BACKGROUND

The effects of vitamin D in regulating bone mineralization are well documented. The action of vitamin D as a key link between Toll-like receptor activation and antibacterial responses in innate immunity has recently been shown. The data suggest that differences in the ability of human populations to produce vitamin D may contribute to susceptibility to microbial infection.

OBJECTIVE

We aimed to explore whether an association exists between vitamin D insufficiency and acute respiratory tract infection in young Finnish men.

METHODS

Young Finnish men (n = 800) serving on a military base in Finland were enrolled for this study. Their serum 25-hydroxyvitamin [25(OH)D] concentrations were measured in July 2002. They were followed for 6 mo, and the number of days of absence from duty due to respiratory infection were counted.

RESULTS

The mean (+/- SD) serum 25(OH)D concentrations were 80.2 +/- 29.3 nmol/L (n = 756). Subjects with serum 25(OH)D concentrations < 40 nmol/L (n = 24) had significantly (P = 0.004) more days of absence from duty due to respiratory infection (median: 4; quartile 1-quartile 3: 2-6) than did control subjects (2; 0-4; n = 628; incidence rate ratio 1.63; 95% CI: 1.15, 2.24). We found a significant (P = 0.004) association between serum 25(OH)D concentrations and the amount of physical exercise before induction into military service. We also found significantly (P < 0.001) lower serum 25(OH)D concentrations in subjects who smoked (72.8 +/- 26.6 nmol/L; n = 192) than in control subjects (82.9 +/- 29.7 nmol/L; n = 537).

CONCLUSIONS

Clinical trials of vitamin D supplementation are needed to investigate whether it enhances immunity to microbial infections.

To determine whether the cardiovascular effect of 1,25(OH)(2)D is dependent on calcium and/or phosphorus, mice with targeted deletion of the 25(OH)D 1alpha-hydroxylase and their wild-type littermates were fed a normal diet or a diet to rescue the ambient serum calcium and phosphorus levels. Mice on the normal diet were treated daily with vehicle or 1,25(OH)(2)D(3) while mice on the rescue diet received vehicle, captopril or losartan. After four weeks the vehicle-treated knockout mice developed hypertension, cardiac hypertrophy and impaired cardiac function along with an up-regulation of the renin-angiotensin system in both renal and cardiac tissues. Although the serum calcium and phosphorus levels were normalized in knockout mice on the rescue diet, abnormalities in blood pressure, cardiac structure-function and the renin-angiotensin system remained. In contrast, 1,25(OH)(2)D(3) not only normalized serum calcium and phosphorus levels but also normalized blood pressure, cardiac structure-function and the renin-angiotensin system. Treatment of the knockout mice with either captopril or losartan normalized blood pressure and cardiac structure and function although renin expression remained elevated. This study shows that 1,25(OH)2D plays a protective role in the cardiovascular system by repressing the renin-angiotensin system independent of extracellular calcium or phosphorus.

OBJECTIVE

To quantitatively assess the strength and shape of the association between blood 25-hydroxy vitamin D [25(OH)D] levels and incident risk of type 2 diabetes.

METHODS

A systematic search of the MEDLINE and Embase databases and a hand search of references from original reports were conducted up to 31 October 2012. Prospective observational studies that assessed the association between blood levels of 25(OH)D and risk of incident type 2 diabetes were included for meta-analysis. DerSimonian and Laird's random-effects model was used. A quadratic spline regression analysis was used to examine the shape of the association with a generalized least-squares trend test performed for the dose-response relation.

RESULTS

A total of 21 prospective studies involving 76,220 participants and 4,996 incident type 2 diabetes cases were included for meta-analysis. Comparing the highest to the lowest category of 25(OH)D levels, the summary relative risk for type 2 diabetes was 0.62 (95% CI 0.54-0.70). A spline regression model showed that higher 25(OH)D levels were monotonically associated with a lower diabetes risk. This inverse association did not differ by sex, duration of follow-up, study sample size, diabetes diagnostic criteria, or 25(OH)D assay method. A linear trend analysis showed that each 10 nmol/L increment in 25(OH)D levels was associated with a 4% lower risk of type 2 diabetes (95% CI 3-6; P for linear trend < 0.0001).

CONCLUSIONS

Our meta-analysis showed an inverse and significant association between circulating 25(OH)D levels and risk of type 2 diabetes across a broad range of blood 25(OH)D levels in diverse populations.

