipid destruction in cell membranes. In vivo studies have demonstrated that melatonin is remarkably potent in protecting against free radical damage induced by a variety of means. Thus, DNA damage resulting from either the exposure of animals to the chemical carcinogen safrole or to ionizing radiation is markedly reduced when melatonin is co-administered. Likewise, the induction of cataracts, generally accepted as being a consequence of free radical attack on lenticular macromolecules, in newborn rats injected with a GSH-depleting drug are prevented when the animals are given daily melatonin injections. Also, paraquat-induced lipid peroxidation in the lungs of rats is overcome when they also receive melatonin during the exposure period. Paraquat is a highly toxic herbicide that inflicts at least part of its damage by generating free radicals.(ABSTRACT TRUNCATED AT 400 WORDS)

The increasing number of roles discovered for Al3+ in physiological processes demands an understanding of how Al3+ interacts with compounds in biological systems. Al3+ is expected to complex with oxygen donor ligands, especially phosphates, and it does so in soils, in the gastrointestinal tract, and in cells. The stability of Al3+ complexes has generally been misjudged because of lack of recognition that free, aqueous Al3+ is not the dominant form in neutral solutions and that the solubility of Al(OH)3 limits the free Al3+ at the plasma pH 7.4 to less than 10(-11) mol/L. In the presence of inorganic phosphate, the permitted free Al3+ is decreased further, through formation of insoluble aluminum phosphate. This precipitate facilitates the elimination of Al3+ from the body. In contrast, citrate solubilizes Al3+, and an appreciable fraction occurs as a neutral complex that may pass through membranes and provide a vehicle for Al3+ absorption into the body. In the blood plasma the most likely small-molecule complex is that with citrate, while the only competitive protein complex is that with transferrin, a protein built to transport Fe3+ but whose sites are only 30% occupied.

The purpose of the study was to determine the effect of vitamin D supplementation on bone turnover and bone loss in elderly women. Three hundred forty-eight women, ages 70 yr and older, were randomized to receive 400 IU vitamin D3 per day (n = 177) or placebo (n = 171), double-blind, for a period of 2 yr. Main outcome measures were bone mineral density of both hips (femoral neck and trochanter) and the distal radius, as well as biochemical markers of bone turnover. The effect of vitamin D supplementation was expressed as the difference in mean (percentage) change between the placebo group and the vitamin D group. The measurements were repeated in 283 women after 1 yr and in 248 women after 2 yr. Vitamin D supplementation significantly increased serum 25-hydroxyvitamin D (250HD) (+35 nmol/L) and 1,25-dehydroxyvitamin D [1,25-(OH)2D] (+7.0 pmol/L) levels and urinary calcium/creatinine ratios (+0.5%) and significantly decreased PTH(1-84) secretion (-0.74 pmol/L) after 1 yr. No effect was found for the parameters of bone turnover. The effect on the bone mineral density of the left femoral neck was +1.8% in the first yr, +0.2% in the second yr, and +1.9% during the whole period (95% confidence interval 0.4, 3.4%). At the right femoral neck the effects were +1.5%, +1.1%, and +2.6% (confidence interval 1.1, 4.0%), respectively. No effect was found at the femoral trochanter and the distal radius. Supplementation with 400 IU vitamin D3 daily in elderly women slightly decreases PTH secretion and increases bone mineral density at the femoral neck.

A global understanding of the actions of the nuclear hormone 1α,25-dihydroxyvitamin D(3) (1α,25(OH)(2)D(3)) and its vitamin D receptor (VDR) requires a genome-wide analysis of VDR binding sites. In THP-1 human monocytic leukemia cells we identified by ChIP-seq 2340 VDR binding locations, of which 1171 and 520 occurred uniquely with and without 1α,25(OH)(2)D(3) treatment, respectively, while 649 were common. De novo identified direct repeat spaced by 3 nucleotides (DR3)-type response elements (REs) were strongly associated with the ligand-responsiveness of VDR occupation. Only 20% of the VDR peaks diminishing most after ligand treatment have a DR3-type RE, in contrast to 90% for the most growing peaks. Ligand treatment revealed 638 1α,25(OH)(2)D(3) target genes enriched in gene ontology categories associated with immunity and signaling. From the 408 upregulated genes, 72% showed VDR binding within 400 kb of their transcription start sites (TSSs), while this applied only for 43% of the 230 downregulated genes. The VDR loci showed considerable variation in gene regulatory scenarios ranging from a single VDR location near the target gene TSS to very complex clusters of multiple VDR locations and target genes. In conclusion, ligand binding shifts the locations of VDR occupation to DR3-type REs that surround its target genes and occur in a large variety of regulatory constellations.

