OBJECTIVE

To assess the genetic and nongenetic correlates of circulating measures of vitamins K and D status in a community-based sample of men and women.

METHODS

A cross-sectional study of 1762 participants of the Framingham Offspring Study (919 women; mean age 59 years). Vitamin K status was measured as plasma phylloquinone and serum percent undercarboxylated osteocalcin (ucOC), and vitamin D was measured using plasma 25-hydroxyvitamin D (25(OH)D). Associations between vitamin K status and vitamin D status with biologically plausible nongenetic factors were assessed using stepwise regression. Heritability and linkage were determined using Sequential Oligogenic Linkage Analysis Routines (SOLAR).

RESULTS

Nongenetic factors accounted for 20.1 and 12.3% of the variability in plasma phylloquinone in men and women respectively, with triglycerides and phylloquinone intake being the primary correlates. In men 12.2% and in women 14.6% of the variability in %ucOC was explained by nongenetic factors in our models. Heritability estimates for these vitamin K status biomarkers were nonsignificant. Season, vitamin D intake, high-density lipoprotein (HDL) cholesterol and waist circumference explained 24.7% (men) and 24.2% (women) of the variability in plasma 25(OH)D. Of the three vitamins examined, only 25(OH)D was significantly heritable (heritability estimate=28.8%, P<0.01), but linkage analysis of 25(OH)D did not achieve genome-wide significance.

CONCLUSIONS

Variability in biomarkers of vitamin K status was attributed to nongenetic factors, whereas plasma 25(OH)D was found to be significantly heritable. Further studies are warranted to investigate genetic loci influencing vitamin D status.

The initiation factor 4E for eukaryotic translation (eIF4E) binds the messenger RNA 5'-cap structure and is important in the regulation of protein synthesis. Mammalian eIF4E activity is inhibited when the initiation factor binds to the translational repressors, the 4E-binding proteins (4E-BPS). Here we show that the Drosophila melanogaster 4E-BP (d4E-BP) is a downstream target of the phosphatidylinositol-3-OH kinase (PI(3)K) signal-transduction cascade, which affects the interaction of d4E-BP with eIF4E. Ectopic expression of a highly active d4E-BP mutant in wing-imaginal discs causes a reduction of wing size, brought about by a decrease in cell size and number. A marked reduction in cell size was also observed in post-mitotic cells. Expression of d4E-BP in the eye and wing together with PI(3)K or dAkt1, the serine/threonine kinase downstream of PI(3)K, resulted in suppression of the growth phenotype elicited by these kinases. Our results support a role for d4E-BP as an effector of cell growth.

This review looks at the critical role of vitamin D in improving barrier function, production of antimicrobial peptides including cathelicidin and some defensins, and immune modulation. The function of vitamin D in the innate immune system and in the epithelial cells of the oral cavity, lung, gastrointestinal system, genito-urinary system, skin and surface of the eye is discussed. Clinical conditions are reviewed where vitamin D may play a role in the prevention of infections or where it may be used as primary or adjuvant treatment for viral, bacterial and fungal infections. Several conditions such as tuberculosis, psoriasis, eczema, Crohn's disease, chest infections, wound infections, influenza, urinary tract infections, eye infections and wound healing may benefit from adequate circulating 25(OH)D as substrate. Clinical diseases are presented in which optimization of 25(OH)D levels may benefit or cause harm according to present day knowledge. The safety of using larger doses of vitamin D in various clinical settings is discussed.

