Vitamin D and its receptor during late development.

Expression of the vitamin D receptor (VDR) is widespread but may vary depending on the developmental stage of the animal, and therefore may differentially influence phenotypic function. Thus, the major role of the 1,25-dihydroxyvitamin D [1,25(OH)2D]/VDR system is to regulate mineral and skeletal homeostasis, although mainly after birth. Post-natally, under conditions of low dietary calcium, the 1,25(OH)2D/VDR system enhances intestinal transcellular transport of calcium and possibly paracellular calcium entry by regulating genes that are critical for these functions. This process, by providing adequate calcium, is essential for normal development of the skeletal growth plate and mineralization of bone. Furthermore, blood calcium and phosphorus homeostasis is maintained by an interplay between feedback loops of the 1,25(OH)2D/VDR system with parathyroid hormone and with fibroblast-growth factor (FGF) 23 respectively. The 1,25(OH)2D/VDR system can also modulate the expression of genes involved in both bone formation and resorption post-natally. Ligand independent activity of the VDR normally influences mammalian hair cycling after birth by potentiating Wnt and bone morphogenetic protein (BMP) signaling. Nevertheless ligand bound VDR may also modulate epidermal cell proliferation/differentiation by regulating the balance in function of c-MYC and its antagonist the transcriptional repressor MAD1/MXD1 in skin epithelia. The 1,25(OH)2D/VDR system can also modulate innate immune cells and promote a more tolerogenic immunological status and may therefore influence inflammation and the development of autoimmunity; whether this impacts the fetus is uncertain. This article is part of a Special Issue entitled: Nuclear receptors in animal development.

Differential effect of vitamin D on NOD2- and TLR-induced cytokines in Crohn's disease.

Accumulating evidence implicates defective innate immunity in the pathogenesis of Crohn's disease (CD). Ineffectual NOD2 (nucleotide-binding oligomerization domain 2) is the most common susceptibility gene contributing to CD. Vitamin D (vD), a potent modulator of innate and adaptive immunity, induces NOD2 gene expression and its downstream function. We hypothesized that the hormonal form of vD (1,25D) could beneficially modulate innate immune function in CD. Using peripheral mononuclear cells and monocyte-derived dendritic cells (Mo-DCs) from CD, it was found that 1,25D decreased Toll-like receptor (TLR)-induced cytokine production and enhanced cytokine levels induced by muramyl dipeptide (MDP), the NOD2 ligand. 1,25D increased the synergistic effect provided by NOD2 and TLR co-activation on interleukin (IL)-10, IL-23, and tumor necrosis factor-alpha (TNF-α). Whereas 1,25D inhibits Mo-DC TLR-induced cytokines, co-stimulation of NOD2 results in increased IL-10 and IL-23. IL-12p70 was completely abrogated by 1,25D. 1,25D similarly modulated cytokine production by immune cells in ulcerative colitis patients and healthy controls. Mo-DCs from CD patients heterozygous for NOD2 mutations had a response similar to those from patients without NOD2 mutations. Immune cells from patients homozygous for the 1007 fs mutation were unresponsive to MDP and 1,25D. Our in vitro data support 1,25D as a potential modulator of immunity. However, these results cannot be extrapolated to CD patients without further controlled studies.

Induction of kinase suppressor of RAS-1(KSR-1) gene by 1, alpha25-dihydroxyvitamin D3 in human leukemia HL60 cells through a vitamin D response element in the 5'-flanking region.

Differentiation therapy is being developed as an additional therapeutic option for the treatment of several forms of cancer, including myeloid leukemia. In model systems, the physiologically active form of vitamin D, 1, alpha25-dihydroxyvitamin D3 (1,25D), induces monocytic differentiation of human myeloid cells, but the mechanism is not clear. We report here, the first direct connection between the signal provided by 1,25D and the molecular circuitry known to be involved in monocytic differentiation. Specifically, we show that 1,25D selectively increases the expression of the gene encoding kinase suppressor of Ras-1 (KSR-1) in HL60 cells, while other differentiation-inducing agents such as 12-O-tetradecanoylphorbol-13-acetate, retinoic acid or dimethyl sulfoxide do not significantly increase KSR-1 expression. Further, the upregulation of KSR-1 gene by 1,25D is competed by ZK159222, an antagonist of vitamin D receptor (VDR) action, and can occur in the presence of protein synthesis inhibitor cycloheximide, showing that the effect is direct. Most importantly, we have identified a vitamin D responsive element (VDRE) in the promoter region of the human KSR-1 gene, to which VDR binds in a 1,25D-dependent manner, in vitro and in vivo. This binding is paralleled by increased association of RNA polymerase II with the transcription start site of KSR-1 gene, and the VDRE is functional in reporter assays. Our findings offer a potential mechanism for a signaling pathway that contributes to 1,25D-induced monocytic differentiation of human myeloid leukemia cells.

Rituxan (anti-CD20 antibody)-induced translocation of CD20 into lipid rafts is crucial for calcium influx and apoptosis.