This article describes the information currently available in the National Nutrition Monitoring System that is relevant to assessing the vitamin D status of US population groups, the strengths and limitations of this information, and selected results of vitamin D nutritional status assessments. The National Health and Nutrition Examination Survey (NHANES) provides information on vitamin D intakes only from 1988 to 1994. NHANES collected information on supplement use and circulating 25-hydroxyvitamin D [25(OH)D] concentrations from 1988 through current surveys. The National Nutrient Database for Standard Reference started providing limited data on the vitamin D content of foods in 2002 and continues to update these values. The Food Label and Package Survey provides 2006-2007 label information on vitamin D fortification of marketed foods. Despite limitations in the available data and controversies about appropriate criteria for evaluating vitamin D status among population groups, we can make some useful comparisons of vitamin D status among life-stage groups. In general, males have higher vitamin D intakes and 25(OH)D concentrations than do females. Children tend to have higher vitamin D status than adults. The increasing use of multivitamin-mineral dietary supplements in younger to older adults is not associated with a corresponding increase in serum 25(OH)D concentrations. In general, leaner individuals have higher circulating concentrations of 25(OH)D and supplement use than do heavier individuals. Finally, non-Hispanic whites tend to have higher vitamin D status than do non-Hispanic blacks and Mexican Americans.

Embryonic stem (ES) cells are pluripotent cells derived from early mammalian embryos. Their immortality and rapid growth make them attractive sources for stem cell therapies; however, they produce tumours (teratomas) when transplanted, which could preclude their therapeutic usage. Why ES cells, which lack chromosomal abnormalities, possess tumour-like properties is largely unknown. Here we show that mouse ES cells specifically express a Ras-like gene, which we have named ERas. We show that human HRasp, which is a recognized pseudogene, does not contain reported base substitutions and instead encodes the human orthologue of ERas. This protein contains amino-acid residues identical to those present in active mutants of Ras and causes oncogenic transformation in NIH 3T3 cells. ERas interacts with phosphatidylinositol-3-OH kinase but not with Raf. ERas-null ES cells maintain pluripotency but show significantly reduced growth and tumorigenicity, which are rescued by expression of ERas complementary DNA or by activated phosphatidylinositol-3-OH kinase. We conclude that the transforming oncogene ERas is important in the tumour-like growth properties of ES cells.

Chemical modifications have been incorporated into short interfering RNAs (siRNAs) without reducing their ability to inhibit gene expression in mammalian cells grown in vitro. In this study, we begin to assess the potential utility of 2'-modified siRNAs in mammals. We demonstrate that siRNA modified with 2'-fluoro (2'-F) pyrimidines are functional in cell culture and have a greatly increased stability and a prolonged half-life in human plasma as compared to 2'-OH containing siRNAs. Moreover, we show that the 2'-F containing siRNAs are functional in mice and can inhibit the expression of a target gene in vivo. However, even though the modified siRNAs have greatly increased resistance to nuclease degradation in plasma, this increase in stability did not translate into enhanced or prolonged inhibitory activity of target gene reduction in mice following tail vein injection. Thus, this study shows that 2'-F modified siRNAs are functional in vivo, but that they are not necessarily more potent than unmodified siRNAs in animals.

An involvement of the mesolimbic dopamine (DA) system in mediating the motivational effects of opioids has been suggested. Accordingly, the present study employed the technique of in vivo microdialysis to examine the effects of selective mu-, delta-, and kappa- opioids on DA release in the nucleus accumbens (NAC) of anesthetized rats. Microdialysis probes were inserted into the NAC and perfusates were analyzed for DA and its metabolites, dihydroxyphenylacetic acid (DO-PAC) and homovanillic acid (HVA), using a reverse-phase HPLC system with electrochemical detection for separation and quantification. Intracerebroventricular (i.c.v.) administration of selective mu-opioid [D-Ala2, N-methyl-Phe4, Gly5-ol]-enkephalin (DAMGO) or delta-opioid [D-Pen2, D-Pen5]-enkephalin (DPDPE) agonists, at doses that function as positive reinforcers in rats, resulted in an immediate and significant increase in extracellular DA. DOPAC and HVA levels were also significantly increased. The effects of DAMGO were blocked by the selective mu-antagonist D-Pen-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP) whereas those of DPDPE were blocked by the delta-antagonist allyl2-Tyr-Aib-Aib-Phe-Leu-OH (ICI 174,864). In contrast to mu- and delta-agonists, the kappa-agonist N-CH3-Tyr-Gly-Gly-Phe-Leu-Arg-N-CH3-Arg-D-Leu-NHC2H5 (E-2078), a dynorphin analog that produces aversive states, decreased DA release in a biphasic manner. Norbinaltorphimine, a selective kappa-antagonist, could block this effect. These results demonstrate that mu-, delta-, and kappa-opioid agonists differentially affect DA release in the NAC and this action is centrally mediated.

