The stability of a new type of DNA mimic, peptide nucleic acid (PNA) in human blood serum, Eschericia coli and Micrococcus luteus extracts and nuclear and cytoplasmic extracts from mouse Ehrlich ascites tumor cells was investigated using HPLC analysis. Under conditions that caused complete cleavage of a control peptide, adrenocorticotropic hormone fragment 4-10, no significant degradation of the PNAs, H-T10-LysNH2 and H-TGTACGTCACAACTA-NH2, could be detected. Similarly, PNA H-T5-LysNH2 was found to resist attack by fungal proteinase K or porcine intestinal mucosa peptidase at concentrations exceeding those necessary to completely degrade a control peptide, H-Phe-Trp-Tyr-Cys-Phe-Trp-Tyr-Lys-Phe-Trp-Tyr-Lys-OH, by at least 1000- and 30-fold, respectively. Thus PNA is expected to have sufficient biostability to be used as a drug.

Activated B and T lymphocytes from normal human subjects are known to have the specific high-affinity receptor for 1,25-dihydroxyvitamin D3 (1,25-(OH)2-D3). In an attempt to determine a functional role for the sterol in such cells, we studied the effect of 1,25-(OH)2-D3 on DNA synthesis and Ig production by normal human peripheral blood mononuclear (PBM) cells activated in vitro by the polyclonal lymphocyte activators pokeweed mitogen and phytohemagglutinin, and the specific antigen dermatophyton O. A dose-dependent inhibition of [3H]thymidine incorporation was observed in cells incubated with 1,25-(OH)2-D3 in concentrations ranging from 10(-10) to 10(-7) M. Production of IgG and IgM, determined by enzyme-linked immunosorbent assay, was similarly inhibited by increasing concentrations of 1,25-(OH)2-D3. Half-maximal inhibition of DNA and Ig synthesis was found at 10(-10) to 10(-9) M 1,25-(OH)2-D3. This suppressive effect was specific for 1,25-(OH)2-D3; of the other vitamin D metabolites examined, only 10(-7) M 24R,25 dihydroxyvitamin D3 (24,25-(OH)2-D3) had a similar inhibitory effect. 1,25-(OH)2-D3 was not cytotoxic and did not affect unactivated PBMs. These data demonstrate that 1,25-(OH)2-D3 is a potent inhibitor of human PBM Ig production in vitro and suggest that this action is mediated through the hormone's antiproliferative effect on Ig-producing B cells and/or helper T cells.

Chemokines trigger rapid integrin-dependent lymphocyte arrest to vascular endothelium. We show that the chemokines SLC, ELC, and SDF-1alpha rapidly induce lateral mobility and transient increase of affinity of the beta2 integrin LFA-1. Inhibition of phosphatidylinositol 3-OH kinase (PI(3)K) activity blocks mobility but not affinity changes and prevents lymphocyte adhesion to ICAM-1 immobilized at low but not high densities, suggesting that mobility enhances the frequency of encounters between high-affinity integrin and ligand but that at higher ligand density affinity changes are sufficient for arrest. Thus, chemokines trigger, through distinct signaling pathways, both a high-affinity state and lateral mobility of LFA-1 that can coordinately determine the vascular arrest of circulating lymphocytes under physiologic conditions.

The human immunodeficiency virus (HIV) replicates its genome and mutates at exceptionally high rates. As a result, the virus is able to evade immunological and chemical antiviral agents. We tested the hypothesis that a further increase in the mutation rate by promutagenic nucleoside analogs would abolish viral replication. We evaluated deoxynucleoside analogs for lack of toxicity to human cells, incorporation by HIV reverse transcriptase, resistance to repair when incorporated into the DNA strand of an RNA.DNA hybrid, and mispairing at high frequency. Among the candidates tested, 5-hydroxydeoxycytidine (5-OH-dC) fulfilled these criteria. In seven of nine experiments, the presence of this analog resulted in the loss of viral replicative potential after 9-24 sequential passages of HIV in human CEM cells. In contrast, loss of viral replication was not observed in 28 control cultures passaged in the absence of the nucleoside analog, nor with other analogs tested. Sequence analysis of a portion of the HIV reverse transcriptase gene demonstrated a disproportionate increase in G ->> A substitutions, mutations predicted to result from misincorporation of 5-OH-dC into the cDNA during reverse transcription. Thus, "lethal mutagenesis" driven by the class of deoxynucleoside analogs represented by 5-OH-dC could provide a new approach to treating HIV infections and, potentially, other viral infections.

