Oxidative damage has been implicated in the pathology of Parkinson's disease (PD), e.g., rises in the level of the DNA damage product, 8-hydroxy-2'-deoxyguanosine, have been reported. However, many other products result from oxidative DNA damage, and the pattern of products can be diagnostic of the oxidizing species. Gas chromatography/mass spectrometry was used to examine products of oxidation and deamination of all four DNA bases in control and PD brains. Products were detected in all brain regions examined, both normal and PD. Analysis showed that levels of 8-hydroxyguanine (8-OHG) tended to be elevated and levels of 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FAPy guanine) tended to be decreased in PD. The most striking difference was a rise in 8-OHG in PD substantia nigra (p = 0.0002); rises in other base oxidation/deamination products were not evident, showing that elevation in 8-OHG is unlikely to be due to peroxynitrite (ONOO-) or hydroxyl radicals (OH.), or to be a prooxidant effect of treatment with L-Dopa. However, some or all of the rise in 8-OHG could be due to a change in 8-OHG/FAPy guanine ratios rather than to an increase in total oxidative guanine damage.

The active form of vitamin D, 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), has potent immunomodulatory properties that have promoted its potential use in the prevention and treatment of infectious disease and autoimmune conditions. A variety of immune cells, including macrophages, dendritic cells, and activated T cells express the intracellular vitamin D receptor and are responsive to 1,25(OH)(2)D(3.) Despite this, how 1,25(OH)(2)D(3) regulates adaptive immunity remains unclear and may involve both direct and indirect effects on the proliferation and function of T cells. To further clarify this issue, we have assessed the effects of 1,25(OH)(2)D(3) on human CD4(+)CD25(-) T cells. We observed that stimulation of CD4(+)CD25(-) T cells in the presence of 1,25(OH)(2)D(3) inhibited production of proinflammatory cytokines including IFN- gamma, IL-17, and IL-21 but did not substantially affect T cell division. In contrast to its inhibitory effects on inflammatory cytokines, 1,25(OH)(2)D(3) stimulated expression of high levels of CTLA-4 as well as FoxP3, the latter requiring the presence of IL-2. T cells treated with 1,25(OH)(2)D(3) could suppress proliferation of normally responsive T cells, indicating that they possessed characteristics of adaptive regulatory T cells. Our results suggest that 1,25(OH)(2)D(3) and IL-2 have direct synergistic effects on activated T cells, acting as potent anti-inflammatory agents and physiologic inducers of adaptive regulatory T cells.

Vitamin D3 is made in the skin from 7-dehydrocholesterol under the influence of UV light. Vitamin D2 (ergocalciferol) is derived from the plant sterol ergosterol. Vitamin D is metabolized first to 25 hydroxyvitamin D (25OHD), then to the hormonal form 1,25-dihydroxyvitamin D (1,25(OH)2D). CYP2R1 is the most important 25-hydroxylase; CYP27B1 is the key 1-hydroxylase. Both 25OHD and 1,25(OH)2D are catabolized by CYP24A1. 1,25(OH)2D is the ligand for the vitamin D receptor (VDR), a transcription factor, binding to sites in the DNA called vitamin D response elements (VDREs). There are thousands of these binding sites regulating hundreds of genes in a cell-specific fashion. VDR-regulated transcription is dependent on comodulators, the profile of which is also cell specific. Analogs of 1,25(OH)2D are being developed to target specific diseases with minimal side effects. This review will examine these different aspects of vitamin D metabolism, mechanism of action, and clinical application.