Cell extension in the growing zone of plant roots typically takes place with a maximum local growth rate of 50% length increase per hour. The biochemical mechanism of this dramatic growth process is still poorly understood. Here we test the hypothesis that the wall-loosening reaction controlling root elongation is effected by the production of reactive oxygen intermediates, initiated by a NAD(P)H oxidase-catalyzed formation of superoxide radicals (O(2)(.-)) at the plasma membrane and culminating in the generation of polysaccharide-cleaving hydroxyl radicals ((.)OH) by cell wall peroxidase. The following results were obtained using primary roots of maize (Zea mays) seedlings as experimental material. (1) Production of O(2)(.-), H(2)O(2), and (.)OH can be demonstrated in the growing zone using specific histochemical assays and electron paramagnetic resonance spectroscopy. (2) Auxin-induced inhibition of growth is accompanied by a reduction of O(2)(.-) production. (3) Experimental generation of (.)OH in the cell walls with the Fenton reaction causes wall loosening (cell wall creep), specifically in the growing zone. Alternatively, wall loosening can be induced by (.)OH produced by endogenous cell wall peroxidase in the presence of NADH and H(2)O(2). (4) Inhibition of endogenous (.)OH formation by O(2)(.-) or (.)OH scavengers, or inhibitors of NAD(P)H oxidase or peroxidase activity, suppress elongation growth. These results show that juvenile root cells transiently express the ability to generate (.)OH, and to respond to (.)OH by wall loosening, in passing through the growing zone. Moreover, inhibitor studies indicate that (.)OH formation is essential for normal root growth.

BACKGROUND

Vitamin D deficiency is prevalent in India, a finding that is unexpected in a tropical country with abundant sunshine. Vitamin D deficiency during pregnancy has important implications for the newborn and infant. There are few data from India about the prevalence of hypovitaminosis D in pregnancy and in the newborn.

OBJECTIVE

Our aim was to determine the prevalence of osteomalacia and hypovitaminosis D in pregnancy and in cord blood and to correlate maternal 25-hydroxyvitamin D [25(OH)D] status with sun exposure, daily calcium intake (dietary plus supplemental), and intact parathyroid hormone (PTH) concentrations.

METHODS

Serum calcium, inorganic phosphorus, 25(OH)D, heat-labile alkaline phosphatase, and PTH were studied in 207 urban and rural pregnant subjects at term. Alkaline phosphatase and 25(OH)D were measured in the cord blood of 117 newborns.

RESULTS

Mean maternal serum 25(OH)D was 14 +/- 9.3 ng/mL, and cord blood 25(OH)D was 8.4 +/- 5.7 ng/mL. PTH rose above the normal range when 25(OH)D was <22.5 ng/mL. Eighty-four percent of women (84.3% of urban and 83.6% of rural women) had 25(OH)D values below that cutoff. Fourteen percent of the subjects had elevated alkaline phosphatase (17% of urban and 7% of rural subjects). Calcium intake was uniformly low, although higher in urban (842 +/- 459 mg/d) than in rural (549 +/- 404 mg/d) subjects (P < 0.001). Maternal serum 25(OH)D correlated positively with cord blood 25(OH)D (r = 0.79, P < 0.001) and negatively with PTH (r = -0.35, P < 0.001).

CONCLUSIONS

We observed a high prevalence of physiologically significant hypovitaminosis D among pregnant women and their newborns, the magnitude of which warrants public health intervention.

