OBJECTIVE

Experimental evidence suggests that 1,25-dihydroxyvitamin D and its precursor, 25-hydroxyvitamin D [25(OH)D], may aid in the prevention of colorectal cancer. We therefore examined risk in relation to plasma concentrations of these vitamin D metabolites.

METHODS

In a nested case-control study among women in the Nurses' Health Study, we identified 193 colorectal cancer cases, ages 46 to 78 years, diagnosed up to 11 years after blood collection. Two controls were matched per case on year of birth and month of blood draw. Odds ratios (OR) for risk of colorectal cancer were calculated using conditional logistic regression adjusted for body mass index, physical activity, smoking, family history, use of hormone replacement therapy, aspirin use, and dietary intakes.

RESULTS

We found a significant inverse linear association between plasma 25(OH)D and risk of colorectal cancer (P = 0.02). Among women in the highest quintile, the OR (95% confidence interval) was 0.53 (0.27-1.04). This inverse association remained strong when limited to women>> or =60 years at blood collection (P = 0.006) but was not apparent among the younger women (P = 0.70). Benefit from higher 25(OH)D concentrations was observed for cancers at the distal colon and rectum (P = 0.02) but was not evident for those at the proximal colon (P = 0.81). In contrast to 25(OH)D, we did not observe an association between 1,25-dihydroxyvitamin D and colorectal cancer, although risk was elevated among the women in the highest quintile if they were also in the lower half of the 25(OH)D distribution (OR, 2.52; 95% confidence interval, 1.04-6.11).

CONCLUSIONS

From these results and supporting evidence from previous studies, we conclude that higher plasma levels of 25(OH)D are associated with a lower risk of colorectal cancer in older women, particularly for cancers at the distal colon and rectum.

The hormone, 1,25-dihydroxyvitamin D3 (1,25-[OH]2-D3), inhibits lymphocyte activation in vitro. We studied the ability of the vitamin D metabolite to interfere in vivo with a primary T cell-mediated model of autoimmunity, murine experimental autoimmune encephalomyelitis (EAE). Within 2 wk of antigenic challenge, immunized animals will develop acute paralysis with central nervous tissue inflammation. If mice survive, a rise in antibody titer develops within a month. The administration of 0.1 microgram 1,25-(OH)2-D3 i.p. given every other day for 15 d, starting 3 d before immunization, significantly prevented the development of EAE. The rise in antibody titer to myelin basic protein was also abrogated. Histopathologic lesions of EAE were inhibited by treatment with the sterol. These results suggest a potent immunosuppressive role for 1,25-(OH)2-D3 in vivo in the modulation of a cell-mediated model of autoimmunity.

During normal cellular activities, various processes inside of cells produce reactive oxygen species (ROS). Some of the most common ROS are hydrogen peroxide (H(2)O(2)), superoxide ion (O(2)(-)), and hydroxide radical (OH(-)). These compounds, when present in a high enough concentration, can damage cellular proteins and lipids or form DNA adducts that may promote carcinogenic activity. The purpose of antioxidants in a physiological setting is to prevent ROS concentrations from reaching a high-enough level within a cell that damage may occur. Cellular antioxidants may be enzymatic (catalase, glutathione peroxidase, superoxide dismutase) or nonenzymatic (glutathione, thiols, some vitamins and metals, or phytochemicals such as isoflavones, polyphenols, and flavanoids). Reactive oxygen species are a potential double-edged sword in disease prevention and promotion. Whereas generation of ROS once was viewed as detrimental to the overall health of the organism, advances in research have shown that ROS play crucial roles in normal physiological processes including response to growth factors, the immune response, and apoptotic elimination of damaged cells. Notwithstanding these beneficial functions, aberrant production or regulation of ROS activity has been demonstrated to contribute to the development of some prevalent diseases and conditions, including cancer and cardiovascular disease (CVD). The topic of antioxidant usage and ROS is currently receiving much attention because of studies linking the use of some antioxidants with increased mortality in primarily higher-risk populations and the lack of strong efficacy data for protection against cancer and heart disease, at least in populations with adequate baseline dietary consumption. In normal physiological processes, antioxidants effect signal transduction and regulation of proliferation and the immune response. Reactive oxygen species have been linked to cancer and CVD, and antioxidants have been considered promising therapy for prevention and treatment of these diseases, especially given the tantalizing links observed between diets high in fruits and vegetables (and presumably antioxidants) and decreased risks for cancer.