Serum calcium levels are tightly controlled by an integrated hormone-controlled system that involves active vitamin D [1,25(OH)(2)D], which can elicit calcium mobilization from bone when intestinal calcium absorption is decreased. The skeletal adaptations, however, are still poorly characterized. To gain insight into these issues, we analyzed the consequences of specific vitamin D receptor (Vdr) inactivation in the intestine and in mature osteoblasts on calcium and bone homeostasis. We report here that decreased intestinal calcium absorption in intestine-specific Vdr knockout mice resulted in severely reduced skeletal calcium levels so as to ensure normal levels of calcium in the serum. Furthermore, increased 1,25(OH)(2)D levels not only stimulated bone turnover, leading to osteopenia, but also suppressed bone matrix mineralization. This resulted in extensive hyperosteoidosis, also surrounding the osteocytes, and hypomineralization of the entire bone cortex, which may have contributed to the increase in bone fractures. Mechanistically, osteoblastic VDR signaling suppressed calcium incorporation in bone by directly stimulating the transcription of genes encoding mineralization inhibitors. Ablation of skeletal Vdr signaling precluded this calcium transfer from bone to serum, leading to better preservation of bone mass and mineralization. These findings indicate that in mice, maintaining normocalcemia has priority over skeletal integrity, and that to minimize skeletal calcium storage, 1,25(OH)(2)D not only increases calcium release from bone, but also inhibits calcium incorporation in bone.

Reactive oxygen species (ROS: superoxide radical, O2.-; hydrogen peroxide, H2O2; hydroxyl radical, OH.), which arise from the univalent reduction of dioxygen are formed in mitochondria. We summarize here results which indicate that ROS, and also the radical nitrogen monoxide ('nitric oxide', NO), act as physiological modulators of some mitochondrial functions, but may also damage mitochondria. Hydrogen peroxide, which originates in mitochondria predominantly from the dismutation of superoxide, causes oxidation of mitochondrial pyridine nucleotides and thereby stimulates a specific Ca2+ release from intact mitochondria. This release is prevented by cyclosporin A (CSA). Hydrogen peroxide thus contributes to the maintenance of cellular Ca2+ homeostasis. A stimulation of mitochondrial ROS production followed by an enhanced Ca2+ release and re uptake (Ca2+ 'cycling') by mitochondria causes apoptosis and necrosis, and contributes to hypoxia/reperfusion injury. These kinds of cell injury can be attenuated at the mitochondrial level by CSA. When ROS are produced in excessive amounts in mitochondria nucleic acids, proteins, and lipids are extensively modified by oxidation. Physiological (sub-micromolar) concentrations of NO potently and reversibly deenergize mitochondria at oxygen tensions that prevail in cells by transiently binding to cytochrome oxidase. This is paralleled by mitochondrial Ca2+ release and uptake. Higher NO concentrations or prolonged exposure of cells to NO causes their death. It is concluded that ROS and NO are important physiological reactants in mitochondria and become toxic only when present in excessive amounts.

Flavonol glycosides constitute one of the most prominent plant natural product classes that accumulate in the model plant Arabidopsis thaliana. To date there are no reports of functionally characterized flavonoid glycosyltransferases in Arabidopsis, despite intensive research efforts aimed at both flavonoids and Arabidopsis. In this study, flavonol glycosyltransferases were considered in a functional genomics approach aimed at revealing genes involved in determining the flavonol-glycoside profile. Candidate glycosyltransferase-encoding genes were selected based on homology to other known flavonoid glycosyltransferases and two T-DNA knockout lines lacking flavonol-3-O-rhamnoside-7-O-rhamnosides (ugt78D1) and quercetin-3-O-rhamnoside-7-O-glucoside (ugt73C6 and ugt78D1) were identified. To confirm the in planta results, cDNAs encoding both UGT78D1 and UGT73C6 were expressed in vitro and analyzed for their qualitative substrate specificity. UGT78D1 catalyzed the transfer of rhamnose from UDP-rhamnose to the 3-OH position of quercetin and kaempferol, whereas UGT73C6 catalyzed the transfer of glucose from UDP-glucose to the 7-OH position of kaempferol-3-O-rhamnoside and quercetin-3-O-rhamnoside, respectively. The present results suggest that UGT78D1 and UGT73C6 should be classified as UDP-rhamnose:flavonol-3-Orhamnosyltransferase and UDP-glucose:flavonol-3-O-glycoside-7-O-glucosyltransferase, respectively.