The hepatitis C virus (HCV) H strain polyprotein is cleaved to produce at least nine distinct products: NH2-C-E1-E2-NS2-NS3-NS4A-NS4B-NS5A-NS5B-CO OH. In this report, a series of C-terminal truncations and fusion with a human c-myc epitope tag allowed identification of a tenth HCV-encoded cleavage product, p7, which is located between the E2 and NS2 proteins. As determined by N-terminal sequence analysis, p7 begins with position 747 of the HCV H strain polyprotein. p7 is preceded by a hydrophobic sequence at the C terminus of E2 which may direct its translocation into the endoplasmic reticulum, allowing cleavage at the E2/p7 site by host signal peptidase. This hypothesis is supported by the observation that cleavage at the E2/p7 and p7/NS2 sites in cell-free translation studies was dependent upon the addition of microsomal membranes. However, unlike typical cotranslational signal peptidase cleavages, pulse-chase experiments indicate that cleavage at the E2/p7 site is incomplete, leading to the production of two E2-specific species, E2 and E2-p7. Possible roles of p7 and E2-p7 in the HCV life cycle are discussed.

The active form of vitamin D, 1alpha,25-dihydroxyvitamin D [1alpha,25(OH)2D], is synthesized from its precursor 25 hydroxyvitamin D [25(OH)D] via the catalytic action of the 25(OH)D-1alpha-hydroxylase [1alpha(OH)ase] enzyme. Many roles in cell growth and differentiation have been attributed to 1,25(OH)2D, including a central role in calcium homeostasis and skeletal metabolism. To investigate the in vivo functions of 1,25(OH)2D and the molecular basis of its actions, we developed a mouse model deficient in 1alpha(OH)ase by targeted ablation of the hormone-binding and heme-binding domains of the 1alpha(OH)ase gene. After weaning, mice developed hypocalcemia, secondary hyperparathyroidism, retarded growth, and the skeletal abnormalities characteristic of rickets. These abnormalities are similar to those described in humans with the genetic disorder vitamin D dependent rickets type I [VDDR-I; also known as pseudovitamin D-deficiency rickets (PDDR)]. Altered non-collagenous matrix protein expression and reduced numbers of osteoclasts were also observed in bone. Female mutant mice were infertile and exhibited uterine hypoplasia and absent corpora lutea. Furthermore, histologically enlarged lymph nodes in the vicinity of the thyroid gland and a reduction in CD4- and CD8-positive peripheral T lymphocytes were observed. Alopecia, reported in vitamin D receptor (VDR)-deficient mice and in humans with VDDR-II, was not seen. The findings establish a critical role for the 1alpha(OH)ase enzyme in mineral and skeletal homeostasis as well as in female reproduction and also point to an important role in regulating immune function.

This Review summarizes recent advances in understanding copper-transporting ATPase 1 (ATP7A), and examines the neurological phenotypes associated with dysfunction of this protein. Involvement of ATP7A in axonal outgrowth, synapse integrity and neuronal activation underscores the fundamental importance of copper metabolism to neurological function. Defects in ATP7A cause Menkes disease, an infantile-onset, lethal condition. Neonatal diagnosis and early treatment with copper injections enhance survival in patients with this disease, and can normalize clinical outcomes if mutant ATP7A molecules retain small amounts of residual activity. Gene replacement rescues a mouse model of Menkes disease, suggesting a potential therapeutic approach for patients with complete loss-of-function ATP7A mutations. Remarkably, a newly discovered ATP7A disorder-isolated distal motor neuropathy-has none of the characteristic clinical or biochemical abnormalities of Menkes disease or its milder allelic variant occipital horn syndrome (OHS), instead resembling Charcot-Marie-Tooth disease type 2. These findings indicate that ATP7A has a crucial but previously unappreciated role in motor neuron maintenance, and that the mechanism underlying ATP7A-related distal motor neuropathy is distinct from Menkes disease and OHS pathophysiology. Collectively, these insights refine our knowledge of the neurology of ATP7A-related copper transport diseases and pave the way for further progress in understanding ATP7A function.

BACKGROUND

Cardiovascular disease is the leading cause of death among those with diabetes mellitus. Vitamin D deficiency is associated with an increased risk of cardiovascular disease in this population. To determine the mechanism by which vitamin D deficiency mediates accelerated cardiovascular disease in patients with diabetes mellitus, we investigated the effects of active vitamin D on macrophage cholesterol deposition.