Although researchers first identified the fat-soluble vitamin cholecalciferol almost a century ago and studies have now largely elucidated the transcriptional mechanism of action of its hormonal form, 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)], we know surprisingly little about mechanisms of vitamin D toxicity. The lipophilic nature of vitamin D explains its adipose tissue distribution and its slow turnover in the body (half-life approximately 2 mo). Its main transported metabolite, 25-hydroxyvitamin D(3) [25(OH)D(3)], shows a half-life of approximately 15 d and circulates at a concentration of 25-200 nmol/L, whereas the hormone 1alpha,25(OH)(2)D(3) has a half-life of approximately 15 h. Animal experiments involving vitamin D(3) intoxication have established that 25(OH)D(3) can reach concentrations up to 2.5 mumol/L, at which it is accompanied by hypercalcemia and other pathological sequelae resulting from a high Ca/PO(4) product. The rise in 25(OH)D(3) is accompanied by elevations of its precursor, vitamin D(3), as well as by rises in many of its dihydroxy- metabolites [24,25(OH)(2)D(3); 25,26(OH)(2)D(3); and 25(OH)D(3)-26,23-lactone] but not 1alpha,25(OH)(2)D(3). Early assumptions that 1alpha,25(OH)(2)D(3) might cause hypercalcemia in vitamin D toxicity have been replaced by the theories that 25(OH)D(3) at pharmacologic concentrations can overcome vitamin D receptor affinity disadvantages to directly stimulate transcription or that total vitamin D metabolite concentrations displace 1alpha,25(OH)(2)D from vitamin D binding, increasing its free concentration and thus increasing gene transcription. Occasional anecdotal reports from humans intoxicated with vitamin D appear to support the latter mechanism. Although current data support the viewpoint that the biomarker plasma 25(OH)D concentration must rise above 750 nmol/L to produce vitamin D toxicity, the more prudent upper limit of 250 nmol/L might be retained to ensure a wide safety margin.

The crystal structure of the binary complex tRNA(Asp)-aspartyl tRNA synthetase from yeast was solved with the use of multiple isomorphous replacement to 3 angstrom resolution. The dimeric synthetase, a member of class II aminoacyl tRNA synthetases (aaRS's) exhibits the characteristic signature motifs conserved in eight aaRS's. These three sequence motifs are contained in the catalytic site domain, built around an antiparallel beta sheet, and flanked by three alpha helices that form the pocket in which adenosine triphosphate (ATP) and the CCA end of tRNA bind. The tRNA(Asp) molecule approaches the synthetase from the variable loop side. The two major contact areas are with the acceptor end and the anticodon stem and loop. In both sites the protein interacts with the tRNA from the major groove side. The correlation between aaRS class II and the initial site of aminoacylation at 3'-OH can be explained by the structure. The molecular association leads to the following features: (i) the backbone of the GCCA single-stranded portion of the acceptor end exhibits a regular helical conformation; (ii) the loop between residues 320 and 342 in motif 2 interacts with the acceptor stem in the major groove and is in contact with the discriminator base G and the first base pair UA; and (iii) the anticodon loop undergoes a large conformational change in order to bind the protein. The conformation of the tRNA molecule in the complex is dictated more by the interaction with the protein than by its own sequence.

The three-dimensional structures of avian H5 and swine H9 influenza hemagglutinins (HAs) from viruses closely related to those that caused outbreaks of human disease in Hong Kong in 1997 and 1999 were determined bound to avian and human cell receptor analogs. Emerging influenza pandemics have been accompanied by the evolution of receptor-binding specificity from the preference of avian viruses for sialic acid receptors in alpha2,3 linkage to the preference of human viruses for alpha2,6 linkages. The four new structures show that HA binding sites specific for human receptors appear to be wider than those preferring avian receptors and how avian and human receptors are distinguished by atomic contacts at the glycosidic linkage. alpha2,3-Linked sialosides bind the avian HA in a trans conformation to form an alpha2,3 linkage-specific motif, made by the glycosidic oxygen and 4-OH of the penultimate galactose, that is complementary to the hydrogen-bonding capacity of Gln-226, an avian-specific residue. alpha2,6-Linked sialosides bind in a cis conformation, exposing the glycosidic oxygen to solution and nonpolar atoms of the receptor to Leu-226, a human-specific residue. The new structures are compared with previously reported crystal structures of HA/sialoside complexes of the H3 subtype that caused the 1968 Hong Kong Influenza virus pandemic and analyzed in relation to HA sequences of all 15 subtypes and to receptor affinity data to make clearer how receptor-binding sites of HAs from avian viruses evolve as the virus adapts to humans.