Silent information regulator 2 (Sir2) enzymes catalyze NAD+-dependent protein/histone deacetylation, where the acetyl group from the lysine epsilon-amino group is transferred to the ADP-ribose moiety of NAD+, producing nicotinamide and the novel metabolite O-acetyl-ADP-ribose. Sir2 proteins have been shown to regulate gene silencing, metabolic enzymes, and life span. Recently, nicotinamide has been implicated as a direct negative regulator of cellular Sir2 function; however, the mechanism of nicotinamide inhibition was not established. Sir2 enzymes are multifunctional in that the deacetylase reaction involves the cleavage of the nicotinamide-ribosyl, cleavage of an amide bond, and transfer of the acetyl group ultimately to the 2'-ribose hydroxyl of ADP-ribose. Here we demonstrate that nicotinamide inhibition is the result of nicotinamide intercepting an ADP-ribosyl-enzyme-acetyl peptide intermediate with regeneration of NAD+ (transglycosidation). The cellular implications are discussed. A variety of 3-substituted pyridines was found to be substrates for enzyme-catalyzed transglycosidation. A Brönsted plot of the data yielded a slope of +0.98, consistent with the development of a nearly full positive charge in the transition state, and with basicity of the attacking nucleophile as a strong predictor of reactivity. NAD+ analogues including beta-2'-deoxy-2'-fluororibo-NAD+ and a His-to-Ala mutant were used to probe the mechanism of nicotinamide-ribosyl cleavage and acetyl group transfer. We demonstrate that nicotinamide-ribosyl cleavage is distinct from acetyl group transfer to the 2'-OH ribose. The observed enzyme-catalyzed formation of a labile 1'-acetylated-ADP-fluororibose intermediate using beta-2'-deoxy-2'-fluororibo-NAD+ supports a mechanism where, after nicotinamide-ribosyl cleavage, the carbonyl oxygen of acetylated substrate attacks the C-1' ribose to form an initial iminium adduct.

A range of applications of the alkaline comet assay is covered, from investigations of the physicochemical behaviour of DNA, through studies of cellular responses to DNA damage, to biomonitoring of human populations. The underlying principles of this assay are discussed, and new evidence presented which supports the concept of relaxation of supercoiled loops, rather than alkaline unwinding, as the primary reason for comet tail formation. DNA-damaging agents that do not induce strand breaks directly can be detected when cellular repair processes convert lesions to transient strand breaks; an approach is outlined here which maximises this effect and thus widens the scope of the assay. Purified repair enzymes, applied to DNA during the course of the comet assay procedure, greatly increase the sensitivity and specificity of the assay; recent developments with formamidopyrimidine glycosylase (recognising 8-OH-gua and other damaged purines) and uvrABC (for bulky lesions) are presented. The kinetics of cellular repair after low doses of oxidative damage have been followed with this modified comet assay. Finally, the successful measurement of biomarkers of oxidative damage in human populations establishes the comet assay as a valuable tool in molecular epidemiology.