RESULTS

We obtained macrophages from 76 obese, diabetic, hypertensive patients with vitamin D deficiency (25-hydroxyvitamin D <80 nmol/L; group A) and 4 control groups: obese, diabetic, hypertensive patients with normal vitamin D (group B; n=15); obese, nondiabetic, hypertensive patients with vitamin D deficiency (group C; n=25); and nonobese, nondiabetic, nonhypertensive patients with vitamin D deficiency (group D; n=10) or sufficiency (group E; n=10). Macrophages from the same patients in all groups were cultured in vitamin D-deficient or 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] -supplemented media and exposed to modified low-density lipoprotein cholesterol. 1,25(OH)(2)D(3) suppressed foam cell formation by reducing acetylated or oxidized low-density lipoprotein cholesterol uptake in diabetic subjects only. Conversely, deletion of the vitamin D receptor in macrophages from diabetic patients accelerated foam cell formation induced by modified LDL. 1,25(OH)(2)D(3) downregulation of c-Jun N-terminal kinase activation reduced peroxisome proliferated-activated receptor-gamma expression, suppressed CD36 expression, and prevented oxidized low-density lipoprotein-derived cholesterol uptake. In addition, 1,25(OH)(2)D(3) suppression of macrophage endoplasmic reticulum stress improved insulin signaling, downregulated SR-A1 expression, and prevented oxidized and acetylated low-density lipoprotein-derived cholesterol uptake.

CONCLUSIONS

These results identify reduced vitamin D receptor signaling as a potential mechanism underlying increased foam cell formation and accelerated cardiovascular disease in diabetic subjects.

Insulin stimulates glucose uptake in muscle and adipocytes by signalling the translocation of GLUT4 glucose transporters from intracellular membranes to the cell surface. The translocation of GLUT4 may involve signalling pathways that are both independent of and dependent on phosphatidylinositol-3-OH kinase (PI(3)K). This translocation also requires the actin cytoskeleton, and the rapid movement of GLUT4 along linear tracks may be mediated by molecular motors. Here we report that the unconventional myosin Myo1c is present in GLUT4-containing vesicles purified from 3T3-L1 adipocytes. Myo1c, which contains a motor domain, three IQ motifs and a carboxy-terminal cargo domain, is highly expressed in primary and cultured adipocytes. Insulin enhances the localization of Myo1c with GLUT4 in cortical tubulovesicular structures associated with actin filaments, and this colocalization is insensitive to wortmannin. Insulin-stimulated translocation of GLUT4 to the adipocyte plasma membrane is augmented by the expression of wild-type Myo1c and inhibited by a dominant-negative cargo domain of Myo1c. A decrease in the expression of endogenous Myo1c mediated by small interfering RNAs inhibits insulin-stimulated uptake of 2-deoxyglucose. Thus, myosin Myo1c functions in a PI(3)K-independent insulin signalling pathway that controls the movement of intracellular GLUT4-containing vesicles to the plasma membrane.

The pharmacological characteristics of the binding of [3H]8-OH-DPAT ([3H]8-hydroxy-2(di-n-propylamino)tetralin, [125I]CYP ((-)[125I]iodocyanopindolol) (in the presence of 30 microM (-)isoprenaline) and [3H]mesulergine to 5-HT1 recognition sites were studied in rat and pig brain membranes. [3H]8-OH-DPAT bound in rat and pig cortex to the 5-HT1A recognition site characterized by high affinity for 5-CT (5-carboxamido-tryptamine), 8-OH-DPAT, 5-HT (5-hydroxytryptamine, serotonin) and low affinity for pirenperone, ketanserin and mesulergine. [125I]CYP bound in rat but not in pig cortex to the 5-HT1B site which shows high affinity for (-)21-009 (4[3-ter-butyl-amino-2-hydroxy-propoxy]indol-2-carbonic acid isopropyl ester), (+/-)ICYP (3-I-cyanopindolol), 5-HT, RU 24969 (5-methoxy-3-[1,2,3,6-tetrahydropyridon-4-yl]1H-indole) and low affinity for 8-OH-DPAT, mesulergine and pirenperone. [3H]Mesulergine bound in pig choroid plexus and in rat cortex (besides binding to 5-HT2 sites in rat cortex) to the 5-HT1C recognition site characterized by high affinity for metergoline, mesulergine, 5-HT and methergine and low affinity for (-)21-009, ICYP, 8-OH-DPAT and spiroperidol. The pharmacological profile of 5-HT1A sites in rat and pig cortex appears to be identical; 5-HT1C sites in pig choroid plexus and rat cortex show no differences. In contrast, it was not possible to label 5-HT1B sites with [125I]CYP in pig brain membranes indicating that like 5-HT2 receptors, 5-HT1 recognition sites show species differences. The pharmacological profiles of the three 5-HT1 recognition sites are clearly different from one another. Furthermore, the pharmacological profile of each individual 5-HT1 recognition site is also different from that of the 5-HT2 receptors labelled with [3H]ketanserin in rat cortex membranes although some similarities exist between 5-HT2 and 5-HT1C sites. Finally, the beta-adrenoceptor antagonist (-)21-009 which has different affinities for 5-HT1A, 5-HT1B and 5-HT1C recognition sites, yielded triphasic competition curves for [3H]5-HT binding in rat cortex membranes providing evidence that [3H]5-HT labels three distinct 5-HT1 sites in these membranes.