The structural proteins of murine type C retroviruses are proteolytic cleavage products of two different precursor polyproteins coded by the viral gag and env genes. To further investigate the nature and number of proteolytic cleavages involved in virus maturation, we quantitatively isolated the structural proteins of the Rauscher and Moloney strains of type C murine leukemia virus (R-MuLV and M-MuLV, respectively) by reversed-phase high-pressure liquid chromatography. Proteins and polypeptides isolated from R-MuLV included p10, p12, p15, p30, p15(E), gp69, and gp71 and three previously undescribed virus components designated here as p10', p2(E), and p2(E). Homologous proteins and polypeptides were isolated from M-MuLV. Complete or partial amino acid sequences of all the proteins listed above were either determined in this study or were available in previous reports from this laboratory. These data were compared with those from the translation of the M-MuLV proviral DNA sequence (Shinnick et al., Nature [London] 293:543-548, 1981) to determine the exact nature of proteolytic cleavages for all the structural proteins described above and to determine the origin of p10' and p2(E)s. The results showed that, during proteolytic processing of gp80env from M-MuLV (M-gp 80env), a single Arg residue was excised between gp70 and p15(E) and a single peptide bond was cleaved between p15(E) and p2(E). The structure of M-gPr80env is gp70-(Arg)-p15(E)-p2(E). The data suggest that proteolytic cleavage sites in R-gp85env are identical to corresponding cleavage sites in M-gp80env. The p2(E)s are shown to be different genetic variants of p2(E) present in the uncloned-virus preparations. The data for R- and M-p10's shows that they are cleavage products of the gag precursor with the structure p10-Thr-Leu-Asp-Asp-OH. The complete structure of Pr65gag is p15-p12-p30-p10'. Stoichiometries of the gag and env cleavage products in mature R- and M-MuLV were determined. In each virus, gag cleavage products (p15, p12, p30, and p10 plus p10') were found in equimolar amounts and p15(E)s were equimolar with p2(E)s. The stoichiometry of gag to env cleavage products was 4:1. These data are consistent with the proposal that proteolytic processing of precursor polyproteins occurs after virus assembly and that the C-terminal portion of Pr15(E) [i.e., p15(E)-p2(E)] is located on the inner side of the lipid bilayer of the virus.

Radiation therapy is a mainstay in the treatment of glioblastomas, but these tumors are often associated with radioresistance. Activation of the phosphatidylinositol-3-OH kinase (PI3K)/Akt pathway, which occurs frequently in glioblastomas due to inactivation of the tumor suppressor phosphatase and tensin homologue (PTEN), correlates with radioresistance. To directly test the link between Akt activation and radioresistance, we utilized PTEN-deficient U251 glioblastoma cells engineered to inducibly restore PTEN upon exposure to doxycycline. These cells showed high basal levels of Akt activation (i.e. high levels of phospho-Akt), but induction of PTEN led to substantially decreased phospho-Akt and was associated with radiosensitization. To investigate whether the PTEN-induced radiosensitization was attributable to impaired sensing versus repair of DNA damage, we assessed levels of gamma-H2AX after ionizing radiation in U251 cells induced for PTEN. Initial post-radiation levels of gamma-H2AX foci were not decreased in PTEN-induced cells; however, the resolution of these foci was significantly delayed. In contrast to these results, induction of phosphatase-dead PTEN showed no appreciable effect. Finally, exposure of cells to the PI3K inhibitor LY294002 did not decrease the occurrence of gamma-H2AX foci after irradiation but did markedly delay their resolution. These results together support a direct link between Akt activation, repair of DNA damage, and radioresistance in glioblastoma. Targeting the PI3K/Akt pathway may modulate DNA repair to improve the efficacy of radiation therapy.

We systematically investigated the association of 48 SNPS in four vitamin D metabolizing genes [CYP27A1, GC, CYP27B1 and CYP24A1] with serum 25-hydroxyvitamin D [25(OH)D] and 1,25-dihydroxyvitamin D [1,25(OH)(2)D] levels and the association of these SNPS and an additional 164 SNPS in eight downstream mediators of vitamin D signaling [VDR, RXRA, RXRB, PPAR, NCOA1, NCOA2, NCOA3 and SMAD3] with prostate cancer risk in the 749 incident prostate cancer cases and 781 controls of the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial. 25(OH)D (all cases and controls) and 1,25(OH)(2)D (a subset of 150 controls) levels were measured by radioimmunoassay and SNP data were genotyped as part of a genome-wide scan. Among investigated SNPS, only four tag SNPS in GC, the major serum 25(OH)D carrier, were associated with 25(OH)D levels; no SNPS were associated with 1,25(OH)(2)D levels. None of the 212 SNPS examined were associated with cancer risk overall. Among men in the lowest tertile of serum 25(OH)D (<48.9 nmol/l), however, prostate cancer risk was related to tag SNPS in or near the 3' untranslated region (UTR) of VDR, with the strongest association for rs11574143 [odds ratio (95% confidence interval) for risk allele carriers versus wild-type: 2.49 (1.51-4.11), P = 0.0007]; the genotype associations were null among men in tertile 2 and tertile 3. Results from the most comprehensive evaluation of serum vitamin D and its related genes to date suggest that tag SNPS in the 3' UTR of VDR may be associated with risk of prostate cancer in men with low vitamin D status.