Considering that the vitamin D receptor as well as the 1-α-hydroxylase enzyme that converts 25-hydroxyvitamin D (25(OH)D) to its active form 1,25-dihydroxyvitamin D have been found in tissues throughout the body, it is likely that vitamin D is important for more than the calcium balance. Accordingly, low serum levels of 25(OH)D have been associated with mortality, cardiovascular disease, type 2 diabetes, hypertension and obesity. Low serum levels of 25(OH)D have also been associated with an unfavourable lipid profile, which could possible explain the relation with cardiovascular disease and mortality. However, the relation between vitamin D and lipids have so far received little attention and is therefore the main focus of the present review. A PubMed search identified 22 cross-sectional studies where serum levels of 25(OH)D and lipids were related and that included a minimum of 500 subjects, and 10 placebo-controlled double-blind intervention studies with vitamin D where more than 50 subjects were included. In all the cross-sectional studies serum 25(OH)D was positively associated with high-density lipoprotein cholesterol (HDL-C) resulting in a favourable low-density lipoprotein cholesterol (LDL-C) (or total cholesterol) to HDL-C ratio. There was also a uniform agreement between studies on a negative relation between serum 25(OH)D and triglycerides (TG). On the other hand, the intervention studies gave divergent results, with some showing a positive and some a negative effect of vitamin D supplementation. However, none of the intervention studies were specifically designed for evaluating the relation between vitamin D and lipids, none had hyperlipemia as an inclusion criterion, and none were sufficiently powered. In only one study was a significant effect seen with an 8% (0.28 mmol/L) increase in serum LDL-C and a 16% (0.22 mmol/L) decrease in serum TG in those given vitamin D as compared to the placebo group. Accordingly, the effect of vitamin D supplementation on serum lipids is at present uncertain. Considering the numerous other promising vitamins and minerals that when properly tested have been disappointing, one should wait for the results of forthcoming vitamin D intervention studies before drawing conclusions on potential beneficial effects of vitamin D.

1. Intracellular pH was recorded fluorimetrically by using carboxy-SNARF-1, AM-loaded into superfused ventricular myocytes isolated from guinea-pig heart. Intracellular acid and base loads were induced experimentally and the changes of pHi used to estimate intracellular buffering power (beta). The rate of pHi recovery from acid or base loads was used, in conjunction with the measurements of beta, to estimate sarcolemmal transporter fluxes of acid equivalents. A combination of ion substitution and pharmacological inhibitors was used to dissect acid effluxes carried on Na+-H+ exchange (NHE) and Na+-HCO3- cotransport (NBC), and acid influxes carried on Cl--HCO3- exchange (AE) and Cl--OH- exchange (CHE). 2. The intracellular intrinsic buffering power (betai), estimated under CO2/HCO3--free conditions, varied inversely with pHi in a manner consistent with two principal intracellular buffers of differing concentration and pK. In CO2/HCO3--buffered conditions, intracellular buffering was roughly doubled. The size of the CO2-dependent component (betaCO2) was consistent with buffering in a cell fully open to CO2. Because the full value of betaCO2 develops slowly (2.5 min), it had to be measured under equilibrium conditions. The value of betaCO2 increased monotonically with pHi. 3. In 5 % CO2/HCO3--buffered conditions (pHo 7.40), acid extrusion on NHE and NBC increased as pHi was reduced, with the greater increase occurring through NHE at pHi < 6.90. Acid influx on AE and CHE increased as pHi was raised, with the greater increase occurring through AE at pHi>> 7.15. At resting pHi (7.04-7.07), all four carriers were activated equally, albeit at a low rate (about 0.15 mM min-1). 4. The pHi dependence of flux through the transporters, in combination with the pHi and time dependence of intracellular buffering (betai + betaCO2), was used to predict mathematically the recovery of pHi following an intracellular acid or base load. Under several conditions the mathematical predictions compared well with experimental recordings, suggesting that the model of dual acid influx and acid efflux transporters is sufficient to account for pHi regulation in the cardiac cell. Key properties of the pHi control system are discussed.

Biophysical evidence has placed the binding site for the naturally occurring marine toxins tetrodotoxin (TTX) and saxitoxin (STX) in the external mouth of the Na+ channel ion permeation pathway. We developed a molecular model of the binding pocket for TTX and STX, composed of antiparallel beta-hairpins formed from peptide segments of the four S5-S6 loops of the voltage-gated Na+ channel. For TTX the guanidinium moiety formed salt bridges with three carboxyls, while two toxin hydroxyls (C9-OH and C10-OH) interacted with a fourth carboxyl on repeats I and II. This alignment also resulted in a hydrophobic interaction with an aromatic ring of phenylalanine or tyrosine residues for the brainII and skeletal Na+ channel isoforms, but not with the cysteine found in the cardiac isoform. In comparison to TTX, there was an additional interaction site for STX through its second guanidinium group with a carboxyl on repeat IV. This model satisfactorily reproduced the effects of mutations in the S5-S6 regions and the differences in affinity by various toxin analogs. However, this model differed in important ways from previously published models for the outer vestibule and the selectivity region of the Na+ channel pore. Removal of the toxins from the pocket formed by the four beta-hairpins revealed a structure resembling a funnel that terminated in a narrowed region suitable as a candidate for the selectivity filter of the channel. This region contained two carboxyls (Asp384 and Glu942) that substituted for molecules of water from the hydrated Na+ ion. Simulation of mutations in this region that have produced Ca2+ permeation of the Na+ channel created a site with three carboxyls (Asp384, Glu942, and Glu1714) in proximity.