The antioxidant and prooxidant behavior of flavonoids and the related activity-structure relationships were investigated in this study using the oxygen radical absorbance capacity assay. Three different reactive species were used in the assay: 2,2'-azobis(2-amidino-propane) dihydrochloride, a peroxyl radical generator; Cu(2+)-H2O2, mainly a hydroxyl radical generator; and Cu2+, a transition metal. Flavonoids including flavones, isoflavones, and flavanones acted as antioxidants against peroxyl and hydroxyl radicals and served as prooxidants in the presence of Cu2+. Both the antioxidant and the copper-initiated prooxidant activities of a flavonoid depend upon the number of hydroxyl substitutions in its backbone structure, which has neither antioxidant nor prooxidant action. In general, the more hydroxyl substitutions, the stronger the antioxidant and prooxidant activities. The flavonoids that contain multiple hydroxyl substitutions showed antiperoxyl radical activities several times stronger than Trolox, an alpha-to copherol analogue. The single hydroxyl substitution at position 5 provides no activity, whereas the di-OH substitution at 3' and 4' is particularly important to the peroxyl radical absorbing activity of a flavonoid. The conjugation between rings A and B does not affect the antioxidant activity but is very important for the copper-initiated prooxidant action of a flavonoid. The O-methylation of the hydroxyl substitutions inactivates both the antioxidant and the prooxidant activities of the flavonoids.

Here we report the structural characterization of the product formed from the reaction between hydroethidine (HE) and superoxide (O(2)(.-)). By using mass spectral and NMR techniques, the chemical structure of this product was determined as 2-hydroxyethidium (2-OH-E(+)). By using an authentic standard, we developed an HPLC approach to detect and quantitate the reaction product of HE and O(2)(.-) formed in bovine aortic endothelial cells after treatment with menadione or antimycin A to induce intracellular reactive oxygen species. Concomitantly, we used a spin trap, 5-tert-butoxycarbonyl-5-methyl-1-pyrroline N-oxide (BMPO), to detect and identify the structure of reactive oxygen species formed. BMPO trapped the O(2)(.-) that formed extracellularly and was detected as the BMPO-OH adduct during use of the EPR technique. BMPO, being cell-permeable, inhibited the intracellular formation of 2-OH-E(+). However, the intracellular BMPO spin adduct was not detected. The definitive characterization of the reaction product of O(2)(.-) with HE described here forms the basis of an unambiguous assay for intracellular detection and quantitation of O(2)(.-). Analysis of the fluorescence characteristics of ethidium (E(+)) and 2-OH-E(+) strongly suggests that the currently available fluorescence methodology is not suitable for quantitating intracellular O(2)(.-). We conclude that the HPLC/fluorescence assay using HE as a probe is more suitable [corrected] for detecting intracellular O(2)(.-).

OBJECTIVE

In small case-control studies, obesity was associated with worse vitamin D status. Our aim was to assess the association of adiposity (anthropometric measures as well as dual energy x-ray absorptiometry) with serum 25-hydroxyvitamin D (25-OH-D) and serum PTH levels in a large population-based study including older men and women.

METHODS

Subjects were participants of the Longitudinal Aging Study Amsterdam and were aged 65 yr and older. In 453 participants, serum 25-OH-D and PTH were determined, and body mass index, waist circumference, waist to hip ratio, sum of skin folds, and total body fat percentage by dual energy x-ray absorptiometry were measured.

RESULTS

After adjustment for potential confounders, higher body mass index, waist circumference, and sum of skin folds were statistically significantly associated with lower 25-OH-D (standardized beta values were -0.136, -0.137, and -0.140, respectively; all P < 0.05) and with higher PTH (0.166, 0.113, and 0.114, respectively; all P < 0.05). Total body fat percentage was more strongly associated with 25-OH-D and PTH (-0.261 and 0.287, respectively; both P < 0.001) compared with anthropometric measures. Total body fat percentage remained associated with 25-OH-D after adjustment for PTH, and with PTH after adjustment for 25-OH-D.

CONCLUSIONS

Precisely measured total body fat is inversely associated with 25-OH-D levels and is positively associated with PTH levels. The associations were weaker if anthropometric measures were used, indicating a specific role of adipose tissue. Regardless of the possible underlying mechanisms, it may be relevant to take adiposity into account when assessing vitamin D requirements.