1. Xanthine oxidase acting aerobically upon acetaldehyde was found to cause the peroxidation of linolenate. This was demonstrated by increased absorbance at 233 nm due to diene conjugation and by the detection of a lipid peroxide spot on the thin layer chromatograms. 2. Superoxide dismutase inhibited this lipid peroxidation, as did catalase, thus indicating that both O2- and H2O2 were essential intermediates. Scavengers of singlet oxygen also inhibited the peroxidation of linolenate, whereas scavengers of hydroxyl radical did not. These effects, which were observed in the absence of iron salts, led to the proposal that O2- and H2O2 can directly give rise to a singlet oxygen, as follows: O2- + H2O2 leads to OH- + OH. + O2. 3. This proposal was further supported through the use of 2,5-dimethylfuran, as an indicating scavenger of singlet oxygen. Thus, when this compound was exposed to a known source of singlet oxygen, it gave a product which was detectable by thin layer chromatography. This product was also observed when 2,5-dimethylfuran was exposed to the xanthine oxidase system, in which case its accumulation was prevented by superoxide dismutase or by catalase, but not by scavengers of hydroxyl radical.

BACKGROUND

We examined the relationship between overweight in preschool children and three environmental factors--the proximity of the children's residences to playgrounds and to fast food restaurants and the safety of the children's neighborhoods. We hypothesized that children who lived farther from playgrounds, closer to fast food restaurants, and in unsafe neighborhoods were more likely to be overweight.

METHODS

This was a cross-sectional study of 7,020 low-income children, 36 through 59 months of age living in Cincinnati, OH. Overweight was defined as a measured body mass index>> or =95th percentile. The distance between each child's residence and the nearest public playground and fast food restaurant was determined with geographic information systems. Neighborhood safety was defined by the number of police-reported crimes per 1,000 residents per year in each of 46 city neighborhoods.

RESULTS

Overall, 9.2% of the children were overweight, 76% black, and 23% white. The mean (+/- SD) distances from a child's home to the nearest playground and fast food restaurant were 0.31 (+/- 0.22) and 0.70 (+/- 0.38) miles, respectively. There was no association between child overweight and proximity to playgrounds, proximity to fast food restaurants, or level of neighborhood crime. The association between child overweight and playground proximity did not differ by neighborhood crime level.

CONCLUSIONS

Within a population of urban low-income preschoolers, overweight was not associated with proximity to playgrounds and fast food restaurants or with the level of neighborhood crime.

Butanol is an aliphatic saturated alcohol having the molecular formula of C(4)H(9)OH. Butanol can be used as an intermediate in chemical synthesis and as a solvent for a wide variety of chemical and textile industry applications. Moreover, butanol has been considered as a potential fuel or fuel additive. Biological production of butanol (with acetone and ethanol) was one of the largest industrial fermentation processes early in the 20th century. However, fermentative production of butanol had lost its competitiveness by 1960s due to increasing substrate costs and the advent of more efficient petrochemical processes. Recently, increasing demand for the use of renewable resources as feedstock for the production of chemicals combined with advances in biotechnology through omics, systems biology, metabolic engineering and innovative process developments is generating a renewed interest in fermentative butanol production. This article reviews biotechnological production of butanol by clostridia and some relevant fermentation and downstream processes. The strategies for strain improvement by metabolic engineering and further requirements to make fermentative butanol production a successful industrial process are also discussed.

The mechanism of action of macrophage colony-stimulating factor (M-CSF) in osteoclast development was examined in a co-culture system of mouse osteoblastic cells and spleen cells. In this co-culture, osteoclast-like multinucleated cells (MNCs) were formed within 6 d in response to 10 nM 1 alpha,25(OH)2D3 added only for the final 2 d of culture. Simultaneously adding hydroxyurea for the final 2 d completely inhibited proliferation of cultured cells without affecting 1 alpha,25(OH)2D3-stimulated MNC formation. Autoradiographic examination using [3H]-thymidine revealed that osteoclast progenitors primarily proliferated during the first 4 d, whereas their differentiation into MNCs occurred predominantly during the final 2 d of culture in response to 1 alpha,25(OH)2D3. When anti-M-CSF antibody or anti-M-CSF receptor antibody was added either for the first 4 d or for the final 2 d, the MNC formation was similarly inhibited. In co-cultures of normal spleen cells and osteoblastic cells obtained from op/op mice, which cannot produce functionally active M-CSF, the lack of M-CSF either for the first 4 d or for the final 2 d failed to form MNCs in response to 1 alpha,25(OH)2D3 added for the last 2 d. These results clearly indicate that M-CSF is indispensable for both proliferation of osteoclast progenitors and their differentiation into mature osteoclasts.