A total of 342 previously untreated eligible children were entered into the first Intergroup Ewing's Sarcoma Study (IESS) between May 1973 and November 1978. In group I institutions, patients were randomized between treatment 1 (radiotherapy to primary lesion plus cyclophosphamide, vincristine, dactinomycin, and Adriamycin [doxorubicin; Adria Laboratories, Columbus, OH] [VAC plus ADR]) or treatment 2 (same as treatment 1 without ADR), and group II institutions randomized patients between treatment 2 or treatment 3 (same as treatment 2 plus bilateral pulmonary radiotherapy [VAC plus BPR]). The percentages of patients relapse-free and surviving (RFS) at 5 years for treatments 1, 2, and 3 were 60%, 24%, and 44%, respectively. There was strong statistical evidence of a significant advantage in RFS for treatment 1 (VAC plus ADR) versus 2 (VAC alone) (P less than .001) and 3 (P less than .05) and also of treatment 3 versus 2 (P less than .001). Similar significant results were observed with respect to overall survival. Patients with disease at pelvic sites have significantly poorer survival at 5 years than those with disease at nonpelvic sites (34% v 57%; P less than .001). Among pelvic cases, there was no evidence of differing survival by treatment (P = .81), but among nonpelvic cases, there was strong evidence of differing survival by treatment (P less than .001). The overall percentage of patients developing metastatic disease was 44%; the percentages by treatments 1, 2, and 3 were 30%, 72%, and 42%, respectively. The overall incidence of local recurrence was 15%, and there was no evidence that local recurrence rate differed by treatment. Patient characteristics related to prognosis, both with respect to RFS and overall survival experience, were primary site (nonpelvic patients were most favorable) and patient age (younger patients were more favorable).

Molecular epidemiologic studies of vitamin D and risk of cancer and other health outcomes usually involve a single measurement of the biomarker 25-hydroxyvitamin D [25(OH)D] in serum or plasma. However, the extent to which 25(OH)D concentration at a single time point is representative of an individual's long-term vitamin D status is unclear. To address this question, we evaluated within-person variability in 25(OH)D concentrations across serum samples collected at three time points over a 5-year period among 29 participants in the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial. Blood collection took place year-round, although samples for a given participant were collected in the same month each year. The within-person coefficient of variation and intraclass correlation coefficient were calculated using variance components estimated from random effects models. Spearman rank correlation coefficients were calculated to evaluate agreement between measurements at different collection times (baseline, +1 year, +5 years). The within-subject coefficient of variation was 14.9% [95% confidence interval (CI), 12.4-18.1%] and the intraclass correlation coefficient was 0.71 (95% CI, 0.63-0.88). Spearman rank correlation coefficients comparing baseline to +1 year, +1 year to +5 years, and baseline to +5 years were 0.65 (95% CI, 0.37-0.82), 0.61 (0.29-0.81), and 0.53 (0.17-0.77), respectively. Slightly stronger correlations were observed after restricting to non-Hispanic Caucasian subjects. These findings suggest that serum 25(OH)D concentration at a single time point may be a useful biomarker of long-term vitamin D status in population-based studies of various diseases.

OBJECTIVE

Vitamin D deficiency is common among the elderly and may contribute to cerebrovascular diseases. We aimed to elucidate whether low vitamin D levels are predictive for fatal stroke.