The three-dimensional structure of the chemotactic peptide N-formyl-l-Met-l-Leu-l-Phe-OH was determined by using solid-state NMR (SSNMR). The set of SSNMR data consisted of 16 (13)C-(15)N distances and 18 torsion angle constraints (on 10 angles), recorded from uniformly (13)C,(15)N- and (15)N-labeled samples. The peptide's structure was calculated by means of simulated annealing and a newly developed protocol that ensures that all of conformational space, consistent with the structural constraints, is searched completely. The result is a high-quality structure of a molecule that has thus far not been amenable to single-crystal diffraction studies. The extensions of the SSNMR techniques and computational methods to larger systems appear promising.

Phosphoinositide-3-OH kinase (PI(3)K), activated through growth factor stimulation, generates a lipid second messenger, phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3). PtdIns(3,4,5)P3 is instrumental in signalling pathways that trigger cell activation, cytoskeletal rearrangement, survival and other reactions. However, some targets of PtdIns(3,4,5)P3 are yet to be discovered. We demonstrate that SWAP-70, a unique signalling protein, specifically binds PtdIns(3,4,5)P3. On stimulation by growth factors, cytoplasmic SWAP-70, which is dependent on PI(3)K but independent of Ras, moved to cell membrane rearrangements known as ruffles. However, mutant SWAP-70 lacking the ability to bind PtdIns(3,4,5)P3 blocked membrane ruffling induced by epidermal growth factor or platelet-derived growth factor. SWAP-70 shows low homology with Rac-guanine nucleotide exchange factors (GEFs), and catalyses PtdIns(3,4,5)P3-dependent guanine nucleotide exchange to Rac. SWAP-70-deficient fibroblasts showed impaired membrane ruffling after stimulation with epidermal growth factor, and failed to activate Rac fully. We conclude that SWAP-70 is a new type of Rac-GEF which, independently of Ras, transduces signals from tyrosine kinase receptors to Rac.

Mitochondrial dysfunction and oxidative damage may play a role in the pathogenesis of Huntington's disease (HD). We examined concentrations of 8-hydroxy-2-deoxyguanosine (OH(8)dG), a well-established marker of oxidative damage to DNA, in a transgenic mouse model of HD (R6/2). Increased concentrations of OH(8)dG were found in the urine, plasma and striatal microdialysates of the HD mice. Increased concentrations were also observed in isolated brain DNA at 12 and 14 weeks of age. Immunocytochemistry showed increased OH(8)dG staining in late stages of the illness. These results suggest that oxidative damage may play a role in the pathogenesis of neuronal degeneration in the R6/2 transgenic mouse model of HD.

Reactive oxygen species (ROS) can attack a diverse range of targets to exert antimicrobial activity, which accounts for their versatility in mediating host defense against a broad range of pathogens. Most ROS are formed by the partial reduction in molecular oxygen. Four major ROS are recognized comprising superoxide (O2•-), hydrogen peroxide (H2O2), hydroxyl radical (•OH), and singlet oxygen ((1)O2), but they display very different kinetics and levels of activity. The effects of O2•- and H2O2 are less acute than those of •OH and (1)O2, because the former are much less reactive and can be detoxified by endogenous antioxidants (both enzymatic and nonenzymatic) that are induced by oxidative stress. In contrast, no enzyme can detoxify •OH or (1)O2, making them extremely toxic and acutely lethal. The present review will highlight the various methods of ROS formation and their mechanism of action. Antioxidant defenses against ROS in microbial cells and the use of ROS by antimicrobial host defense systems are covered. Antimicrobial approaches primarily utilizing ROS comprise both bactericidal antibiotics and nonpharmacological methods such as photodynamic therapy, titanium dioxide photocatalysis, cold plasma, and medicinal honey. A brief final section covers reactive nitrogen species and related therapeutics, such as acidified nitrite and nitric oxide-releasing nanoparticles.

OBJECTIVE

It remains unclear whether vitamin D insufficiency, which is common in individuals with multiple sclerosis (MS), has an adverse effect on MS outcomes.

OBJECTIVE

To determine whether serum concentrations of 25-hydroxyvitamin D (25[OH]D), a marker of vitamin D status, predict disease activity and prognosis in patients with a first event suggestive of MS (clinically isolated syndrome).