OBJECTIVE

Adjuvant chemotherapy improves disease-free survival (DFS) for patients with osteogenic sarcoma (OS). We reviewed our experience with OS to determine prognostic factors, the role of preoperative chemotherapy and subsequent histologic response, and the role of salvage chemotherapy after poor initial response.

METHODS

From 1975 to 1984, we saw 279 patients with previously untreated OS without metastasis. All patients received intensive chemotherapy and underwent surgical resection of primary tumor. Chemotherapy included high-dose methotrexate; Adriamycin (doxorubicin; Adria Laboratories, Columbus, OH); and bleomycin, cyclophosphamide, and dactinomycin (BCD). Selected patients also received cisplatin.

RESULTS

DFS was not affected by use of preoperative chemotherapy versus immediate surgery, by use of limb-sparing surgery versus amputation, age, sex, or dose intensity of chemotherapy. DFS did correlate with serum lactate dehydrogenase (LDH), alkaline phosphatase, primary tumor site, race, and histologic response to preoperative chemotherapy. There was no difference in DFS for patients with a poor histologic response who did or did not receive cisplatin, although patients who did receive cisplatin had a longer time to relapse. The 5-year DFS was 76% for patients aged less than or equal to 21 years who had extremity primary tumor and were treated with the T10 protocol.

CONCLUSIONS

Intensive chemotherapy can achieve DFS for a high proportion of patients with OS. Although it is a powerful predictor of DFS, histologic response to preoperative chemotherapy cannot be assessed at diagnosis. We have not shown an ability to salvage patients with an unfavorable response. We need to increase the proportion of patients with a favorable response, identify the patients who will have an unfavorable response, and develop novel treatments to salvage poor responders.

25-Hydroxyvitamin D (25[OH]D) can potentially interfere with inflammatory response and fibrogenesis. Its role in disease progression in chronic hepatitis C (CHC) and its relation with histological and sustained virological response (SVR) to therapy are unknown. One hundred ninety-seven patients with biopsy-proven genotype 1 (G1) CHC and 49 healthy subjects matched by age and sex were consecutively evaluated. One hundred sixty-seven patients underwent antiviral therapy with pegylated interferon plus ribavirin. The 25(OH)D serum levels were measured by high-pressure liquid chromatography. Tissue expression of cytochrome (CY) P27A1 and CYP2R1, liver 25-hydroxylating enzymes, were assessed by immunochemistry in 34 patients with CHC, and in eight controls. The 25(OH)D serum levels were significantly lower in CHC than in controls (25.07 +/- 9.92 microg/L versus 43.06 +/- 10.19; P < 0.001). Lower levels of 25(OH)D were independently linked to female sex (P = 0.007) and necroinflammation (P = 0.04) by linear regression analysis. CYP27A1, but not CYP2R1, was directly related to 25(OH)D levels (P = 0.01), and inversely to necroinflammation (P = 0.01). Low 25(OH)D (odds ratio [OR], 0.942; 95% confidence interval [CI], 0.893-0.994) and cholesterol (OR, 0.981; 95%CI, 0.969-0.992) levels, older age (OR, 1.043; 95%CI, 1.002-1.085), high ferritin (OR, 1.003; 95%CI, 1.001-1.005), and necroinflammation (OR, 2.235; 95%CI, 1.014-4.929) were independently associated with severe fibrosis (F3-F4) by multivariate logistic analysis. Seventy patients (41%) achieved SVR. By multivariate analysis, hepatic steatosis (OR, 0.971; 95%CI, 0.944-0.999), lower cholesterol (OR, 1.009; 95% CI, 1.000-1.018), and 25(OH)D levels (OR, 1.039; 95%CI, 1.002-1.077) were independently associated with no SVR.