METHODS

The LUdwigshafen RIsk and Cardiovascular Health (LURIC) study includes 3316 patients who were referred to coronary angiography at baseline between 1997 and 2000. 25-Hydroxyvitamin D [25(OH)D] and 1,25-dihydroxyvitamin D [1,25(OH)2D] were measured in 3299 and 3315 study participants, respectively. To account for the seasonal variation of vitamin D metabolites, we calculated z values for the 25(OH)D and 1,25(OH)2D concentrations within each month of blood draw.

RESULTS

During a median follow-up time of 7.75 years, 769 patients died, including 42 fatal (ischemic and hemorrhagic) strokes. When compared with survivors in binary logistic-regression analyses, the odds ratios (with 95% CIs) for fatal stroke were 0.58 (0.43 to 0.78; P<0.001) per z value of 25(OH)D and 0.62 (0.47 to 0.81; P<0.001) per z value of 1,25(OH)2D. After adjustment for several possible confounders, these odds ratios remained significant for 25(OH)D at 0.67 (0.46 to 0.97; P=0.032) and for 1,25(OH)2D at 0.72 (0.52 to 0.99; P=0.047). Z values of 25(OH)D and 1,25(OH)2D were also reduced in the 274 patients who had a history of previous cerebrovascular disease events at baseline.

CONCLUSIONS

Low levels of 25(OH)D and 1,25(OH)2D are independently predictive for fatal strokes, suggesting that vitamin D supplementation is a promising approach in the prevention of strokes.

1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) is an immunomodulatory agent inducing dendritic cells (DCs) to become tolerogenic. To further understand its mechanisms of action, we have examined the effects of 1,25(OH)(2)D(3) on tolerogenic properties of blood myeloid (M-DCs) and plasmacytoid (P-DCs) human DC subsets. Exposure of M-DCs to 1,25(OH)(2)D(3) up-regulated production of CCL22, a chemokine attracting regulatory T cells, whereas production of CCL17, the other CCR4 ligand, was reduced. 1,25(OH)(2)D(3) also decreased IL-12p75 production by M-DCs, as expected, and inhibited CCR7 expression. 1,25(OH)(2)D(3) treatment markedly increased CD4(+) suppressor T cell activity while decreasing the capacity of M-DCs to induce Th1 cell development. Surprisingly, 1,25(OH)(2)D(3) did not exert any discernible effect on tolerogenic properties of P-DCs, and even their high production of IFN-alpha was not modulated. In particular, the intrinsically high capacity of P-DCs to induce CD4(+) suppressor T cells was unaffected by 1,25(OH)(2)D(3). Both DC subsets expressed similar levels of the vitamin D receptor, and its ligation by 1,25(OH)(2)D(3) similarly activated the primary response gene cyp24. Interestingly, 1,25(OH)(2)D(3) inhibited NF-kappaB p65 phosphorylation and nuclear translocation in M-DCs but not P-DCs, suggesting a mechanism for the inability of 1,25(OH)(2)D(3) to modulate tolerogenic properties in P-DCs.

OBJECTIVE

Obesity is a well-known risk factor for vitamin D deficiency. We evaluated the interrelationship between vitamin D status, body size, and glucose homeostasis, measured by HbA1c (A1C).

METHODS

Data are from the survey of the 45-year-old 1958 British birth cohort (2002-2004). Information on A1C, 25-hydroxyvitamin D [25(OH)D; an indicator of vitamin D status], and BMI was collected from 7,198 Caucasian subjects.

RESULTS

25(OH)D was < 75 nmol/l in 80% of the obese subjects (BMI>> or = 30 kg/m2) versus 68% of the other subjects (P < 0.0001). Serum 25(OH)D decreased and A1C increased by increasing BMI (P < 0.0001 for both comparisons). There was a nonlinear association between 25(OH)D and A1C: a steep linear decrease in A1C by 25(OH)D until 65 nmol/l and only smaller decreases with further increases. There was evidence for effect modification by BMI in the association between 25(OH)D and A1C (P < 0.0001), and differences appeared stronger for participants with higher compared with lower BMIs. After adjustment for sex, season, geographical location, physical activity, and social class, percent change in A1C by 10-nmol/l increase in 25(OH)D was -0.21 (95% CI -0.31 to -0.11) for BMI < 25 kg/m2, -0.25 (-0.37 to -0.13) for BMI 25-29.9 kg/m2, -0.65 (-0.95 to -0.34) for BMI 30-34.9 kg/m2, and -1.37 (-2.09 to -0.64) for BMI>> or = 35 kg/m2.