METHODS

The Betaferon/Betaseron in Newly Emerging multiple sclerosis For Initial Treatment study was a randomized trial originally designed to evaluate the impact of early vs delayed interferon beta-1b treatment in patients with clinically isolated syndrome. Serum 25(OH)D concentrations were measured at baseline and 6, 12, and 24 months. A total of 465 of the 468 patients randomized had at least 1 25(OH)D measurement, and 334 patients had them at both the 6- and 12-month (seasonally asynchronous) measurements. Patients were followed up for 5 years clinically and by magnetic resonance imaging.

METHODS

New active lesions, increased T2 lesion volume, and brain volume on magnetic resonance imaging, as well as MS relapses and disability (Expanded Disability Status Scale score).

RESULTS

Higher 25(OH)D levels predicted reduced MS activity and a slower rate of progression. A 50-nmol/L (20-ng/mL) increment in average serum 25(OH)D levels within the first 12 months predicted a 57% lower rate of new active lesions (P < .001), 57% lower relapse rate (P = .03), 25% lower yearly increase in T2 lesion volume (P < .001), and 0.41% lower yearly loss in brain volume (P = .07) from months 12 to 60. Similar associations were found between 25(OH)D measured up to 12 months and MS activity or progression from months 24 to 60. In analyses using dichotomous 25(OH)D levels, values greater than or equal to 50 nmol/L (20 ng/mL) at up to 12 months predicted lower disability (Expanded Disability Status Scale score, -0.17; P = .004) during the subsequent 4 years.

CONCLUSIONS

Among patients with MS mainly treated with interferon beta-1b, low 25(OH)D levels early in the disease course are a strong risk factor for long-term MS activity and progression.

The steroid hormone 1 alpha,25(OH)(2)-vitamin D(3) (1,25D) regulates gene transcription through a nuclear receptor [vitamin D receptor (VDR)] and initiation of rapid cellular responses through a putative plasma membrane-associated receptor (VDR(mem)). This study characterized the VDR(mem) present in a caveolae-enriched membrane fraction (CMF), a site of accumulation of signal transduction agents. Saturable and specific [(3)H]-1,25D binding in vitro was found in CMF of chick, rat, and mouse intestine; mouse lung and kidney; and human NB4 leukemia and rat ROS 17/2.8 osteoblast-like cells; in all cases the 1,25D K(D) binding dissociation constant = 1-3 nM. Our data collectively support the classical VDR being the VDR(mem) in caveolae: 1) VDR antibody immunoreactivity was detected in CMF of all tissues tested; 2) competitive binding of [(3)H]-1,25D by eight analogs of 1,25D was significantly correlated between nuclei and CMF (r(2) = 0.95) but not between vitamin D binding protein (has a different ligand binding specificity) and CMF; 3) confocal immunofluorescence microscopy of ROS 17/2.8 cells showed VDR in close association with the caveolae marker protein, caveolin-1, in the plasma membrane region; 4) in vivo 1,25D pretreatment reduced in vitro [(3)H]-1,25D binding by 30% in chick and rat intestinal CMF demonstrating in vivo occupancy of the CMF receptor by 1,25D; and 5) comparison of [(3)H]-1,25D binding in VDR KO and WT mouse kidney tissue showed 85% reduction in VDR KO CMF and 95% reduction in VDR KO nuclear fraction. This study supports the presence of VDR as the 1,25D-binding protein associated with plasma membrane caveolae.

OBJECTIVE

Vitamin D deficiency (25-hydroxyvitamin D [25(OH)D] <50 nmol/L) is commonly reported in both children and adults worldwide, and growing evidence indicates that vitamin D deficiency is associated with many extraskeletal chronic disorders, including the autoimmune diseases type 1 diabetes and multiple sclerosis.

METHODS

We measured 25(OH)D concentrations in 720 case and 2,610 control plasma samples and genotyped single nucleotide polymorphisms from seven vitamin D metabolism genes in 8,517 case, 10,438 control, and 1,933 family samples. We tested genetic variants influencing 25(OH)D metabolism for an association with both circulating 25(OH)D concentrations and disease status.