The molecular mechanisms mediating cell surface trafficking of caveolae are unknown. Caveolae bud from plasma membranes to form free carrier vesicles through a "pinching off" or fission process requiring cytosol and driven by GTP hydrolysis (Schnitzer, J.E., P. Oh, and D.P. McIntosh. 1996. Science. 274:239-242). Here, we use several independent techniques and functional assays ranging from cell-free to intact cell systems to establish a function for dynamin in the formation of transport vesicles from the endothelial cell plasma membrane by mediating fission at the neck of caveolae. This caveolar fission requires interaction with cytosolic dynamin as well as its hydrolysis of GTP. Expression of dynamin in cytosol as well as purified recombinant dynamin alone supports GTP-induced caveolar fission in a cell-free assay whereas its removal from cytosol or the addition to the cytosol of specific antibodies for dynamin inhibits this fission. Overexpression of mutant dynamin lacking normal GTPase activity not only inhibits GTP-induced fission and budding of caveolae but also prevents caveolae-mediated internalization of cholera toxin B chain in intact and permeabilized endothelial cells. Analysis of endothelium in vivo by subcellular fractionation and immunomicroscopy shows that dynamin is concentrated on caveolae, primarily at the expected site of action, their necks. Thus, through its ability to oligomerize, dynamin appears to form a structural collar around the neck of caveolae that hydrolyzes GTP to mediate internalization via the fission of caveolae from the plasma membrane to form free transport vesicles.

Formalin-fixed archival samples are known to be poor materials for molecular biological applications. We conducted a series of experiments to understand the alterations in RNA in fixed tissue. We found that formalin-fixed tissue was resistant to solubilization by chaotropic agents. However, proteinase K completely solubilized the fixed tissue and enabled the extraction of almost the same amount of RNA as from a fresh sample. The extracted RNA did not show apparent degradation. However, as reported, successful PCR amplification was limited to short targets. The nature of such 'fixed' RNA was analyzed using synthetic homo-oligo RNAs. The heterogeneous increase in molecular weight of the RNAs, measured by MALDI-TOF mass spectrometry, showed that all four bases showed addition of mono-methylol (-CH(2)OH) groups at various rates. The modification rate varied from 40% for adenine to 4% for uracil. In addition, some adenines underwent dimerization through methylene bridging. The majority of the methylol groups, however, could be removed from bases by simply elevating the temperature in formalin-free buffer. This demodification proved effective in restoring the template activity of RNA from fixed tissue. The improvement in PCR results suggested that more than half of the modification was removed by this demodification.

Vitamin D is an important immune system regulator. The active form of vitamin D, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], has been shown to inhibit the development of autoimmune diseases, including inflammatory bowel disease (IBD). Paradoxically, other immune system-mediated diseases (experimental asthma) and immunity to infectious organisms were unaffected by 1,25(OH)2D3 treatment. There are similar paradoxical effects of vitamin D deficiency on various immune system functions. Vitamin D and vitamin D receptor (VDR) deficiency resulted in accelerated IBD. Experimental asthma was unaffected by 1,25(OH)2D3 treatment and was less severe among VDR-deficient mice. Vitamin D is a selective regulator of the immune system, and the outcome of 1,25(OH)2D3 treatment, vitamin D deficiency, or VDR deficiency depends on the nature of the immune response (eg, infectious disease, asthma, or autoimmune disease). An additional factor that determines the effect of vitamin D status on immune function is dietary calcium. Dietary calcium has independent effects on IBD severity. Vitamin D-deficient mice on low-calcium diets developed the most severe IBD, and 1,25(OH)2D3 treatment of mice on low-calcium diets improved IBD symptoms. However, the best results for IBD were observed when the calcium concentration was high and 1,25(OH)2D3 was administered. Both the type of immune response and the calcium status of the host determine the effects of vitamin D status and 1,25(OH)2D3 on immunity.

Emerging evidence supports a pathological link between vitamin D deficiency and the risk of inflammatory bowel disease (IBD). To explore the mechanism we used the dextran sulfate sodium (DSS)-induced colitis model to investigate the role of the vitamin D receptor (VDR) in mucosal barrier homeostasis. While VDR(+/+) mice were mostly resistant to 2.5% DSS, VDR(-/-) mice developed severe diarrhea, rectal bleeding, and marked body weight loss, leading to death in 2 wk. Histological examination revealed extensive ulceration and impaired wound healing in the colonic epithelium of DSS-treated VDR(-/-) mice. Severe ulceration in VDR(-/-) mice was preceded by a greater loss of intestinal transepithelial electric resistance (TER) compared with VDR(+/+) mice. Confocal and electron microscopy (EM) revealed severe disruption in epithelial junctions in VDR(-/-) mice after 3-day DSS treatment. Therefore, VDR(-/-) mice were much more susceptible to DSS-induced mucosal injury than VDR(+/+) mice. In cell cultures, 1,25-dihydroxy-vitamin D(3) [1,25(OH)(2)D(3)] markedly enhanced tight junctions formed by Caco-2 monolayers by increasing junction protein expression and TER and preserved the structural integrity of tight junctions in the presence of DSS. VDR knockdown with small interfering (si)RNA reduced the junction proteins and TER in Caco-2 monolayers. 1,25(OH)(2)D(3) can also stimulate epithelial cell migration in vitro. These observations suggest that VDR plays a critical role in mucosal barrier homeostasis by preserving the integrity of junction complexes and the healing capacity of the colonic epithelium. Therefore, vitamin D deficiency may compromise the mucosal barrier, leading to increased susceptibility to mucosal damage and increased risk of IBD.