CONCLUSIONS

G1 CHC patients had low 25(OH)D serum levels, possibly because of reduced CYP27A1 expression. Low vitamin D is linked to severe fibrosis and low SVR on interferon (IFN)-based therapy.

The balance between oxidation and antioxidation is believed to be critical in maintaining healthy biological systems. Under physiological conditions, the human antioxidative defense system including e.g., superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione (GSH) and others, allows the elimination of excess reactive oxygen species (ROS) including, among others superoxide anions (O2(·-)), hydroxyl radicals (OH·), alkoxyl radicals (RO·) and peroxyradicals (ROO·). However, our endogenous antioxidant defense systems are incomplete without exogenous originating reducing compounds such as vitamin C, vitamin E, carotenoids and polyphenols, playing an essential role in many antioxidant mechanisms in living organisms. Therefore, there is continuous demand for exogenous antioxidants in order to prevent oxidative stress, representing a disequilibrium redox state in favor of oxidation. However, high doses of isolated compounds may be toxic, owing to prooxidative effects at high concentrations or their potential to react with beneficial concentrations of ROS normally present at physiological conditions that are required for optimal cellular functioning. This review aims to examine the double-edged effects of dietary originating antioxidants with a focus on the most abundant compounds, especially polyphenols, vitamin C, vitamin E and carotenoids. Different approaches to enrich our body with exogenous antioxidants such as via synthetic antioxidants, diets rich in fruits and vegetables and taking supplements will be reviewed and experimental and epidemiological evidences discussed, highlighting that antioxidants at physiological doses are generally safe, exhibiting interesting health beneficial effects.

Proton chemical shifts of a series of disordered linear peptides (H-Gly-Gly-X-Gly-Gly-OH, with X being one of the 20 naturally occurring amino acids) have been obtained using 1D and 2D 1H NMR at pH 5.0 as a function of temperature and solvent composition. The use of 2D methods has allowed some ambiguities in side-chain assignments in previous studies to be resolved. An additional benefit of the temperature data is that they can be used to obtain 'random coil' amide proton chemical shifts at any temperature between 278 and 318 K by interpolation. Changes of chemical shift as a function of trifluoroethanol concentration have also been determined at a variety of temperatures for a subset of peptides. Significant changes are found in backbone and side-chain amide proton chemical shifts in these 'random coil' peptides with increasing amounts of trifluoroethanol, suggesting that caution is required when interpreting chemical shift changes as a measure of helix formation in peptides in the presence of this solvent. Comparison of the proton chemical shifts obtained here for H-Gly-Gly-X-Gly-Gly-OH with those for H-Gly-Gly-X-Ala-OH [Bundi, A. and Wüthrich, K., (1979) Biopolymers, 18, 285-297] and for Ac-Gly-Gly-X-Ala-Gly-Gly-NH2 [Wishart, D.S., Bigam, C.G., Holm, A., Hodges, R.S. and Sykes, B.D. (1995) J. Biomol. NMR, 5, 67-81] generally shows good agreement for CH protons, but reveals significant variability for NH protons. Amide proton chemical shifts appear to be highly sensitive to local sequence variations and probably also to solution conditions. Caution must therefore be exercised in any structural interpretation based on amide proton chemical shifts.

As a dendritic cell (DC) matures, it becomes more potent as an antigen-presenting cell. This functional change is accompanied by a change in DC immunophenotype. The signal transduction events underlying this process are poorly characterized. In this study, we have investigated the signal transduction pathways involved in the lipopolysaccharide (LPS)-induced maturation of human monocyte-derived DCs (MoDCs) in vitro. We show that exposure of immature MoDCs to LPS activates the p38 stress-activated protein kinase (p38SAPK), extracellular signal-regulated protein kinase (ERK), phosphoinositide 3-OH kinase (PI3 kinase)/Akt, and nuclear factor (NF)-kappaB pathways. Studies using inhibitors demonstrate that PI3 kinase/Akt but not the other pathways are important in maintaining survival of LPS-stimulated MoDCs. Inhibiting p38SAPK prevented activation of the transcription factors ATF-2 and CREB and significantly reduced the LPS-induced up-regulation of CD80, CD83, and CD86, but did not have any significant effect on the LPS-induced changes in macropinocytosis or HLA-DR, CD40, and CD1a expression. Inhibiting the NF-kappaB pathway significantly reduced the LPS-induced up-regulation of HLA-DR as well as CD80, CD83, and CD86. Inhibiting the p38SAPK and NF-kappaB pathways simultaneously had variable effects depending on the cell surface marker studied. It thus appears that different aspects of LPS-induced MoDC maturation are regulated by different and sometimes overlapping pathways.