Rab5 is a small GTPase involved in the control of intracellular trafficking, both at the level of receptor endocytosis and endosomal dynamics. The finding that Rab5 can be activated by receptor tyrosine kinases (RTK) raised the question of whether it also participates in effector pathways emanating from these receptors. Here we show that Rab5 is indispensable for a form of RTK-induced actin remodelling, called circular ruffling. Three independent signals, originating from Rab5, phosphatidylinositol-3-OH kinase and Rac, respectively, are simultaneously required for the induction of circular ruffles. Rab5 signals to the actin cytoskeleton through RN-tre, a previously identified Rab5-specific GTPase-activating protein (GAP). Here we demonstrate that RN-tre has the dual function of Rab5-GAP and Rab5 effector. We also show that RN-tre is critical for macropinocytosis, a process previously connected to the formation of circular ruffles. Finally, RN-tre interacts with both F-actin and actinin-4, an F-actin bundling protein. We propose that RN-tre establishes a three-pronged connection with Rab5, F-actin and actinin-4. This may aid crosslinking of actin fibres into actin networks at the plasma membrane. Thus, we have shown that Rab5 is a signalling GTPase and have elucidated the major molecular elements of its downstream pathway.

Protonated amino acids and derivatives RCH(NH2)C(+O)X · H(+) (X = OH, NH2, OCH3) do not form stable acylium ions on loss of HX, but rather the acylium ion eliminates CO to form the immonium ion RCH = NH 2 (+) . By contrast, protonated dipeptide derivatives H2NCH(R)C(+O)NHCH(R')C(+O)X · H(+) [X = OH, OCH3, NH2, NHCH(R″)COOH] form stable B2 ions by elimination of HX. These B2 ions fragment on the metastable ion time scale by elimination of CO with substantial kinetic energy release (T 1/2 = 0.3-0.5 eV). Similarly, protonated N-acetyl amino acid derivatives CH3C(+O)NHCH(R')C(+O)X · H(+) [X = OH, OCH3, NH2, NHCH(R″)COOH] form stable B ions by loss of HX. These B ions also fragment unimolecularly by loss of CO with T 1/2 values of ∼ 0.5 eV. These large kinetic energy releases indicate that a stable configuration of the B ions fragments by way of activation to a reacting configuration that is higher in energy than the products, and some of the fragmentation exothermicity of the final step is partitioned into kinetic energy of the separating fragments. We conclude that the stable configuration is a protonated oxazolone, which is formed by interaction of the developing charge (as HX is lost) with the N-terminus carbonyl group and that the reacting configuration is the acyclic acylium ion. This conclusion is supported by the similar fragmentation behavior of protonated 2-phenyl-5-oxazolone and the B ion derived by loss of H-Gly-OH from protonated C6H5C(+O)-Gly-Gly-OH. In addition, ab initio calculations on the simplest B ion, nominally HC(+O)NHCH2CO(+), show that the lowest energy structure is the protonated oxazolone. The acyclic acylium isomer is 1.49 eV higher in energy than the protonated oxazolone and 0.88 eV higher in energy than the fragmentation products, HC(+O)N(+)H = CH2 + CO, which is consistent with the kinetic energy releases measured.

Stem cell self-renewal implies proliferation under continued maintenance of multipotency. Small changes in numbers of stem cells may lead to large differences in differentiated cell numbers, resulting in significant physiological consequences. Proliferation is typically regulated in the G1 phase, which is associated with differentiation and cell cycle arrest. However, embryonic stem (ES) cells may lack a G1 checkpoint. Regulation of proliferation in the 'DNA damage' S/G2 cell cycle checkpoint pathway is known for its role in the maintenance of chromatin structural integrity. Here we show that autocrine/paracrine gamma-aminobutyric acid (GABA) signalling by means of GABA(A) receptors negatively controls ES cell and peripheral neural crest stem (NCS) cell proliferation, preimplantation embryonic growth and proliferation in the boundary-cap stem cell niche, resulting in an attenuation of neuronal progenies from this stem cell niche. Activation of GABA(A) receptors leads to hyperpolarization, increased cell volume and accumulation of stem cells in S phase, thereby causing a rapid decrease in cell proliferation. GABA(A) receptors signal through S-phase checkpoint kinases of the phosphatidylinositol-3-OH kinase-related kinase family and the histone variant H2AX. This signalling pathway critically regulates proliferation independently of differentiation, apoptosis and overt damage to DNA. These results indicate the presence of a fundamentally different mechanism of proliferation control in these stem cells, in comparison with most somatic cells, involving proteins in the DNA damage checkpoint pathway.