The technique of small-probe contact dot surfaces is described as a method for calculating and displaying the detailed atomic contacts inside or between molecules. It allows one both to measure and to visualize directly the goodness-of-fit of packing interactions. It requires both highly accurate structures and also the explicit inclusion of all hydrogen atoms and their van der Waals interactions. A reference dataset of 100 protein structures was chosen on the basis of resolution (1.7 A or better), crystallographic R-value, non-homology, and the absence of any unusual problems. Hydrogen atoms were added in standard geometry and, where needed, with rotational optimization of OH, SH, and NH+3 positions. Side-chain amide orientations were corrected where required by NH van der Waals clashes, as described in the accompanying paper. It was determined that, in general, methyl groups pack well in the default staggered conformation, except for the terminal methyl groups of methionine residues, which required rotational optimization. The distribution of serious clashes (i.e. non-H-bond overlap of>>/=0.4 A) was studied as a function of resolution, alternate conformations, and temperature factor (B), leading to the decision that packing and other structural features would not be analyzed for residues in 'b' alternate conformations or with B-factors of 40 or above. At the level of the fine details analyzed here, structural accuracy improves quite significantly over the range from 1.7 to 1.0 A resolution. These high-resolution structures show impressively well-fitted packing interactions, with some regions thoroughly interdigitated and other regions somewhat sparser. Lower-resolution structures or model structures could undoubtedly be improved in accuracy by the incorporation of this additional information: for example, nucleic acid structures in non-canonical conformations are often very accurate for the bases and much less reliable for the backbone, whose conformation could be specified better by including explicit H atom geometry and contacts. The contact dots are an extremely sensitive method of finding problem areas, and often they can suggest how to make improvements. They can also provide explanations for structural features that have been described only as empirical regularities, which is illustrated by showing that the commonest rotamer of methionine (a left-handed spiral, with all chi values near -60 degrees) is preferred because it provides up to five good H atom van der Waals contacts. This methodology is thus applicable in two different ways: (1) for finding and correcting errors in structure models (either experimental or theoretical); and (2) for analyzing interaction patterns in the molecules themselves.

The need, safety, and effectiveness of vitamin D supplementation during pregnancy remain controversial. In this randomized, controlled trial, women with a singleton pregnancy at 12 to 16 weeks' gestation received 400, 2000, or 4000 IU of vitamin D(3) per day until delivery. The primary outcome was maternal/neonatal circulating 25-hydroxyvitamin D [25(OH)D] concentration at delivery, with secondary outcomes of a 25(OH)D concentration of 80 nmol/L or greater achieved and the 25(OH)D concentration required to achieve maximal 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] production. Of the 494 women enrolled, 350 women continued until delivery: Mean 25(OH)D concentrations by group at delivery and 1 month before delivery were significantly different (p < 0.0001), and the percent who achieved sufficiency was significantly different by group, greatest in 4000-IU group (p < 0.0001). The relative risk (RR) for achieving a concentration of 80 nmol/L or greater within 1 month of delivery was significantly different between the 2000- and the 400-IU groups (RR = 1.52, 95% CI 1.24-1.86), the 4000- and the 400-IU groups (RR = 1.60, 95% CI 1.32-1.95) but not between the 4000- and. 2000-IU groups (RR = 1.06, 95% CI 0.93-1.19). Circulating 25(OH)D had a direct influence on circulating 1,25(OH)(2)D(3) concentrations throughout pregnancy (p < 0.0001), with maximal production of 1,25(OH)(2)D(3) in all strata in the 4000-IU group. There were no differences between groups on any safety measure. Not a single adverse event was attributed to vitamin D supplementation or circulating 25(OH)D levels. It is concluded that vitamin D supplementation of 4000 IU/d for pregnant women is safe and most effective in achieving sufficiency in all women and their neonates regardless of race, whereas the current estimated average requirement is comparatively ineffective at achieving adequate circulating 25(OH)D concentrations, especially in African Americans.