CONCLUSIONS

Body size was a strong determinant for 25(OH)D, with concentrations being suboptimal in most obese participants. Randomized controlled trials [using dosages sufficient to improve 25(OH)D also for the obese] are required to determine whether clinically relevant improvements in glucose metabolism can be obtained by vitamin D supplementation.

OBJECTIVE

Single-center studies suggest [corrected] that hypovitaminosis D is widespread. Our objective was to determine the serum levels of 25-hydroxyvitamin D (25[OH]D) in a nationally representative sample of U.S. [corrected] children ages 1-11 [corrected] years.

METHODS

Data were obtained from the 2001-2006 National Health and Nutrition Examination Survey (NHANES). [corrected] Serum 25(OH)D levels was [corrected] determined by radioimmunoassay and categorized as <25 nmol/L, [corrected] <50 nmol/L, [corrected] and <75 nmol/L. National estimates were obtained by using assigned patient visit weights and reported with 95% confidence intervals (95% CI). [corrected]

RESULTS

During [corrected] 2001-2006, the mean serum 25(OH)D level for U.S. children ages 1 to 11 years was 68 nmol/L (95% CI, [corrected] 66-70). Children ages 6-11 [corrected] years had lower mean levels of 25(OH)D (66 nmol/L 95% CI, [corrected] 64-68) compared to [corrected] children ages 1-5 [corrected] years (70 nmol/L 95% [corrected] CI, 68-73). [corrected] Overall, the prevalence of <25 nmol/L [corrected] was 1% (95% CI, 0.7-1.4), <50 nmol/L was 18% (95% CI, [corrected] 16-21), and <75 nmol/L was 69% (95% CI, [corrected] 65-73). The prevalence of [corrected] 25(OH)D [corrected] <75 nmol/L was higher among ages [corrected] 6-11 [corrected] years (73%) compared to ages [corrected] 1-5 [corrected] years (63%); females [corrected] (71%) compared to males [corrected] (67%); and non-Hispanic black (92%) and Hispanic (80%) [corrected] compared to [corrected] non-Hispanic whites [corrected] (59%).

CONCLUSIONS

Based on [corrected] a nationally representative sample of U.S. children aged 1-11 [corrected] years, millions of children may have suboptimal levels of 25(OH)D, especially non-Hispanic black and Hispanic children. More data in children are needed not only to understand better the health implications of specific serum levels of 25(OH)D but also to determine the appropriate vitamin D supplement requirements for children.

Many of the transcriptional and growth regulating activities of 1α,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] in the intestine and colon are recapitulated in the human colorectal cancer cell LS180. We therefore used this line together with chromatin immunoprecipitation-seq and gene expression analyses to identify the vitamin D receptor (VDR)/retinoid X receptor (RXR) and transcription factor 7-like 2 (TCF7L2/TCF4)/β-catenin cistromes and the genes that they regulate. VDR and RXR colocalized to predominantly promoter distal, vitamin D response element-containing sites in a largely ligand-dependent manner. These regulatory sites control the expression of both known as well as novel 1,25-(OH)(2)D(3) target genes. TCF4 and β-catenin cistromes partially overlapped, contained TCF/lymphoid enhancer-binding factor consensus elements, and were only modestly influenced by 1,25-(OH)(2)D(3). However, the two heterodimer complexes colocalized at sites near a limited set of genes that included c-FOS and c-MYC; the expression of both genes was modulated by 1,25-(OH)(2)D(3). At the c-FOS gene, both VDR/RXR and TCF4/β-catenin bound to a single distal enhancer located 24 kb upstream of the transcriptional start site. At the c-MYC locus, however, binding was noted at a cluster of sites between -139 and -165 kb and at a site located -335 kb upstream. Examined as isolated enhancer fragments, these regions exhibited basal and 1,25-(OH)(2)D(3)-inducible activities that were interlinked to both VDR and β-catenin activation. These data reveal additional complexity in the regulation of target genes by 1,25-(OH)(2)D(3) and support a direct action of both VDR and the TCF4/β-catenin regulatory complex at c-FOS and c-MYC.