The chemical diversity of antioxidants makes it difficult to separate and quantify antioxidants from the vegetable matrix. Therefore, it is desirable to establish a method that can measure the total antioxidant activity level directly from vegetable extracts. The current literature clearly states that there is no "total antioxidant" as a nutritional index available for food labeling because of the lack of standard quantitation methods. Thus, this work reports the development of a simple, widely applicable antioxidant capacity index for dietary polyphenols and vitamins C and E, utilizing the copper(II)-neocuproine [Cu(II)-Nc] reagent as the chromogenic oxidizing agent. Because the copper(II) (or cupric) ion reducing ability of polyphenols is measured, the method is named by our research group "cupric reducing antioxidant capacity" abbreviated as the CUPRAC method. This method should be advantageous over the ferric reducing antioxidant power (FRAP) method because the redox chemistry of copper(II)-as opposed to that of ferric ion-involves faster kinetics. The method comprises mixing of the antioxidant solution (directly or after acid hydrolysis) with a copper(II) chloride solution, a neocuproine alcoholic solution, and an ammonium acetate aqueous buffer at pH 7 and subsequent measurement of the developed absorbance at 450 nm after 30 min. Because the color development is fast for compounds such as ascorbic acid, gallic acid, and quercetin but slow for naringin and naringenin, the latter compounds were assayed after incubation at 50 degrees C on a water bath for 20 min [after Cu(II)-Nc reagent addition] so as to force the oxidation reaction to reach completion. The flavonoid glycosides were hydrolyzed to their corresponding aglycons by refluxing in 1.2 M HCl-containing 50% MeOH so as to exert maximal reducing power toward Cu(II)-Nc. Certain compounds also needed incubation after acid hydrolysis to fully exhibit their reducing capability. The CUPRAC antioxidant capacities of synthetic mixtures of antioxidants were experimentally measured as Trolox equivalents and compared to those theoretically found by making use of the principle of additivity of absorbances assuming no chemical interaction between the mixture constituents. Because ascorbic acid is not resistant to elevated temperature incubation, it should be assayed initially by measuring the absorbance (at 450 nm) difference of original and ascorbate oxidase-added mixture solutions at the end of 1 min of Cu(II)-Nc reagent addition. Thus, the total CUPRAC antioxidant capacity of a mixture containing various antioxidants should be that finally measured after a suitable combination of hydrolysis and incubation procedures, added to the initially measured capacity due to ascorbate. The antioxidant polyphenolic compounds tested demonstrate that the highest capacities in the CUPRAC method were observed for epicatechin gallate, epigallocatechin gallate, quercetin, fisetin, epigallocatechin, catechin, and caffeic acid in this order, in accordance with theoretical expectations, because the number and position of the hydroxyl groups as well as the degree of conjugation of the whole molecule are important. The antioxidant potency of flavonoids is nearly proportional to the total number of -OH groups and is positively affected by the presence of an o-dihydroxy moiety in the B-ring. beta-Carotene, which did not react with the CUPRAC reagent in alcoholic aqueous medium, could be assayed in dichloromethane solvent. Linear calibration curves for ascorbic acid and flavonoids were redrawn in synthetic solutions containing a mixture of antioxidants, and also in real matrices such as grape and orange juices, green tea, and blackberry tea, showing an initial nonzero absorbance with the CUPRAC reagent. The parallellism of the linear calibration curves of pure compounds in a given complex matrix effectively demonstrated that there were no interferent chemical interactions among the solution constituents and that the antioxidant capacities of the tested antioxidants were additive. The CUPRAC reagent is reasonably selective, stable, easily accessible, and sensitive toward thiol-type oxidants, unlike the FRAP method. The reaction is carried out at nearly physiological pH as opposed to the unrealistic acidic pH of FRAP.