We define previously unrecognized in vivo pathways of vitamin D(3) (D3) metabolism generating novel D3-hydroxyderivatives different from 25-hydroxyvitamin D(3) [25(OH)D3] and 1,25(OH)(2)D3. Their novel products include 20-hydroxyvitamin D(3) [20(OH)D3], 22(OH)D3, 20,23(OH)(2)D3, 20,22(OH)(2)D3, 1,20(OH)(2)D3, 1,20,23(OH)(3)D3, and 17,20,23(OH)(3)D3 and were produced by placenta, adrenal glands, and epidermal keratinocytes. We detected the predominant metabolite [20(OH)D3] in human serum with a relative concentration ∼20 times lower than 25(OH)D3. Use of inhibitors and studies performed with isolated mitochondria and purified enzymes demonstrated involvement of the steroidogenic enzyme cytochrome P450scc (CYP11A1) as well as CYP27B1 (1α-hydroxylase). In placenta and adrenal glands with high CYP11A1 expression, the predominant pathway was D3 → 20(OH)D3 → 20,23(OH)(2)D3 → 17,20,23(OH)(3)D3 with further 1α-hydroxylation, and minor pathways were D3 → 25(OH)D3 → 1,25(OH)(2)D3 and D3 → 22(OH)D3 → 20,22(OH)(2)D3. In epidermal keratinocytes, we observed higher proportions of 22(OH)D3 and 20,22(OH)(2)D3. We also detected endogenous production of 20(OH)D3, 22(OH) D3, 20,23(OH)(2)D3, 20,22(OH)(2)D3, and 17,20,23(OH)(3)D3 by immortalized human keratinocytes. Thus, we provide in vivo evidence for novel pathways of D3 metabolism initiated by CYP11A1, with the product profile showing organ/cell type specificity and being modified by CYP27B1 activity. These findings define the pathway intermediates as natural products/endogenous bioregulators and break the current dogma that vitamin D is solely activated through the sequence D3 → 25(OH)D3 → 1,25(OH)(2)D3.

In recent years, there have been reports suggesting a high prevalence of low vitamin D intakes and vitamin D deficiency or inadequate vitamin D status in Europe. Coupled with growing concern about the health risks associated with low vitamin D status, this has resulted in increased interest in the topic of vitamin D from healthcare professionals, the media and the public. Adequate vitamin D status has a key role in skeletal health. Prevention of the well-described vitamin D deficiency disorders of rickets and osteomalacia are clearly important, but there may also be an implication of low vitamin D status in bone loss, muscle weakness and falls and fragility fractures in older people, and these are highly significant public health issues in terms of morbidity, quality of life and costs to health services in Europe. Although there is no agreement on optimal plasma levels of vitamin D, it is apparent that blood 25-hydroxyvitamin D [25(OH)D] levels are often below recommended ranges for the general population and are particularly low in some subgroups of the population, such as those in institutions or who are housebound and non-Western immigrants. Reported estimates of vitamin D status within different European countries show large variation. However, comparison of studies across Europe is limited by their use of different methodologies. The prevalence of vitamin D deficiency [often defined as plasma 25(OH)D <25 nmol/l] may be more common in populations with a higher proportion of at-risk groups, and/or that have low consumption of foods rich in vitamin D (naturally rich or fortified) and low use of vitamin D supplements. The definition of an adequate or optimal vitamin D status is key in determining recommendations for a vitamin D intake that will enable satisfactory status to be maintained all year round, including the winter months. In most European countries, there seems to be a shortfall in achieving current vitamin D recommendations. An exception is Finland, where dietary survey data indicate that recent national policies that include fortification and supplementation, coupled with a high habitual intake of oil-rich fish, have resulted in an increase in vitamin D intakes, but this may not be a suitable strategy for all European populations. The ongoing standardisation of measurements in vitamin D research will facilitate a stronger evidence base on which policies can be determined. These policies may include promotion of dietary recommendations, food fortification, vitamin D supplementation and judicious sun exposure, but should take into account national, cultural and dietary habits. For European nations with supplementation policies, it is important that relevant parties ensure satisfactory uptake of these particularly in the most vulnerable groups of the population.

We have shown previously that the AKT2 pathway is essential for cell survival and important in malignant transformation. In this study, we demonstrate elevated kinase levels of AKT2 and phosphatidylinositol-3-OH kinase (PI3K) in 32 of 80 primary breast carcinomas. The majority of the cases with the activation are estrogen receptor alpha (ERalpha) positive, which prompted us to examine whether AKT2 regulates ERalpha activity. We found that constitutively activated AKT2 or AKT2 activated by epidermal growth factor or insulin-like growth factor-1 promotes the transcriptional activity of ERalpha. This effect occurred in the absence or presence of estrogen. Activated AKT2 phosphorylates ERalpha in vitro and in vivo, but it does not phosphorylate a mutant ERalpha in which ser-167 was replaced by Ala. The PI3K inhibitor, wortmannin, abolishes both the phosphorylation and transcriptional activity of ERalpha induced by AKT2. However, AKT2-induced ERalpha activity was not inhibited by tamoxifen but was completely abolished by ICI 164,384, implicating that AKT2-activated ERalpha contributes to tamoxifen resistance. Moreover, we found that ERalpha binds to the p85alpha regulatory subunit of PI3K in the absence or presence of estradiol in epithelial cells and subsequently activates PI3K/AKT2, suggesting ERalpha regulation of PI3K/AKT2 through a nontranscriptional and ligand-independent mechanism. These data indicate that regulation between the ERalpha and PI3K/AKT2 pathway (ERalpha-PI3K/AKT2-ERalpha) may play an important role in pathogenesis of human breast cancer and could contribute to ligand-independent breast cancer cell growth.