RESULTS

Type 1 diabetic patients have lower circulating levels of 25(OH)D than similarly aged subjects from the British population. Only 4.3 and 18.6% of type 1 diabetic patients reached optimal levels (≥75 nmol/L) of 25(OH)D for bone health in the winter and summer, respectively. We replicated the associations of four vitamin D metabolism genes (GC, DHCR7, CYP2R1, and CYP24A1) with 25(OH)D in control subjects. In addition to the previously reported association between type 1 diabetes and CYP27B1 (P = 1.4 × 10(-4)), we obtained consistent evidence of type 1 diabetes being associated with DHCR7 (P = 1.2 × 10(-3)) and CYP2R1 (P = 3.0 × 10(-3)).

CONCLUSIONS

Circulating levels of 25(OH)D in children and adolescents with type 1 diabetes vary seasonally and are under the same genetic control as in the general population but are much lower. Three key 25(OH)D metabolism genes show consistent evidence of association with type 1 diabetes risk, indicating a genetic etiological role for vitamin D deficiency in type 1 diabetes.

We determined the quantitative relationships between graded oral dosing with vitamin D3, 25(OH)D3, and 1,25(OH)2D3 for short treatment periods and changes in circulating levels of these substances. The subjects were 116 healthy men (mean age, 28 +/- 4 years, with usual milk consumption of < or = 0.47 l/day and mean serum 25(OH)D of 67 +/- 25 nmol/l). They were distributed among nine open-label treatment groups: vitamin D3 (25, 250 or 1250 micrograms/day for 8 weeks), 25(OH)D3 (10, 20 or 50 micrograms/day for 4 weeks) and 1,25(OH)2D3 (0.5, 1.0 or 1.0 microgram/day for 2 weeks). All treatment occurred between January 3 and April 3. We measured fasting serum, calcium, parathyroid hormone, vitamin D3, 25(OH)D and 1,25(OH)2D immediately before and after treatment. In the three groups treated with vitamin D3, mean values for circulating vitamin D3 increased by 13, 137 and 883 nmol/l and serum 25(OH)D increased by 29, 146 and 643 nmol/l for the three dosage groups, respectively. Treatment with 25(OH)D3 increased circulating 25(OH)D by 40, 76 and 206 nmol/l, respectively. Neither compound changed serum 1,25(OH)2D levels. However, treatment with 1,25(OH)2D3 increased circulating 1,25(OH)2D by 10, 46 and 60 pmol/l, respectively. Slopes calculated from these data allow the following estimates of mean treatment effects for typical dosage units in healthy 70-kg adults: an 8-week course of vitamin D3 at 10 micrograms/day (400 IU/day) would raise serum vitamin D by 9 nmol/l and serum 25(OH)D by 11 nmol/l; a 4-week course of 25(OH)D3 at 20 micrograms/day would raise serum 25(OH)D by 94 nmol/l; and a 2-week course of 1,25(OH)2D3 at 0.5 microgram/day would raise serum 1,25(OH)2D by 17 pmol/l.

The resistance of parathyroid cells to 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in uremic hyperparathyroidism is thought to be caused, in part, by a 1,25(OH)2D3 receptor (VDR) deficiency in the parathyroids. However, results of biochemical studies addressing VDR numbers in the parathyroids are controversial. Several studies have found VDR content to be decreased in the parathyroids of uremic patients and animals, while others have found no such decrease in the parathyroids of uremic animals. To clarify the role of VDR, we investigated VDR distribution in surgically-excised parathyroids obtained from chronic dialysis patients by immunohistochemistry. We classified the parathyroids as exhibiting nodular or diffuse hyperplasia. Our studies demonstrated a lower density of VDR in the parathyroids showing nodular hyperplasia than in those showing diffuse hyperplasia. Even in the parathyroids showing diffuse hyperplasia, nodule-forming areas were present; these areas were virtually negative for VDR staining. A significant negative correlation was found between VDR density and the weight of the parathyroids. These findings indicate that the conflicting results of biochemical studies may be caused by the heterogeneous distribution of VDR; the decreased VDR density in parathyroids may contribute to the progression of secondary hyperparathyroidism and to the proliferation of parathyroid cells that is seen in uremia.