Antibacterial activity of zinc oxide nanoparticles (ZnO-NPs) has received significant interest worldwide particularly by the implementation of nanotechnology to synthesize particles in the nanometer region. Many microorganisms exist in the range from hundreds of nanometers to tens of micrometers. ZnO-NPs exhibit attractive antibacterial properties due to increased specific surface area as the reduced particle size leading to enhanced particle surface reactivity. ZnO is a bio-safe material that possesses photo-oxidizing and photocatalysis impacts on chemical and biological species. This review covered ZnO-NPs antibacterial activity including testing methods, impact of UV illumination, ZnO particle properties (size, concentration, morphology, and defects), particle surface modification, and minimum inhibitory concentration. Particular emphasize was given to bactericidal and bacteriostatic mechanisms with focus on generation of reactive oxygen species (ROS) including hydrogen peroxide (H2O2), OH- (hydroxyl radicals), and O2 -2 (peroxide). ROS has been a major factor for several mechanisms including cell wall damage due to ZnO-localized interaction, enhanced membrane permeability, internalization of NPs due to loss of proton motive force and uptake of toxic dissolved zinc ions. These have led to mitochondria weakness, intracellular outflow, and release in gene expression of oxidative stress which caused eventual cell growth inhibition and cell death. In some cases, enhanced antibacterial activity can be attributed to surface defects on ZnO abrasive surface texture. One functional application of the ZnO antibacterial bioactivity was discussed in food packaging industry where ZnO-NPs are used as an antibacterial agent toward foodborne diseases. Proper incorporation of ZnO-NPs into packaging materials can cause interaction with foodborne pathogens, thereby releasing NPs onto food surface where they come in contact with bad bacteria and cause the bacterial death and/or inhibition.

Ribozymes enhance chemical reaction rates using many of the same catalytic strategies as protein enzymes. In the hepatitis delta virus (HDV) ribozyme, site-specific self-cleavage of the viral RNA phosphodiester backbone requires both divalent cations and a cytidine nucleotide. General acid-base catalysis, substrate destabilization and global and local conformational changes have all been proposed to contribute to the ribozyme catalytic mechanism. Here we report ten crystal structures of the HDV ribozyme in its pre-cleaved state, showing that cytidine is positioned to activate the 2'-OH nucleophile in the precursor structure. This observation supports its proposed role as a general base in the reaction mechanism. Comparison of crystal structures of the ribozyme in the pre- and post-cleavage states reveals a significant conformational change in the RNA after cleavage and that a catalytically critical divalent metal ion from the active site is ejected. The HDV ribozyme has remarkable chemical similarity to protein ribonucleases and to zymogens for which conformational dynamics are integral to biological activity. This finding implies that RNA structural rearrangements control the reactivity of ribozymes and ribonucleoprotein enzymes.

OBJECTIVE

To conduct a systematic review of prospective studies assessing the association of vitamin D intake or blood levels of 25-hydroxyvitamin D [25(OH)D] with the risk of colorectal cancer using meta-analysis.

METHODS

Relevant studies were identified by a search of MEDLINE and EMBASE databases before October 2010 with no restrictions. We included prospective studies that reported relative risk (RR) estimates with 95% CIs for the association between vitamin D intake or blood 25(OH)D levels and the risk of colorectal, colon, or rectal cancer. Approximately 1,000,000 participants from several countries were included in this analysis.

RESULTS

Nine studies on vitamin D intake and nine studies on blood 25(OH)D levels were included in the meta-analysis. The pooled RRs of colorectal cancer for the highest versus lowest categories of vitamin D intake and blood 25(OH)D levels were 0.88 (95% CI, 0.80 to 0.96) and 0.67 (95% CI, 0.54 to 0.80), respectively. There was no heterogeneity among studies of vitamin D intake (P = .19) or among studies of blood 25(OH)D levels (P = .96). A 10 ng/mL increment in blood 25(OH)D level conferred an RR of 0.74 (95% CI, 0.63 to 0.89).

CONCLUSIONS

Vitamin D intake and blood 25(OH)D levels were inversely associated with the risk of colorectal cancer in this meta-analysis.

RNA hairpin loops containing a GNRA consensus sequence are the most frequently occurring hairpins in a variety of prokaryotic and eukaryotic RNAs. These tetraloops play important functional roles in RNA folding, in RNA-RNA tertiary interactions and as protein binding sites. Homo and heteronuclear NMR spectroscopy have been used to determine the structures of the most abundant members of the GNRA tetraloop family: the GAGA, GCAA and GAAA loops closed by a C-G base pair. Analysis of the structures of these three hairpin loops reveals a network of heterogeneous hydrogen bonds. The loops contain a G-A base pair, a G base-phosphate hydrogen bond and several 2' OH-base hydrogen bonds. These intramolecular interactions and the extensive base stacking in the loop help explain the high thermodynamic stability and give insight into the diverse biological roles of the GNRA RNA hairpins.