Melatonin (N-acetyl-5-methoxytryptamine), an endogenously produced indole found throughout the animal kingdom, was recently reported, using a variety of techniques, to be a scavenger of a number of reactive oxygen and reactive nitrogen species both in vitro and in vivo. Initially, melatonin was discovered to directly scavenge the high toxic hydroxyl radical (*OH). The methods used to prove the interaction of melatonin with the *OH included the generation of the radical using Fenton reagents or the ultraviolet photolysis of hydrogen peroxide (H202) with the use of spin-trapping agents, followed by electron spin resonance (ESR) spectroscopy, pulse radiolysis followed by ESR, and several spectrofluorometric and chemical (salicylate trapping in vivo) methodologies. One product of the reaction of melatonin with the *OH was identified as cyclic 3-hydroxymelatonin (3-OHM) using high-performance liquid chromatography with electrochemical (HPLC-EC) detection, electron ionization mass spectrometry (EIMS), proton nuclear magnetic resonance (1H NMR) and COSY 1H NMR. Cyclic 3-OHM appears in the urine of humans and other mammals and in rat urine its concentration increases when melatonin is given exogenously or after an imposed oxidative stress (exposure to ionizing radiation). Urinary cyclic 3-OHM levels are believed to be a biomarker (footprint molecule) of in vivo *OH production and its scavenging by melatonin. Although the data are less complete, besides the *OH, melatonin in cell-free systems has been shown to directly scavenge H2O2, singlet oxygen (1O2) and nitric oxide (NO*), with little or no ability to scavenge the superoxide anion radical (O2*-) In vitro, melatonin also directly detoxifies the peroxynitrite anion (ONOO-) and/or peroxynitrous acid (ONOOH), or the activated form of this molecule, ONOOH*; the product of the latter interaction is proposed to be 6-OHM. How these in vitro findings relate to the in vivo antioxidant actions of melatonin remains to be established. The ability of melatonin to scavenge the lipid peroxyl radical (LOO*) is debated. The weight of the evidence is that melatonin is probably not a classic chain-breaking antioxidant, since its ability to scavenge the LOO* seems weak. Its ability to reduce lipid peroxidation may stem from its function as a preventive antioxidant (scavenging initiating radicals), or yet unidentified actions. In sum, in vitro melatonin acts as a direct free radical scavenger with the ability to detoxify both reactive oxygen and reactive nitrogen species; in vivo, it is an effective pharmacological agent in reducing oxidative damage under conditions in which excessive free radical generation is believed to be involved.

The RAS-MAPK, PI (3)K signaling pathways form a network that play a central role in tumorigenesis. The BRAF, KRAS and PI3KCA genes code 3 partners of this network and have been found to be activated by mutation in colorectal cancer; these mutations lead to unrestricted cell growth. We evaluated the clinicopathological features and the prognosis of patients with activated-network colon cancers in a population-based study. A total of 586 colon adenocarcinomas were evaluated using sequencing for mutations of KRAS and PI3KCA, and allelic discrimination for mutation of BRAF. Clinicopathological characteristics were correlated to the risk of bearing a mutation of the network using logistic regression. Three-year survival rates were compared with the Log rank test. A multivariate survival analysis using the Cox model was performed. After adjustment for age and microsatellite instability, activation of the network by mutation of at least 1 of the 3 genes was significantly associated with female sex (p = 0.02) and proximal location (p < 0.001). Lower levels of 3-year survival were associated with activation of the network by mutation of at least 1 of the 3 genes (59.4 and 69.4%, respectively; p = 0.009). These results remained significant in a multivariate analysis adjusted for sex, age, location, stage and microsatellite instability (HR = 1.48; CI CI(95%) = [1.07-2.04]). Our study is the first report to underline the potential role of RAS-MAPK, PI (3)K network mutations on survival in colon cancers. Because of the role of this signaling network on anticancer agents, the evaluation of its mutations could have clinical implications.

Neutrophils play an essential role in host defense against microbial pathogens and in the inflammatory reaction. Upon activation, neutrophils produce superoxide anion (O*2), which generates other reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), hydroxyl radical (OH*) and hypochlorous acid (HOCl), together with microbicidal peptides and proteases. The enzyme responsible for O2* production is called the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase or respiratory burst oxidase. This multicomponent enzyme system is composed of two trans-membrane proteins (p22phox and gp91phox/NOX2, which form the cytochrome b558), three cytosolic proteins (p47phox, p67phox, p40phox) and a GTPase (Rac1 or Rac2), which assemble at membrane sites upon cell activation. NADPH oxidase activation in phagocytes can be induced by a large number of soluble and particulate factors. Three major events accompany NAPDH oxidase activation: (1) protein phosphorylation, (2) GTPase activation, and (3) translocation of cytosolic components to the plasma membrane to form the active enzyme. Actually, the neutrophil NADPH oxidase exists in different states: resting, primed, activated, or inactivated. The resting state is found in circulating blood neutrophils. The primed state can be induced by neutrophil adhesion, pro-inflammatory cytokines, lipopolysaccharide, and other agents and has been characterized as a "ready to go" state, which results in a faster and higher response upon exposure to a second stimulus. The active state is found at the inflammatory or infection site. Activation is induced by the pathogen itself or by pathogen-derived formylated peptides and other agents. Finally, inactivation of NADPH oxidase is induced by anti-inflammatory agents to limit inflammation. Priming is a "double-edged sword" process as it contributes to a rapid and efficient elimination of the pathogens but can also induce the generation of large quantities of toxic ROS by hyperactivation of the NADPH oxidase, which can damage surrounding tissues and participate to inflammation. In order to avoid extensive damage to host tissues, NADPH oxidase priming and activation must be tightly regulated. In this review, we will discuss some of the mechanisms of NADPH oxidase priming in neutrophils and the relevance of this process to physiology and pathology.