Metabolites of vitamin D3 (D3) (cholecalciferol) are recognized as enzymatically formed chemicals in humans that can influence a wide variety of reactions that regulate a large number of cellular functions. The metabolism of D3 has been extensively studied, and a role for three different mitochondrial cytochrome P450s (CYP24A, CYP27A, and CYP27B1) has been described that catalyze the formation of the 24(OH), 25(OH), and 1(OH) metabolites of D3, respectively. The hormone 1,25-dihydroxyvitamin D3 has been most extensively studied and is widely recognized as a regulator of calcium and phosphorous metabolism. Hydroxylated metabolites of D3 interact with the nuclear receptor and thereby influence growth, cellular differentiation, and proliferation. In this article, we describe in vitro experiments using purified mitochondrial cytochrome P450scc (CYP11A1) reconstituted with the iron-sulfer protein, adrenodoxin, and the flavoprotein, adrenodoxin reductase, and show the NADPH and time-dependent formation of two major metabolites of D3 (i.e., 20-hydroxyvitamin D3 and 20,22-dihydroxyvitamin D3) plus two unknown minor metabolites. In addition, we describe the metabolism of 7-dehydrocholesterol by CYP11A1 to a single product identified as 7-dehydropregnenolone. Although the physiological importance of these hydroxylated metabolites of D3 and their in vivo formation and mode of action remain to be determined, the rate with which they are formed by CYP11A1 in vitro suggests an important role.

We have investigated the molecular mechanism whereby 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] inhibits adipogenesis in vitro. 1,25(OH)2D3 blocks 3T3-L1 cell differentiation into adipocytes in a dose-dependent manner; however, the inhibition is ineffective 24-48 h after the differentiation is initiated, suggesting that 1,25(OH)2D3 inhibits only the early events of the adipogenic program. Treatment of 3T3-L1 cells with 1,25(OH)2D3 does not block the mitotic clonal expansion or C/EBPbeta induction; rather, 1,25(OH)2D3 blocks the expression of C/EBPalpha, peroxisome proliferator-activated receptor-gamma (PPARgamma), sterol regulatory element-binding protein-1, and other downstream adipocyte markers. The inhibition by 1,25(OH)2D3 is reversible, since removal of 1,25(OH)2D3 from the medium restores the adipogenic process with only a temporal delay. Interestingly, although the vitamin D receptor (VDR) protein is barely detectable in 3T3-L1 preadipocytes, its levels are dramatically increased during the early phase of adipogenesis, peaking at 4-8 h and subsiding afterward throughout the rest of the differentiation program; 1,25(OH)2D3 treatment appears to stabilize the VDR protein levels. Consistently, adenovirus-mediated overexpression of human (h) VDR in 3T3-L1 cells completely blocks the adipogenic program, confirming that VDR is inhibitory. Inhibition of adipocyte differentiation by 1,25(OH)2D3 is ameliorated by troglitazone, a specific PPARgamma antagonist; conversely, hVDR partially suppresses the transacting activity of PPARgamma but not of C/EBPbeta or C/EBPalpha. Moreover, 1,25(OH)2D3 markedly suppresses C/EBPalpha and PPARgamma mRNA levels in mouse epididymal fat tissue culture. Taken together, these data indicate that the blockade of 3T3-L1 cell differentiation by 1,25(OH)2D3 occurs at the postclonal expansion stages and involves direct suppression of C/EBPalpha and PPARgamma upregulation, antagonization of PPARgamma activity, and stabilization of the inhibitory VDR protein.

We investigated rearrangements of the hydrogen-bond network in water by measuring fluctuations in the OH-stretching frequency of HOD in liquid D2O with femtosecond infrared spectroscopy. Using simulations of an atomistic model of water, we relate these frequency fluctuations to intermolecular dynamics. The model reveals that OH frequency shifts arise from changes in the molecular electric field that acts on the proton. At short times, vibrational dephasing reflects an underdamped oscillation of the hydrogen bond with a period of 170 femtoseconds. At longer times, vibrational correlations decay on a 1.2-picosecond time scale because of collective structural reorganizations.