BACKGROUND

Alpha-Klotho (alphaKl) regulates mineral metabolism such as calcium ion (Ca(2+)) and inorganic phosphate (Pi) in circulation. Defects in mice result in clinical features resembling disorders found in human aging. Although the importance of transmembrane-type alphaKl has been demonstrated, less is known regarding the physiological importance of soluble-type alphaKl (salphaKl) in circulation.

OBJECTIVE

The aims of this study were: (1) to establish a sandwich ELISA system enabling detection of circulating serum salphaKl, and (2) to determine reference values for salphaKl serum levels and relationship to indices of renal function, mineral metabolism, age and sex in healthy subjects.

RESULTS

We successively developed an ELISA to measure serum salphaKl in healthy volunteers (n=142, males 66) of ages (61.1+/-18.5year). The levels (mean+/-SD) in these healthy control adults were as follows: total calcium (Ca; 9.46+/-0.41mg/dL), Pi (3.63+/-0.51mg/dL), blood urea nitrogen (BUN; 15.7+/-4.3mg/dL), creatinine (Cre; 0.69+/-0.14mg/dL), 1,25 dihydroxyvitamin D (1,25(OH)(2)D; 54.8+/-17.7pg/mL), intact parathyroid hormone (iPTH; 49.2+/-20.6pg/mL), calcitonin (26.0+/-12.3pg/mL) and intact fibroblast growth factor (FGF23; 43.8+/-17.6pg/mL). Serum levels of salphaKl ranged from 239 to 1266pg/mL (mean+/-SD; 562+/-146pg/mL) in normal adults. Although salphaKl levels were not modified by gender or indices of mineral metabolism, salphaKl levels were inversely related to Cre and age. However, salphaKl levels in normal children (n=39, males 23, mean+/-SD; 7.1+/-4.8years) were significantly higher (mean+/-SD; 952+/-282pg/mL) than those in adults (mean+/-SD; 562+/-146, P<0.001). A multivariate linear regression analysis including children and adults in this study demonstrated that salphaKl correlated negatively with age and Ca, and positively with Pi. Finally, we measured a serum salphaKl from a patient with severe tumoral calcinosis derived from a homozygous missense mutation of alpha-klotho gene. In this patient, salphaKl level was notably lower than those of age-matched controls.

CONCLUSIONS

We established a detection system to measure human serum salphaKl for the first time. Age, Ca and Pi seem to influence serum salphaKl levels in a normal population. This detection system should be an excellent tool for investigating salphaKl functions in mineral metabolism.

We have demonstrated that cytokine thymic stromal lymphopoietin (TSLP), whose expression is rapidly induced upon keratinocyte-selective ablation of retinoid X receptors (RXRs) -alpha and -beta in the mouse (RXRalphabeta(ep-/-) mice), plays a key role in initiating a skin and systemic atopic dermatitis-like phenotype. We show here that topical application of the physiologically active ligand [1alpha,25-(OH)(2)D(3); calcitriol] of the vitamin D receptor, or of its low-calcemic analog MC903 (calcipotriol; Dovonex), induces TSLP expression in epidermal keratinocytes, which results in an atopic dermatitis-like syndrome mimicking that seen in RXRalphabeta(ep-/-) mutants and transgenic mice overexpressing TSLP in keratinocytes. Furthermore, topical application of retinoic acid receptor RARgamma-selective agonist BMS961 also induces TSLP expression either on its own or synergistically with 1alpha,25-(OH)(2)D(3). Our data demonstrate that RXR/vitamin D receptor and RXR/retinoic acid receptor-gamma heterodimers and their ligands cell-autonomously control the expression of TSLP in epidermal keratinocytes of the mouse. We propose molecular mechanisms through which vitamin D3 and retinoic acid signalings could be involved in the pathogenesis of atopic diseases.