Mitochondrial damage is a well known cause of mitochondria-related diseases. A major mechanism underlying the development of mitochondria-related diseases is thought to be an increase in intracellular oxidative stress produced by impairment of the mitochondrial electron transport chain (ETC). However, clear evidence of intracellular free radical generation has not been clearly provided for mitochondrial DNA (mtDNA)-damaged cells. In this study, using the novel fluorescence dye, 2-[6-(4'-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (HPF), which was designed to detect hydroxyl radicals (*OH), intracellular free radical formation was examined in 143B cells (parental cells), 143B-rho(0) cells (mtDNA-lacking cells), 87 wt (cybrid), and cybrids of 4977-bp mtDNA deletion (common deletion) cells containing the deletion with 0%, 5%, 50% and >99% frequency (HeLacot, BH5, BH50 and BH3.12, respectively), using a laser confocal microscope detection method. ETC inhibitors (rotenone, 3-nitropropionic acid, thenoyltrifluoroacetone, antimycin A and sodium cyanide) were also tested to determine whether inhibitor treatment increased intracellular reactive oxygen species (ROS) generation. A significant increase in ROS for 143B-rho(0) cells was observed compared with 143B cells. However, for the 87 wt cybrid, no increase was observed. An increase was also observed in the mtDNA-deleted cells BH50 and BH3.12. The ETC inhibitors increased intracellular ROS in both 143B and 143B-rho(0) cells. Furthermore, in every fluorescence image, the fluorescence dye appeared localized around the nuclei. To clarify the localization, we double-stained cells with the dye and MitoTracker Red. The resulting fluorescence was consistently located in mitochondria. Furthermore, manganese superoxide dismutase (MnSOD) cDNA-transfected cells had decreased ROS. These results suggest that more ROS are generated from mitochondria in ETC-inhibited and mtDNA-damaged cells, which have impaired ETC.

During the last decades, eating out of home (OH) has gained importance in the diets worldwide. We document the nutritional characteristics of eating OH and its associations with energy intake, dietary quality and socioeconomic status. We carried out a systematic review of peer-reviewed studies in eight databases up to 10 March 2011. Of the 7,319 studies retrieved, 29 met the inclusion criteria and were analysed in this review. The quality of the data was assessed and a sensitivity analysis was conducted by isolating nationally representative or large cohort data from 6 and 11 countries, respectively. OH foods were important sources of energy in all age groups and their energy contribution increased in adolescents and young adults. Eating OH was associated with a higher total energy intake, energy contribution from fat in the daily diet and higher socioeconomic status. Two large studies showed how eating OH was also associated with a lower intake of micronutrients, particularly vitamin C, Ca and Fe. Although the studies were cross-sectional and heterogeneous in the way they classified eating OH, we conclude that eating OH is a risk factor for higher energy and fat intake and lower micronutrient intake.

Fibroblast growth factor (FGF)23 is a phosphaturic hormone that decreases circulating 1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] and elicits hypophosphatemia, both of which contribute to rickets/osteomalacia. It has been shown recently that serum FGF23 increases after treatment with renal 1,25(OH)(2)D(3) hormone, suggesting that 1,25(OH)(2)D(3) negatively feedback controls its levels by inducing FGF23. To establish the tissue of origin and the molecular mechanism by which 1,25(OH)(2)D(3) increases circulating FGF23, we administered 1,25(OH)(2)D(3) to C57BL/6 mice. Within 24 h, these mice displayed a dramatic elevation in serum immunoreactive FGF23, and the expression of FGF23 mRNA in bone was significantly upregulated by 1,25(OH)(2)D(3), but there was no effect in several other tissues. Furthermore, we treated rat UMR-106 osteoblast-like cells with 1,25(OH)(2)D(3), and real-time PCR analysis revealed a dose- and time-dependent stimulation of FGF23 mRNA concentrations. The maximum increase in FGF23 mRNA was 1,024-fold at 10(-7) M 1,25(OH)(2)D(3) after 24-h treatment, but statistically significant differences were observed as early as 4 h after 1,25(OH)(2)D(3) treatment. In addition, using cotreatment with actinomycin D or cycloheximide, we observed that 1,25(OH)(2)D(3) regulation of FGF23 gene expression occurs at the transcriptional level, likely via the nuclear vitamin D receptor, and is dependent on synthesis of an intermediary transfactor. These results indicate that bone is a major site of FGF23 expression and source of circulating FGF23 after 1,25(OH)(2)D(3) administration or physiological upregulation. Our data also establish FGF23 induction by 1,25(OH)(2)D(3) in osteoblasts as a feedback loop between these two hormones that completes a kidney-intestine-bone axis that mediates phosphate homeostasis.