Peroxynitrite [oxoperoxonitrate(1-), ONOO-] may be formed in vivo from superoxide and nitric oxide. The anion is stable, but the acid (pKa = 6.8) decays to nitrate with a rate of 1.3 s-1 at 25 degrees C. The experimental activation parameters of this process are delta H++ = +18 +/- 1 kcal/mol, delta S++ = +3 +/- 2 cal/(mol.K), and delta G++ = +17 +/- 1 kcal/mol. Peroxynitrite (or its protonated form) oxidizes some compounds such as thiols and thioethers in a biomolecular reaction. The reactions with glutathione and cysteine have activation enthalpies of 10.9 and 9.7 kcal/mol, respectively, which are lower than that of the isomerization reaction. Peroxynitrite reacts with other compounds such as dimethyl sulfoxide and deoxyribose in a unimolecular reaction for which the activation of peroxynitrite is rate-limiting. In theory, activation could involve (1) heterolysis to OH- and NO2+ (delta rxn Gzero' = 13 kcal/mol at pH 7) or (2) homolysis to .OH and .NO2 (delta rxn Gzero = 21 kcal/mol), and these processes also could be involved in the isomerization to nitrate. However, thermodynamic and kinetic considerations indicate that neither process is feasible, although binding to metal ions may reduce the large activation energy associated with heterolysis. An intermediate closely related to the transition state for isomerization of ONOOH to HONO2 may be the strongly oxidizing intermediate responsible for hydroxyl radical-like oxidations mediated by ONOOH. Thus, peroxynitrite reacts with different compounds by at least two distinct mechanisms, and the hydroxyl radical is not involved in either.

1alpha,25-Dihydroxyvitamin D3 [1,25(OH)2D3] regulates calcium homeostasis and controls cellular differentiation and proliferation. The vitamin D receptor (VDR) is a ligand-regulated transcription factor that recognizes cognate vitamin D response elements (VDREs) formed by direct or everted repeats of PuG(G/T)TCA motifs separated by 3 or 6 bp (DR3 or ER6). Here, we have identified direct 1,25(OH)2D3 target genes by combining 35,000+ gene microarrays and genome-wide screens for consensus DR3 and ER6 elements, and DR3 elements containing single nucleotide substitutions. We find that the effect of a nucleotide substitution on VDR binding in vitro does not predict VDRE function in vivo, because substitutions that disrupted binding in vitro were found in several functional elements. Hu133A microarray analyses, performed with RNA from human SCC25 cells treated with 1,25(OH)2D3 and protein synthesis inhibitor cycloheximide, identified more than 900 regulated genes. VDREs lying within -10 to +5 kb of 5'-ends were assigned to 65% of these genes, and VDR binding was confirmed to several elements in vivo. A screen of the mouse genome identified more than 3000 conserved VDREs, and 158 human genes containing conserved elements were 1,25(OHOHOH)2D3.

In vitro and animal studies indicate that vitamin D may have anti-cancer benefits, including against progression and metastasis, against a wide spectrum of cancers. Supporting an anti-cancer effect of vitamin D is the ability of many cells to convert 25(OH)D, the primary circulating form of vitamin D, into 1,25(OH)2D, the most active form of this vitamin. No epidemiologic studies have directly measured vitamin D concentrations or intakes on risk of total cancer incidence or mortality. However, higher rates of total cancer mortality in regions with less UV-B radiation, and among African-Americans and overweight and obese people, each associated with lower circulating vitamin D, are compatible with a benefit of vitamin D on mortality. In addition, poorer survival from cancer in individuals diagnosed in the months when vitamin D levels are lowest suggests a benefit of vitamin D against late stages of carcinogenesis. The only individual cancer sites that have been examined directly in relation to vitamin D status are colorectal, prostate and breast cancers. For breast cancer, some data are promising for a benefit from vitamin D but are far too sparse to support a conclusion. The evidence that higher 25(OH)D levels through increased sunlight exposure or dietary or supplement intake inhibit colorectal carcinogenesis is substantial. The biologic evidence for an anti-cancer role of 25(OH)D is also strong for prostate cancer, but the epidemiologic data have not been supportive. Although not entirely consistent, some studies suggest that higher circulating 1,25(OH)2D may be more important than 25(OH)D for protection against aggressive, poorly-differentiated prostate cancer. A possible explanation for these divergent results is that unlike colorectal tumors, prostate cancers lose the ability to hydroxylate 25(OH)D to 1,25(OH)2D, and thus may rely on the circulation as the main source of 1,25(OH)2D. The suppression of circulating 1,25(OH)2D levels by calcium intake could explain why higher calcium and milk intakes appear to increase risk of advanced prostate cancer. Given the potential benefits from vitamin D, further research should be a priority.