The plasma membrane of eukaryotic cells contains several types of lipids displaying high biochemical variability in both their apolar moiety (e.g., the acyl chain of glycerolipids) and their polar head (e.g., the sugar structure of glycosphingolipids). Among these lipids, cholesterol is unique because its biochemical variability is almost exclusively restricted to the oxidation of its polar -OH group. Although generally considered the most rigid membrane lipid, cholesterol can adopt a broad range of conformations due to the flexibility of its isooctyl chain linked to the polycyclic sterane backbone. Moreover, cholesterol is an asymmetric molecule displaying a planar α face and a rough β face. Overall, these structural features open up a number of possible interactions between cholesterol and membrane lipids and proteins, consistent with the prominent regulatory functions that this unique lipid exerts on membrane components. The aim of this review is to describe how cholesterol interacts with membrane lipids and proteins at the molecular/atomic scale, with special emphasis on transmembrane domains of proteins containing either the consensus cholesterol-binding motifs CRAC and CARC or a tilted peptide. Despite their broad structural diversity, all these domains bind cholesterol through common molecular mechanisms, leading to the identification of a subset of amino acid residues that are overrepresented in both linear and three-dimensional membrane cholesterol-binding sites.

The etiology of endemic rickets was discovered a century ago. Vitamin D is the precursor of 25-hydroxyvitamin D and other metabolites, including 1,25(OH)2D, the ligand for the vitamin D receptor (VDR). The effects of the vitamin D endocrine system on bone and its growth plate is primarily indirect and mediated by its effect on intestinal calcium transport and serum calcium and phosphate homeostasis. Rickets and osteomalacia can be prevented by daily supplements of 400 IU of vitamin D. Vitamin D deficiency (serum 25OHD < 50 nmol/l) accelerates bone turnover, bone loss and osteoporotic fractures. These risks can be reduced by 800 IU of vitamin D together with an appropriate calcium intake, given to institutionalized or vitamin D deficient elderly subjects.The VDR and vitamin D metabolic enzymes are widely expressed. Numerous genetic, molecular, cellular and animal studies strongly suggest that vitamin D signaling has many extra-skeletal effects. These include regulation of cell proliferation, immune and muscle function, skin differentiation, and reproduction, as well as vascular and metabolic properties. From observational studies in human subjects, poor vitamin D status is associated with nearly all diseases predicted by these extraskeletal actions. Results of randomized controlled trials and Mendelian randomization studies are supportive of vitamin D supplementation in reducing incidence of some diseases, but, globally, conclusions are mixed. These findings point to a need of continued ongoing and future basic and clinical studies to better define whether vitamin D status can be optimized to improve many aspects of human health.Vitamin D deficiency enhances the risk of osteoporotic fractures and is associated with many diseases. We review what is established and what is plausible regarding the health effects of vitamin D.

1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] stimulates bone resorption in man and other vertebrates, in part, by increasing the number of osteoclasts, the principal resorbing cells of bone. Because osteoclasts are very likely derived from a member(s) of the mononuclear phagocyte family, we determined if 1,25(OH)2D3 promotes maturation of these cells by studying its effects on the human promyelocytic leukemia cell line HL-60. Of the vitamin D3 metabolites tested, only 1,25(OH)2D3, at 10(-10) to 10(-7) M, induces the differentiation of HL60 into mono- and multinucleated macrophage-like cells. Phenotypic change is evident within 24 hr and reaches a plateau between 72 and 96 hr of incubation. The changes are metabolite-specific and include (i) adherence to substrate, (ii) acquisition of the morphological features of mature monocytes, (iii) a 4- to 6-fold enhancement in lysozyme synthesis and secretion, (iv) increase in the fraction of alpha-naphthyl acetate esterase-positive cells from approximately 2% to 100% of the population, and (v) the acquisition of several monocyte-associated cell surface antigens. More importantly, treated HL-60 cells acquire the capacity to bind and degrade bone matrix, two of the essential, functional characteristics of osteoclasts and related bone-resorbing cells. These results, considered together with the reported action of 1,25(OH)2D3 on nontransformed mononuclear cells, are consistent with the view that vitamin D3 enhances bone resorption and osteoclastogenesis in vivo by promoting the differentiation of precursor cells.