We describe the opioid properties of a tridecapeptide, the sequence of which corresponds to the NH2-terminal sequence of dynorphin, a novel porcine pituitary endorphin. It contains [Leu]enkephalin. In the guinea pig ileum longitudinal muscle preparation it is about 700 times more potent than [Leu]enkephalin. Its effects in this tissue are blocked completely by naloxone, but the apparent affinity of naloxone is 1/13th that for blockade of [Leu]enkephalin or normorphine. In the mouse vas deferens, this peptide is 3 times more potent than [Leu]enkephalin. Well-washed rat brain membranes degrade the peptide rapidly, suggesting the presence of a membrane-bound degradative enzyme. The peptide displays considerable immunoreactivity in assays with antisera that have been used for the immunohistochemical localization of [Leu]enkephalin. The remarkable enhancement of the potency of [Leu]enkephalin by the COOH-terminal extension -Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys-OH suggests new interpretations concerning the structure of opiate receptors and the function of the enkephalin pentapeptides.

Metabolism of [3H]25-hydroxyvitamin D3(25-OH-D3) was studied in primary cultures of pulmonary alveolar macrophages (PAM) from seven patients with sarcoidosis and two patients with idiopathic pulmonary fibrosis. Production of a [3H]1,25-dihydroxyvitamin D3 (1,25-[OH]2-D3)-like metabolite of [3H]25-OH-D3 was detected in lipid extracts of cells from five patients with sarcoidosis. Synthesis of this compound in vitro was limited to viable PAM and was greatest in cells derived from a patient with hypercalcemia and an elevated serum concentration of 1,25-dihydroxyvitamin D. The tritiated PAM metabolite coeluted with authentic 1,25-(OH)2-D3 in three different solvent systems on straight-phase high performance liquid chromatography (HPLC) and demonstrated binding to extracted receptor for 1,25-(OH)2-D3, which was identical to that of commercially available [3H]1,25-(OH)2-D3 of comparable specific activity. Incubation of PAM with high concentrations of 25-OH-D3 resulted in production of an unlabeled metabolite that co-chromatographed with the 3H-PAM metabolite on HPLC and that was bound with high affinity by both the specific receptor for 1,25-(OH)2-D3 and antiserum to 1,25-(OH)2-D3.

Self-assembled monolayers (SAMs) of alkanethiols, which can provide flat and chemically well-defined surfaces, were employed as model surfaces to understand cellular interaction with artificial materials. SAMs presenting a wide range of wettabilities were prepared by mixing two kinds of alkanethiols carrying terminal methyl (CH(3)), hydroxyl (OH), carboxylic acid (COOH), or amino (NH(2)) groups. Adhesion behavior of human umbilical vein endothelial cells (HUVECs) and HeLa cells on these mixed SAMs were examined. The number of adhered HUVECs reached a maximum on CH(3)/OH mixed SAMs with a water contact angle of 40 degrees , while cell adhesion increased with decreasing water contact angle up to 60-70 degrees and then leveled off on CH(3)/COOH and CH(3)/NH(2) mixed SAMs. Numbers of adhered HeLa cells showed a maximum on CH(3)/OH and CH(3)/COOH mixed SAMs with a water contact angle of 50 degrees . These facts suggest that cell adhesion is mainly determined by surface wettability, but is also affected by the surface functional group, its surface density, and the kinds of cells. The effect of exchange of adsorbed proteins on cell adhesion was also examined. HUVECs were cultured on the mixed SAMs preadsorbed with albumin. Cell adhesion was effectively prohibited on hydrophobic SAMs pretreated with albumin. Albumin strongly adsorbed and resisted replacement by cell adhesive proteins on hydrophobic SAMs. On the other hand, cells adhered to albumin-adsorbed hydrophilic SAMs. Displacement of preadsorbed albumin with cell adhesive proteins effectively occurs on these hydrophilic SAMs. This effect contributes to induce SAMs with moderate wettability to give suitable surfaces for cell adhesion.