Cell therapy can improve cardiac function in animals and humans after injury, but the mechanism is unclear. We performed cell therapy experiments in genetically engineered mice that permanently express green fluorescent protein (GFP) only in cardiomyocytes after a pulse of 4-OH-tamoxifen. Myocardial infarction diluted the GFP(+) cardiomyocyte pool, indicating refreshment by non-GFP(+) progenitors. Cell therapy with bone marrow-derived c-kit(+) cells, but not mesenchymal stem cells, further diluted the GFP(+) pool, consistent with c-kit(+) cell-mediated augmentation of cardiomyocyte progenitor activity. This effect could not be explained by transdifferentiation to cardiomyocytes by exogenously delivered c-kit(+) cells or by cell fusion. Therapy with c-kit(+) cells but not mesenchymal stem cells improved cardiac function. These findings suggest that stimulation of endogenous cardiogenic progenitor activity is a critical mechanism of cardiac cell therapy.

Platelet-derived growth factor receptor (PDGF-R) phosphorylation at tyrosines 740/751 and insulin receptor phosphorylation of insulin receptor substrate-1 effects the recruitment and activation of phosphatidylinositol-3-OH kinase (PI(3)K). Changes in PI(3)K activity correlate with cell growth but its downstream signal transducers are unknown. Activation of the 70/85K S6 kinases (pp70S6k) by serine phosphorylation results in 40S ribosomal protein S6 phosphorylation and is important for G1 cell-cycle transition in a variety of cells. Although receptor tyrosine kinases activate the microtubule-associated protein kinase cascade through SH2-/SH3-adaptor proteins, Sos and c-Ras, it is unclear how tyrosine kinases are coupled to the pp70S6k phosphorylation cascade. Here we report that PI(3)K mediates PDGF or insulin receptor signalling to pp70S6k. PI(3)K-mediated activation of pp70S6k is independent of conventional protein kinase C isoforms. Additionally, rapamycin blocks pp70S6k activation by all mitogens, without inhibiting PI(3)K, and acts downstream in this signalling system.

More than 3000 type II restriction endonucleases have been discovered. They recognize short, usually palindromic, sequences of 4-8 bp and, in the presence of Mg(2+), cleave the DNA within or in close proximity to the recognition sequence. The orthodox type II enzymes are homodimers which recognize palindromic sites. Depending on particular features subtypes are classified. All structures of restriction enzymes show a common structural core comprising four beta-strands and one alpha-helix. Furthermore, two families of enzymes can be distinguished which are structurally very similar (EcoRI-like enzymes and EcoRV-like enzymes). Like other DNA binding proteins, restriction enzymes are capable of non-specific DNA binding, which is the prerequisite for efficient target site location by facilitated diffusion. Non-specific binding usually does not involve interactions with the bases but only with the DNA backbone. In contrast, specific binding is characterized by an intimate interplay between direct (interaction with the bases) and indirect (interaction with the backbone) readout. Typically approximately 15-20 hydrogen bonds are formed between a dimeric restriction enzyme and the bases of the recognition sequence, in addition to numerous van der Waals contacts to the bases and hydrogen bonds to the backbone, which may also be water mediated. The recognition process triggers large conformational changes of the enzyme and the DNA, which lead to the activation of the catalytic centers. In many restriction enzymes the catalytic centers, one in each subunit, are represented by the PD. D/EXK motif, in which the two carboxylates are responsible for Mg(2+) binding, the essential cofactor for the great majority of enzymes. The precise mechanism of cleavage has not yet been established for any enzyme, the main uncertainty concerns the number of Mg(2+) ions directly involved in cleavage. Cleavage in the two strands usually occurs in a concerted fashion and leads to inversion of configuration at the phosphorus. The products of the reaction are DNA fragments with a 3'-OH and a 5'-phosphate.