Rickets and hyperparathyroidism caused by a defective vitamin D receptor (VDR) can be prevented in humans and animals by high calcium intake, suggesting that intestinal calcium absorption is critical for 1,25(OH)(2) vitamin D [1,25(OH)(2)D(3)] action on calcium homeostasis. We assessed the rate of serum (45)Ca accumulation within 10 min of oral gavage in two strains of VDR-knockout (KO) mice (Leuven and Tokyo KO) and observed a 3-fold lower area under the curve in both KO strains. Moreover, we evaluated the expression of intestinal candidate genes involved in transcellular calcium transport. The calcium transport protein1 (CaT1) was more abundantly expressed at mRNA level than the epithelial calcium channel (ECaC) in duodenum, but both were considerably reduced (CaT1>90%, ECaC>60%) in the two VDR-KO strains on a normal calcium diet. Calbindin-D(9K) expression was decreased only in the Tokyo KO, whereas plasma membrane calcium ATPase (PMCA(1b)) expression was normal in both VDR-KOs. In Leuven wild-type mice, a high calcium diet inhibited (>90%) and 1,25(OH)(2)D(3) injection or low calcium diet induced (6-fold) duodenal CaT1 expression and, to a lesser degree, ECaC and calbindin-D(9K) expression. In Leuven KO mice, however, high or low calcium intake decreased calbindin-D(9K) and PMCA(1b) expression, whereas CaT1 and ECaC expression remained consistently low on any diet. These results suggest that the expression of the novel duodenal epithelial calcium channels (in particular CaT1) is strongly vitamin D-dependent, and that calcium influx, probably interacting with calbindin-D(9K), should be considered as a rate-limiting step in the process of vitamin D-dependent active calcium absorption.

Numerous cross-sectional studies demonstrate an inverse association between plasma 25-hydroxyvitamin D [25(OH)D] and blood pressure or hypertension. Prospective data, however, are limited. Among 1484 women aged 32 to 52 years who did not have hypertension at baseline, we prospectively analyzed the association between plasma levels of 25(OH)D and the odds of incident hypertension using a nested case-control study design. We matched cases and controls on age, race, and month of blood collection and further adjusted for body mass index, physical activity, family history of hypertension, oral contraceptive use, and plasma levels of parathyroid hormone, calcium, phosphorous, creatinine, and uric acid. Median plasma 25(OH)D levels were lower in the cases (25.6 ng/mL) than in the controls (27.3 ng/mL; P<0.001). Women in the lowest compared with highest quartile of plasma 25(OH)D had an adjusted odds ratio for incident hypertension of 1.66 (95% CI: 1.11 to 2.48; P for trend=0.01). Compared with women with sufficient levels, those with vitamin D deficiency (<30 ng/mL; 65.7% of the study population) had a multivariable odds ratio of 1.47 (95% CI: 1.10 to 1.97). Plasma 25(OH)D levels are inversely and independently associated with the risk of developing hypertension.

OBJECTIVE

Serum 25-hydroxyvitamin D [25(OH)D] levels are inversely associated with important cardiovascular disease (CVD) risk factors. However, the association between 25(OH)D levels and prevalent CVD has not been extensively examined in the general population.

METHODS

We performed a cross-sectional analysis of data from the Third National Health and Nutrition Examination Survey (1988-1994) and examined the association between serum 25(OH)D levels and prevalence of CVD in a representative population-based sample of 16,603 men and women aged 18 years or older. Prevalence of CVD was defined as a composite measure inclusive of self-reported angina, myocardial infarction or stroke.

RESULTS

In the whole population, there were 1308 (8%) subjects with self-reported CVD. Participants with CVD had a greater frequency of 25(OH)D deficiency [defined as serum 25(OH)D levels <20 ng/mL] than those without (29.3% vs. 21.4%; p<0.0001). After adjustment for age, gender, race/ethnicity, season of measurement, physical activity, body mass index, smoking status, hypertension, diabetes, elevated low-density lipoprotein cholesterol, hypertriglyceridemia, low high-density lipoprotein cholesterol, chronic kidney disease and vitamin D use, participants with 25(OH)D deficiency had an increased risk of prevalent CVD (odds ratio 1.20 [95% confidence interval (CI) 1.01-1.36; p=0.03]).