Vitamin D functions are not limited to skeletal health benefits and may extend to preservation of insulin secretion and insulin sensitivity. This review summarizes the literature related to potential vitamin D influences on glucose homeostasis and insulin sensitivity. Cross-sectional data provide some evidence that circulating 25-hydroxyvitamin D (25(OH)D) is inversely associated with insulin resistance, although direct measurements of insulin sensitivity are required for confirmation. Reported associations with insulin secretion, however, are contradictory. Available prospective studies support a protective influence of high 25(OH)D concentrations on type 2 diabetes mellitus risk. There is a general lack of consistency in vitamin D intervention outcomes on insulin secretion and sensitivity, likely due to differences in subject populations, length of interventions, and forms of vitamin D supplementation. Vitamin D receptor gene polymorphisms and vitamin D interactions with the insulin like growth factor system may further influence glucose homeostasis. The ambiguity of optimal vitamin D dosing regimens and optimal therapeutic concentrations of serum 25(OH)D limit available intervention studies. Future studies, including cross-sectional and prospective, should be performed in populations at high risk for both vitamin D deficiency and type 2 diabetes mellitus. Well-designed, placebo-controlled, randomized intervention studies are required to establish a true protective influence of vitamin D on glucose homeostasis.

OBJECTIVE

To determine whether vitamin D supplementation decreases the incidence of hip fractures and other peripheral bone fractures.

METHODS

Prospective, double-blind trial.

METHODS

Community setting (Amsterdam and surrounding area).

METHODS

2578 persons (1916 women, 662 men) 70 years of age and older (mean age +/- SD, 80 +/- 6 years) living independently, in apartments for elderly persons, or in homes for elderly persons.

METHODS

Participants were randomly assigned to receive either vitamin D3, 400 IU in one tablet daily, or placebo for a maximum of 3.5 years.

METHODS

Dietary calcium intake and serum 25-hydroxyvitamin D [25(OH)D] were estimated in a subset of participants. During follow-up, attention was concentrated on hip fractures and other peripheral fractures. The maximal follow-up period was 4 years. The results were evaluated by survival analysis.

RESULTS

Mean dietary calcium intake from dairy products was 868 mg/d. Mean serum 25(OH)D concentration in the third year of the study was 23 nmol/L in the placebo group and 60 nmol/L in the vitamin D group. Median follow-up was 3.5 years, and total follow-up was 8450 patient-years. During follow-up, 306 persons in the placebo group and 282 persons in the vitamin D group died (P = 0.20). Hip fractures occurred in 48 persons in the placebo group and 58 persons in the vitamin D group (P = 0.39, intention-to-treat analysis). Other peripheral fractures occurred in 74 persons in the placebo group and 77 persons in the vitamin D group (P = 0.86).

CONCLUSIONS

Our results do not show a decrease in the incidence of hip fractures and other peripheral fractures in Dutch elderly persons after vitamin D supplementation.

Arabidopsis ecotype Columbia (Col-0) seedlings, transformed with a phenylalanine ammonia-lyase 1 promoter (PAL1)-[beta]-glucuronidase (GUS) reporter construct, were inoculated with virulent and avirulent isolates of Peronospora parasitica. The PAL1 promoter was constitutively active in the light in vascular tissue but was induced only in the vicinity of fungal structures in the incompatible interaction. A double-staining procedure was developed to distinguish between GUS activity and fungal structures. The PAL1 promoter was activated in cells undergoing lignification in the incompatible interaction in response to the pathogen. Pretreatment of the seedlings with 2-aminoindan-2-phosphonic acid (AIP), a highly specific PAL inhibitor, made the plants completely susceptible. Lignification was suppressed after AIP treatment, and surprisingly, pathogen-induced PAL1 promoter activity could not be detected. Treatment of the seedlings with 2-hydroxyphenylaminosulphinyl acetic acid (1,1-dimethyl ester) (OH-PAS), a cinnamyl alcohol dehydrogenase inhibitor specific for the lignification pathway, also caused a shift toward susceptibility, but the effect was not as pronounced as it was with AIP. Significantly, although OH-PAS suppressed pathogen-induced lignification, it did not suppress pathogen-induced PAL1 promoter activation. Salicylic acid (SA), supplied to AIP-treated plants, restored resistance and both pathogen-induced lignification and activation of the PAL1 promoter. Endogenous SA levels increased significantly in the incompatible but not in the compatible combination, and this increase was suppressed by AIP but not by OH-PAS. These results provide evidence of the central role of SA in genetically determined plant disease resistance and show that lignification per se, although providing a component of the resistance mechanism, is not the deciding factor between resistance and susceptibility.