Controlled release from biodegradable polymers is a novel approach to replace daily painful injections of protein drugs. A major obstacle to development of these polymers is the need to retain the structure and biological activity of encapsulated proteins during months of incubation under physiological conditions. We encapsulated bovine serum albumin (BSA) in injectable poly(DL-lactide- co-glycolide) (PLGA) 50/50 cylindrical implants and determined the mechanism of BSA instability. Simulations of the polymer microclimate revealed that moisture and acidic pH (<3) triggered unfolding of encapsulated BSA, resulting in peptide bond hydrolysis and noncovalent aggregation. To neutralize the acids liberated by the biodegradable lactic/glycolic acid-based polyester, we coincorporated into the polymer an antacid, Mg(OH)2, which increased microclimate pH and prevented BSA structural losses and aggregation for over one month. We successfully applied this stabilization approach in both cylinder- and microsphere-injectable configurations and for delivery of angiogenic basic fibroblast growth factor and bone-regenerating bone morphogenetic protein-2.

Rac is a member of the Ras superfamily of GTPases and functions as a GDP/GTP-regulated switch. Formation of active Rac-GTP is stimulated by Dbl family guanine nucleotide exchange factors (GEFs), such as Tiam1 (ref. 2). Once activated, Rac stimulates signalling pathways that regulate actin organization, gene expression and cellular proliferation. Rac also functions downstream of the Ras oncoprotein in pathways that stimulate membrane ruffling, growth transformation, activation of the c-Jun amino-terminal kinase (JNK) mitogen-activated protein kinase, activation of the NF-kappa B transcription factor and promotion of cell survival. Although recent studies support phosphatidylinositol 3-OH kinase (PI(3)K)-dependent mechanisms through which Ras might activate Rac (refs 9,10), the precise mechanism remains to be determined. Here we demonstrate that Tiam1, a Rac-specific GEF, preferentially associates with activated GTP-bound Ras through a Ras-binding domain. Furthermore, activated Ras and Tiam1 cooperate to cause synergistic formation of Rac-GTP in a PI(3)K-independent manner. Thus, Tiam1 can function as an effector that directly mediates Ras activation of Rac.

Aptamers are oligonucleotides derived from an in vitro evolution process called SELEX. Aptamers have been evolved to bind proteins which are associated with a number of disease states. Using this method, many powerful antagonists of such proteins have been found. In order for these antagonists to work in animal models of disease and in humans, it is necessary to modify the aptamers. First of all, sugar modifications of nucleoside triphosphates are necessary to render the resulting aptamers resistant to nucleases found in serum. Changing the 2'OH groups of ribose to 2'F or 2'NH2 groups yields aptamers which are long lived in blood. The relatively low molecular weight of aptamers (8000-12000) leads to rapid clearance from the blood. Aptamers can be kept in the circulation from hours to days by conjugating them to higher molecular weight vehicles. When modified, conjugated aptamers are injected into animals, they inhibit physiological functions known to be associated with their target proteins. A new approach to diagnostics is also described. Aptamer arrays on solid surfaces will become available rapidly because the SELEX protocol has been successfully automated. The use of photo-cross-linkable aptamers will allow the covalent attachment of aptamers to their cognate proteins, with very low backgrounds from other proteins in body fluids. Finally, protein staining with any reagent which distinguishes functional groups of amino acids from those of nucleic acids (and the solid support) will give a direct readout of proteins on the solid support.

Partially-reduced forms of dioxygen or "oxy-radicals" (superoxide, O2-/HO2; hydrogen peroxide, H2O2; hydroxyl radical X OH) and oxidants of comparable reactivity are implicated in an increasing number of physiological, toxicological, and pathological states. Transition metal catalysis is recognized as being integral to the generation and the reactions of these activated oxygen species. Factors such as pH and chelation govern the reactivity of the transition metals with dioxygen and "oxy-radicals" and therefore influence the apparent mechanisms by which oxidative damage to phospholipids, DNA, and other biomolecules is initiated. In biological systems the concentrations of redox-active transition metals capable of catalyzing these reactions appears to be relatively low. However, under certain conditions metal storage and transport proteins (ferritin, transferrin, ceruloplasmin, etc.) may furnish additional redox active metals.