The eight catalytic subunits of the mammalian phosphoinositide-3-OH kinase (PI(3)K) family form the backbone of an evolutionarily conserved signalling pathway; however, the roles of most PI(3)K isoforms in organismal physiology and disease are unknown. To delineate the role of p110alpha, a ubiquitously expressed PI(3)K involved in tyrosine kinase and Ras signalling, here we generated mice carrying a knockin mutation (D933A) that abrogates p110alpha kinase activity. Homozygosity for this kinase-dead p110alpha led to embryonic lethality. Mice heterozygous for this mutation were viable and fertile, but displayed severely blunted signalling via insulin-receptor substrate (IRS) proteins, key mediators of insulin, insulin-like growth factor-1 and leptin action. Defective responsiveness to these hormones led to reduced somatic growth, hyperinsulinaemia, glucose intolerance, hyperphagia and increased adiposity in mice heterozygous for the D933A mutation. This signalling function of p110alpha derives from its highly selective recruitment and activation to IRS signalling complexes compared to p110beta, the other broadly expressed PI(3)K isoform, which did not contribute to IRS-associated PI(3)K activity. p110alpha was the principal IRS-associated PI(3)K in cancer cell lines. These findings demonstrate a critical role for p110alpha in growth factor and metabolic signalling and also suggest an explanation for selective mutation or overexpression of p110alpha in a variety of cancers.

Low serum 25-hydroxyvitamin D (25(OH)D) has been shown to correlate with increased risk of type 2 diabetes. Small, observational studies suggest an action for vitamin D in improving insulin sensitivity and/or insulin secretion. The objective of the present study was to investigate the effect of improved vitamin D status on insulin resistance (IR), utilising randomised, controlled, double-blind intervention administering 100 microg (4000 IU) vitamin D(3) (n 42) or placebo (n 39) daily for 6 months to South Asian women, aged 23-68 years, living in Auckland, New Zealand. Subjects were insulin resistant - homeostasis model assessment 1 (HOMA1)>1.93 and had serum 25(OH)D concentration < 50 nmol/l. Exclusion criteria included diabetes medication and vitamin D supplementation >25 microg (1000 IU)/d. The HOMA2 computer model was used to calculate outcomes. Median (25th, 75th percentiles) serum 25(OH)D(3) increased significantly from 21 (11, 40) to 75 (55, 84) nmol/l with supplementation. Significant improvements were seen in insulin sensitivity and IR (P = 0.003 and 0.02, respectively), and fasting insulin decreased (P = 0.02) with supplementation compared with placebo. There was no change in C-peptide with supplementation. IR was most improved when endpoint serum 25(OH)D reached>> or = 80 nmol/l. Secondary outcome variables (lipid profile and high sensitivity C-reactive protein) were not affected by supplementation. In conclusion, improving vitamin D status in insulin resistant women resulted in improved IR and sensitivity, but no change in insulin secretion. Optimal vitamin D concentrations for reducing IR were shown to be 80-119 nmol/l, providing further evidence for an increase in the recommended adequate levels. Registered Trial No. ACTRN12607000642482.

Neutrophils and other phagocytes manufacture O(2)(-) (superoxide) by the one-electron reduction of oxygen at the expense of NADPH. Most of the O(2)(-) reacts with itself to form H(2)O(2) (hydrogen peroxide). From these agents a large number of highly reactive microbicidal oxidants are formed, including HOCl (hypochlorous acid), which is produced by the myeloperoxidase-catalyzed oxidation of Cl(-) by H(2)O(2); OH(*) (hydroxyl radical), produced by the reduction of H(2)O(2) by Fe(++) or Cu(+); ONOO(-) (peroxynitrite), formed by the reaction between O(2)(-) and NO(*); and many others. These reactive oxidants are manufactured for the purpose of killing invading microorganisms, but they also inflict damage on nearby tissues, and are thought to be of pathogenic significance in a large number of diseases. Included among these are emphysema, acute respiratory distress syndrome, atherosclerosis, reperfusion injury, malignancy and rheumatoid arthritis.