The spatial and temporal coordination of the many events required for osteogenic cells to create a mineralized matrix are only partially understood. The complexity of this process, and the nature of the final product, demand that these cells have mechanisms to carefully monitor events in the extracellular environment and have the ability to respond through cellular and molecular changes. The generation of inorganic phosphate during the process of differentiation may be one such signal. In addition to the requirement of inorganic phosphate as a component of hydroxyapatite mineral, Ca(10)(PO(4))(6)(OH)(2), a number of studies have also suggested it is required in the events preceding mineralization. However, contrasting results, physiological relevance, and the lack of a clear mechanism(s) have created some debate as to the significance of elevated phosphate in the differentiation process. More recently, a number of studies have begun to shed light on possible cellular and molecular consequences of elevated intracellular inorganic phosphate. These results suggest a model in which the generation of inorganic phosphate during osteoblast differentiation may in and of itself represent a signal capable of facilitating the temporal coordination of expression and regulation of multiple factors necessary for mineralization. The regulation of protein function and gene expression by elevated inorganic phosphate during osteoblast differentiation may represent a mechanism by which mineralizing cells monitor and respond to the changing extracellular environment.

The binding site for an open-channel blocker, QX-222, at mouse muscle nicotinic acetylcholine receptors was probed using site-directed mutagenesis, oocyte expression, and electrophysiological analysis. The proposed cytoplasmic end of the M2 transmembrane helix is termed position 1'. At position 10' (alpha S252, beta T263, gamma A261, delta A266), Ala residues yield stronger and longer binding of QX-222 than Ser or Thr residues. These effects are opposite and roughly equal (30%-50% per mutation) to previously reported effects at position 6'. The polar end of an anesthetic molecule seems to bind to the position 6' OH groups, which provide a water-like region; the nonpolar moiety is near position 10' and binds more strongly in a nonpolar environment. Interactions with adjacent OH-rich turns of an amphiphilic helix may explain the widespread blocking effects of local anesthetics at the conduction pore of ion channels.

1,25(OH)(2)D(3) has antiproliferative effects and promotes cell differentiation. This consideration has provided the rationale for studies in subtotally nephrectomized rats showing that 1,25(OH)(2)D(3) interfered with glomerulosclerosis. The cellular mechanisms involved have remained obscure, however. It was the purpose of the present study to assess glomerular structure and cellular composition in subtotally nephrectomized (SNX) rats treated with nonpharmacological doses of 1,25(OH)(2)D(3). Male Sprague-Dawley rats were sham operated (sham) or underwent SNX under general anesthesia and received either solvent or 1,25(OH)(2)D(3) (3 ng.100 g body wt(-1).day(-1) sc). Blood pressure (BP) and albuminuria were measured. After 16 wk, the remnant renal tissue was perfusion fixed and morphometric and stereological measurements were carried out. The expression of proliferating cellular antigen (PCNA), cyclin-dependent kinase inhibitor p27, Wilms tumor gene (WT1), and desmin, a marker of early podocyte damage, was investigated by immunohistology. BP, serum creatinine, and urinary albumin excretion were significantly higher in SNX than in sham rats. Albuminuria was significantly lower in SNX+1,25(OH)(2)D(3) compared with SNX+solvent rats. Mean glomerular tuft volume was significantly higher in SNX+solvent (2.69 +/- 0.21 gx 10(6) microm(3)) than in sham rats (1.44 +/- 0.17 and 1.28 +/- 0.14 x 10(6) microm(3)); it was significantly (P < 0.05) lower in SNX+1,25(OH)(2)D(3) rats (1.81 +/- 0.16 x 10(6) microm(3)). The main finding was a significantly higher number of podocytes in SNX+1,25(OH)(2)D(3) (88 +/- 9) and sham (98 +/- 17) compared with SNX+solvent rats (81 +/- 8.7). In parallel, the increase in podocyte volume in SNX+solvent rats was abrogated by treatment with 1,25(OH)(2)D(3), and immunohistochemistry revealed less expression of desmin, PCNA, and p27, suggesting less podocyte injury and activation of the cyclin cascade. This study identifies the podocyte as an important target cell for the renoprotective action of 1,25(OH)(2)D(3). This notion is suggested by less evidence of podocyte injury, decreased podocytes loss, and abrogation of podocyte hypertrophy, findings that may also explain less pronounced albuminuria and glomerulosclerosis.