BACKGROUND

Kidney disease has been identified as a risk factor for vitamin D deficiency in hospitalized patients, and low levels of 25-hydroxyvitamin D have been suggested to be a risk factor for hyperparathyroidism in patients with chronic kidney disease (CKD). However, little is known about the magnitude of vitamin D deficiency in patients with CKD living in the United States.

METHODS

In this regard, we examined the levels of 25(OH)D in 43 patients with CKD and serum creatinine between 1 and 5 mg/dl (calculated glomerular filtration rate 111-11 ml/min per 1.73 m2) as well as in 103 patients undergoing hemodialysis.

RESULTS

In the predialysis patients, we found that 37 of the 43 patients (86%) had suboptimal levels of vitamin D (<30 ng/ml). Regression analysis indicated that there was a negative correlation between 25(OH)D and intact parathyroid hormone (PTH). Alkaline phosphatase showed a similar but less sensitive relationship. Serum albumin levels correlated with 25(OH)D levels. In contrast to findings reported in normal individuals, the levels of calcitriol correlated with those of 25(OH)D in the patients with CKD. In the group undergoing maintenance hemodialyis, we found that 97% of the patients had vitamin D levels in the suboptimal range, and there was no correlation of 25(OH)D levels with either PTH or serum albumin values. These data indicate that vitamin D insufficiency and deficiency are highly prevalent in patients with CKD and may play a role in the development of hyperparathyroidism. The functional significance of low levels of 25(OH)D in patients with stage 5 CKD remains to be determined.

Cleavage maps of colicin E1 plasmid DNA and its smaller derivative, pNT1 DNA, were constructed by using restriction endonucleases. The nucleotide sequence of a region that contains the orgin of replication was determined. The site of the nucleotide from which DNA replication is initiated was determined with 6S L-fragments, the DNA fragment first made on colicin E1 plasmid DNA. The fragments were labeled with [gamma-32P]ATP and polynucleotide 5'-hydroxyl-kinase (ATP:5'-dephosphopolynucleotide 5'-phosphotransferase, EC 2.7.1.78) at the 5'-OH groups which were uncovered by alkali treatment. The site is one of three consecutive nucleotides, dA, dA, and dC, located at a unique position. One or a few rA residues were found to be attached to some of the DNA molecules. The transition from the primer RNA to DNA occurs in a region consisting of a segment of five A residues. Both sides of this segment are rich in G and C.

A particularly important stress for all cells is the one produced by reactive oxygen species (ROS) that are formed as byproducts of cell metabolism. Among DNA damages induced by ROS, 8-hydroxyguanine (8-OH-G) is certainly the product that has retained most of the attention in the past few years. The biological relevance of 8-OH-G in DNA has been unveiled by the study of Escherichia coli and Saccharomyces cerevisiae genes involved in the neutralization of the mutagenic effects of 8-OH-G. These genes, fpg and mutY for E. coli and OGG1 for yeast, code for DNA glycosylases. Inactivation of any of those genes leads to a spontaneous mutator phenotype, characterized by the increase in GC to TA transversions. In yeast, the OGG1 gene encodes a DNA glycosylase/AP lyase that excises 8-OH-G from DNA. In human cells, the OGG1 gene is localized on chromosome 3p25 and encodes two forms of hOgg1 protein which result from an alternative splicing of a single messenger RNA. The alpha-hOgg1 protein has a nuclear localization whereas the beta-hOgg1 is targeted to the mitochondrion. Biochemical studies on the alpha-hOgg1 protein show that it is a DNA glycosylase/AP lyase that excises 8-OH-G and Fapy-G from gamma-irradiated DNA. Several approaches have been used to study the biological role of OGG1 in mammalian cells, ranging from its overexpression in cell lines to the generation of homozygous ogg1-/- null mice. Furthermore, to explore a possible role in the prevention of cancer, the cDNA coding for alpha-hOgg1 has been sequenced in human tumors. All these results point to 8-OH-G as an endogenous source of mutations in eukaryotes and to its likely involvement in the process of carcinogenesis. A review of the recent literature on the mammalian Ogg1 proteins, the main repair system involved in the elimination of this mutagenic lesion, is presented.