Forebrain serotonergic lesions attenuate the ability of d-amphetamine to decrease impulsivity in a delay-discounting paradigm, potentially through interactions between the serotonin (5-HT) and dopamine (DA) systems. Nucleus accumbens (NAC) lesions increase impulsivity, but the extent to which accumbal DA is involved in regulating impulsive choice is unknown. In the current study, the effects of intra-accumbal infusions of 6-hydroxydopamine (6-OHDA) on impulsive choice were evaluated, in combination with d-amphetamine and serotonergic drugs, in order to investigate the importance of 5-HT : DA interactions in the control of impulsive behavior. Following training on a delay-discounting task, animals received intra-NAC 6-OHDA or sham surgery. Postoperatively, subjects received systemic injections of d-amphetamine (0, 0.3, 1.0, 1.5 mg/kg) and the 5-HT(1A) receptor agonist 8-OH-DPAT (0, 0.1, 0.3, 1.0 mg/kg). Intra-NAC 6-OHDA, which reduced local DA and NA levels by 70-75%, had no effect on delay-discounting, but transiently potentiated the d-amphetamine-induced decrease in impulsive choice. 8-OH-DPAT (1.0 mg/kg) increased impulsivity in sham-operated controls, an effect which was blocked by the 5-HT(1A) receptor antagonist WAY 100635. However, 8-OH-DPAT had no effect on impulsivity in 6-OHDA NAC lesioned rats. 8-OH-DPAT (0.3 mg/kg), which did not itself alter task performance, blocked the effect of d-amphetamine in sham-operated controls, while WAY 100635 augmented the effect of amphetamine in all subjects. In an additional experiment, intracerebroventricular administration of the selective serotonergic toxin 5,7-dihydroxytryptamine, which decreased forebrain 5-HT levels by 85-90%, did not block 8-OH-DPAT's ability to increase impulsive choice. These data suggest a significant role for 5-HT : DA interactions within the NAC in the control of impulsivity, and in the mechanism by which amphetamine decreases impulsive choice.

OBJECTIVE

Higher plasma 25-hydroxyvitamin D(3) (25(OH)D) levels are associated with a decreased incidence of colorectal cancer, but the influence of plasma 25(OH)D on the outcome of patients with established colorectal cancer is unknown.

METHODS

We prospectively examined the association between prediagnosis 25(OH)D levels and mortality among 304 participants in the Nurses' Health Study (NHS) and the Health Professionals Follow-Up Study (HPFS) who were diagnosed with colorectal cancer from 1991 to 2002. Participants diagnosed within 2 years of blood collection were excluded. Patients were observed until death, June 2005 (NHS), or January 2005 (HPFS), whichever came first. The primary end point was overall mortality. Cox proportional hazards models were used to calculate hazard ratios (HR) adjusted for other risk factors for cancer survival.

RESULTS

Higher plasma 25(OH)D levels were associated with a significant reduction in overall mortality (P for trend = .02). Compared with the lowest quartile, participants in the highest quartile had an adjusted HR of 0.52 (95% CI, 0.29 to 0.94) for overall mortality. A trend toward improved colorectal cancer-specific mortality was also seen (HR = 0.61; 95% CI, 0.31 to 1.19). The results remained unchanged after excluding patients diagnosed within 5 years of blood collection (P for trend = .04); the multivariate HR for overall mortality comparing extreme quartiles was 0.45 (95% CI, 0.19 to 1.09).

CONCLUSIONS

Among patients with colorectal cancer, higher prediagnosis plasma 25(OH)D levels were associated with a significant improvement in overall survival. Further study of the vitamin D pathway and its influence on colorectal carcinogenesis and cancer progression is warranted.

BACKGROUND

Patients with asthma exhibit variable response to inhaled corticosteroids (ICS). Vitamin D is hypothesized to exert effects on phenotype and glucocorticoid (GC) response in asthma.

OBJECTIVE

To determine the effect of vitamin D levels on phenotype and GC response in asthma.

METHODS

Nonsmoking adults with asthma were enrolled in a study assessing the relationship between serum 25(OH)D (vitamin D) concentrations and lung function, airway hyperresponsiveness (AHR), and GC response, as measured by dexamethasone-induced expression of mitogen-activated protein kinase phosphatase (MKP)-1 by peripheral blood mononuclear cells.

RESULTS

A total of 54 adults with asthma (FEV(1), 82.9 +/- 15.7% predicted [mean +/- SD], serum vitamin D levels of 28.1 +/- 10.2 ng/ml) were enrolled. Higher vitamin D levels were associated with greater lung function, with a 22.7 (+/-9.3) ml (mean +/- SE) increase in FEV(1) for each nanogram per milliliter increase in vitamin D (P = 0.02). Participants with vitamin D insufficiency (<30 ng/ml) demonstrated increased AHR, with a provocative concentration of methacholine inducing a 20% fall in FEV(1) of 1.03 (+/-0.2) mg/ml versus 1.92 (+/-0.2) mg/ml in those with vitamin D of 30 ng/ml or higher (P = 0.01). In ICS-untreated participants, dexamethasone-induced MKP-1 expression increased with higher vitamin D levels, with a 0.05 (+/-0.02)-fold increase (P = 0.02) in MKP-1 expression observed for each nanogram per milliliter increase in vitamin D, a finding that occurred in the absence of a significant increase in IL-10 expression.

CONCLUSIONS

In asthma, reduced vitamin D levels are associated with impaired lung function, increased AHR, and reduced GC response, suggesting that supplementation of vitamin D levels in patients with asthma may improve multiple parameters of asthma severity and treatment response. Clinical trials registered with www.clinicaltrials.gov (NCT00495157, NCT00565266, and NCT00557180).