Phosphatidylinositol 3-OH-kinase (PI3K) and STAT3 are signal transduction molecules activated by leptin in brain areas controlling food intake. To investigate their role in leptin-mediated inhibition of hypothalamic neuropeptide Y (Npy) and agouti-related peptide (Agrp) gene expression, male Sprague-Dawley rats (n = 5/group) were either fed ad libitum or subjected to a 52-h fast. At 12-h intervals, the PI3K inhibitor LY-294002 (LY, 1 nmol) or vehicle was injected intracerebroventricularly (ICV) as a pretreatment, followed 1 h later by leptin (3 microg icv) or vehicle. Fasting increased hypothalamic Npy and Agrp mRNA levels (P < 0.05), and ICV leptin administration prevented this increase. As predicted, LY pretreatment blocked this inhibitory effect of leptin, such that Npy and Agrp levels in LY-leptin-treated animals were similar to fasted controls. By comparison, leptin-mediated activation of hypothalamic STAT3 signaling, as measured by induction of both phospho-STAT3 immunohistochemistry and suppressor of cytokine signaling-3 (Socs3) mRNA, was not significantly attenuated by ICV LY pretreatment. Because NPY/AgRP neurons project to the hypothalamic paraventricular nucleus (PVN), we next investigated whether leptin activation of PVN neurons is similarly PI3K dependent. Compared with vehicle, leptin increased the number of c-Fos positive cells within the parvocellular PVN (P = 0.001), and LY pretreatment attenuated this effect by 35% (P = 0.043). We conclude that leptin requires intact PI3K signaling both to inhibit hypothalamic Npy and Agrp gene expression and activate neurons within the PVN. In addition, these data suggest that leptin activation of STAT3 is insufficient to inhibit expression of Npy or Agrp in the absence of PI3K signaling.

Reactive oxygen intermediates, i.e. the superoxide radical (O*-)(2), hydrogen peroxide (H2O2) and the hydroxyl radical (*OH), are generally regarded as harmful products of oxygenic metabolism causing cell damage in plants, animals and microorganisms. However, oxygen radical chemistry may also play a useful role in polymer breakdown leading to wall loosening during extension growth of plant cells controlled by the phytohormone auxin. Backbone cleavage of cell wall polysaccharides can be accomplished in vitro by (*OH) produced from H2O2 in a Fenton reaction or in a reaction catalyzed by peroxidase supplied with O2 and NADH. Here, we show that coleoptile growth of maize seedlings is accompanied by the release of reactive oxygen intermediates in the cell wall. Auxin promotes release of (O*-)(2) and subsequent generation of (*OH)when inducing elongation growth. Experimental generation of (*OH) in the wall causes an increase in wall extensibility in vitro and replaces auxin in inducing growth. Auxin-induced growth can be inhibited by scavengers of (O*-)(2), H2O2 or (*OH), or inhibitors interfering with the formation of these molecules in the cell wall. These results provide the experimental background for a novel hypothesis on the mechanism of plant cell growth in which (*OH), produced from (O*-)(2) and H2O2 by cell wall peroxidase, acts as a wall-loosening agent.

Dopaminergic mechanisms are thought to be critical in mediating relapse to cocaine-seeking behavior. This study examined the different roles of D1- and D2-like receptor mechanisms in the relapse process. Squirrel monkeys were given extended histories of i. v. cocaine self-administration under conditions in which responding was maintained jointly by response-contingent cocaine injections and a cocaine-paired visual stimulus (second-order schedule). Responding was then extinguished by substituting saline for cocaine injections and omitting presentations of the cocaine-paired stimulus. Subsequently, noncontingent priming injections of cocaine combined with restoration of the cocaine-paired stimulus induced dose-dependent reinstatement of drug-seeking behavior, with response rates approaching those maintained by active cocaine self-administration. The priming effects of cocaine were attenuated by several D1- and D2-like receptor antagonists and low efficacy agonists but not by the D3-preferring antagonists UH 232 and AJ-76. The priming effects of cocaine were mimicked by the D2-like receptor agonists R(-)-propylnorapomorphine hydrochloride (NPA) and quinpirole, less consistently by 7-OH-DPAT, and not by the D1-like receptor agonists SKF-81297 and SKF-82958, the D3-preferring agonist PD-128,907, or any low efficacy agonist. Cotreatment with NPA, PD-128,907, and 7-OH-DPAT did not alter reinstatement of drug-seeking behavior induced by a maximally effective priming dose of cocaine, whereas cotreatment with D1-like receptor agonists attenuated the priming effects of cocaine. The results suggest that D1- and D2-like receptors play fundamentally different roles in the relapse process. Although stimulation of D2-like, but probably not D3-like, receptors appears necessary for induction of relapse, either stimulation or blockade of D1-like receptors appears to be inhibitory with respect to relapse.