CONCLUSIONS

These results indicate a strong and independent relationship of 25(OH)D deficiency with prevalent CVD in a large sample representative of the US adult population.

It has been hypothesized that a deficit in serotonin may be a crucial determinant in the pathophysiology of major depression. Serotonin-1A receptors are located on serotonin cell bodies in the midbrain dorsal raphe (DR) nucleus, and the activation of these receptors inhibits the firing of serotonin neurons and diminishes the release of this neurotransmitter in the prefrontal cortex. Repeated treatment with some antidepressant medications desensitizes serotonin-1A receptors in the rat midbrain. The present study determined whether the binding of [3H]8-hydroxy-2-(di-n-propyl)aminotetralin (8-OH-DPAT), an agonist at serotonin-1A receptors, is altered in the midbrain of suicide victims with major depression. Radiolabeling of the serotonin-1A receptor in the DR varied significantly along the rostral-to-caudal extent of the human midbrain. The binding of [3H]8-OH-DPAT to serotonin-1A receptors was increased significantly in the midbrain DR of suicide victims with major depression as compared with psychiatrically normal control subjects. In suicide victims with major depression, the increase in the binding of [3H]8-OH-DPAT to serotonin-1A receptors was detected in the entire DR and specifically localized to the dorsal and ventrolateral subnuclei. Enhanced radioligand binding of an agonist to inhibitory serotonin-1A autoreceptors in the human DR provides pharmacological evidence to support the hypothesis of diminished activity of serotonin neurons in suicide victims with major depression.

Inactivation of glycogen synthase kinase 3beta (GSK3beta) and the resulting stabilization of free beta-catenin are critical steps in the activation of Wnt target genes. While Akt regulates GSK3alpha/beta in the phosphatidylinositide 3-OH kinase signaling pathway, its role in Wnt signaling is unknown. Here we report that expression of Wnt or Dishevelled (Dvl) increased Akt activity. Activated Akt bound to the Axin-GSK3beta complex in the presence of Dvl, phosphorylated GSK3beta and increased free beta-catenin levels. Furthermore, in Wnt-overexpressing PC12 cells, dominant-negative Akt decreased free beta-catenin and derepressed nerve growth factor-induced differentiation. Therefore, Akt acts in association with Dvl as an important regulator of the Wnt signaling pathway.

This review summarizes the literature describing the molecular mechanisms of arsenic-induced oxidative stress, its relevant biomarkers, and its relation to various diseases, including preventive and therapeutic strategies. Arsenic alters multiple cellular pathways including expression of growth factors, suppression of cell cycle checkpoint proteins, promotion of and resistance to apoptosis, inhibition of DNA repair, alterations in DNA methylation, decreased immunosurveillance, and increased oxidative stress, by disturbing the pro/antioxidant balance. These alterations play prominent roles in disease manifestation, such as carcinogenicity, genotoxicity, diabetes, cardiovascular and nervous systems disorders. The exact molecular and cellular mechanisms involved in arsenic toxicity are rather unrevealed. Arsenic alters cellular glutathione levels either by utilizing this electron donor for the conversion of pentavalent to trivalent arsenicals or directly binding with it or by oxidizing glutathione via arsenic-induced free radical generation. Arsenic forms oxygen-based radicals (OH(•), O(2)(•-)) under physiological conditions by directly binding with critical thiols. As a carcinogen, it acts through epigenetic mechanisms rather than as a classical mutagen. The carcinogenic potential of arsenic may be attributed to activation of redox-sensitive transcription factors and other signaling pathways involving nuclear factor κB, activator protein-1, and p53. Modulation of cellular thiols for protection against reactive oxygen species has been used as a therapeutic strategy against arsenic. N-acetylcysteine, α-lipoic acid, vitamin E, quercetin, and a few herbal extracts show prophylactic activity against the majority of arsenic-mediated injuries in both in vitro and in vivo models. This review also updates the reader on recent advances in chelation therapy and newer therapeutic strategies suggested to treat arsenic-induced oxidative damage.