Rituxan, a chimeric anti-CD20 antibody, is the first antibody approved for immunotherapy in non-Hodgkin's B-cell lymphoma and other B-cell lymphoproliferative disorders. Additionally, efficacy of Rituxan treatment has been reported in nonmalignant autoimmune diseases such as rheumatoid arthritis. Crosslinking of CD20 molecules by Rituxan induces therapeutic B-cell depletion. CD20 is a B-lymphocyte specific integral membrane protein, proposed to function as a store-operated calcium channel, which is activated upon receptor-stimulated calcium depletion of intracellular stores. Crosslinking of CD20 by antibodies has been reported to induce a redistribution of CD20 molecules to specialized microdomains at the plasma membrane known as lipid rafts. Here, we report that in the absence of Rituxan, CD20 exhibits a low affinity to lipid rafts. However, binding of Rituxan significantly increases the affinity of CD20 for lipid rafts resulting in its redistribution to a fraction resistant to Triton X-100 solubilization. Furthermore, we demonstrate that disturbing the raft integrity by cholesterol extraction results in dissociation of CD20 from a Triton X-100 resistant fraction followed by complete inhibition of Rituxan-induced calcium entry and apoptosis. The integrity of lipid rafts seems to play a crucial role for CD20-induced caspase activation. These data show, for the first time, that Rituxan-induced translocation of CD20 to lipid rafts is important for increased intracellular Ca(2+) levels and downstream apoptotic signalling.

Interactions between G-protein-coupled receptors and periplakin: a selective means to regulate G-protein activation.

A substantial number of G-protein-coupled receptor-interacting proteins have been identified initially by the use of yeast two-hybrid screens. Using the C-terminal tail of both opioid receptors and the melanin concentrating hormone receptor-1 as bait, the actin and intermediate filament-binding protein periplakin was isolated. In each case, the site of interaction is within helix VIII of the receptor and periplakin limits agonist-mediated G-protein activation potentially by competing with G-protein for this region of the receptor.

Finite-element analysis of the displacement of closed DNA loops under torsional stress.

Closed DNA loops that contain intrinsic curvature occur in biologically important structures that are formed by bringing together proteins attached at distinct sites. Such loops constitute topological domains that are characterized by a linking number Delta Lk. We calculate, using finite-element analysis, the structural changes induced by small changes in this linking number, Delta Lk. Because of the intrinsic curvature, the slightest change in linking number induces writhe and the loop begins to fold in space. We previously studied the case in which the initial curvature is uniformly distributed along the DNA rod. We found that there are two different folding modes, depending on the amount of intrinsic curvature and the Poisson ratio, a quantity that measures the ratio of bending stiffness to torsional rigidity. For combinations of the Poisson ratio and curvature that lie below a critical curve, called the Fickel curve, the folding is monotonic in the sense that the writhe uniformly increases as Delta Lk increases, until self-contact occurs. For combinations below this curve, the folding is non-monotonic in the sense that as Delta Lk increases the writhe first increases, then decreases back to essentially zero, and then increases uniformly until self-contact occurs. The folding behaviour and the self-contact points in the two folding modes are completely different. In this paper we first review this previous work. We then extend those results to more-complex situations in which the curvature is initially distributed non-uniformly along the DNA rod. We show that the location of the Fickel curve depends upon both the extent of the initial curvature and upon its distribution along the rod. We also show that two DNAs with the same total intrinsic curvature will fold differently depending upon the distribution of that curvature along the DNA axis, and upon the point of the loop at which the applied rotation or change in Delta Lk is introduced.

Imaging of sacral fractures.

The article discusses traumatic, insufficiency and pathological sacral fractures. Special attention is paid to the biomechanics and subsequent classification of traumatic sacral fractures.

Agonist-bound nuclear receptors: not just targets of coactivators.

Members of the nuclear receptor superfamily of ligand-regulated transcription factors are targets of a wide range of lipophilic signaling molecules as well as several drugs and xenobiotics that modulate many aspects of physiology and metabolism. Agonist binding to receptors is associated with recruitment of coactivators, which are essential for activation of target gene transcription. However, several biochemical and molecular genetic studies have shown that a full understanding of the function of agonist-bound receptors must also accommodate the recruitment of corepressors. These factors may attenuate agonist-induced transactivation, act more transiently as part of a cycle of cofactors recruited to target promoters by ligand-bound receptors, or function in hormone-dependent repression of target gene expression.

Effects of a polymer-coated urea product on nitrogen metabolism in lactating Holstein dairy cattle.

The purpose of this study was to evaluate the impact of polymer-coated urea on nitrogen retention, rumen microbial growth, and milk production and composition. Coated urea (CU) that is more slowly hydrolyzed to ammonia than unprotected urea could potentially be used more efficiently by rumen microorganisms. Eight cows were offered each of three diets in a randomized crossover design. Each treatment period consisted of a 14-d adjustment period and a 5-d collection period. Diets were formulated to maintain milk production while reducing plasma urea nitrogen concentrations and urinary nitrogen excretion. Diets consisted of corn silage, mixed grass/legume haylage, chopped alfalfa hay, corn meal, protein, vitamin and mineral supplements, in a total mixed ration and fed ad libitum. The diets contained 17.9%, 18.1%, and 16.4% CP and 0, 0.77%, and 0.77% CU (dry matter basis) and are denoted as CP18-CU, CP18+CU, and CP16+CU, respectively. Individual feed intakes were measured, and total fecal, and urine collections were conducted. Cows were milked twice daily at 0500 and 1700 h, and the milk sampled for composition and milk urea N analysis. Dry matter intake averaged 23.5 +/- 0.2 kg/d and was not altered by diet. Also, milk fat and true protein were not altered by diet and averaged 3.72 and 3.07%, respectively. Milk yield was highest for diets CP18-CU and CP18+CU. Significant differences were observed in N intake and excretion in urine, feces, and milk between dietary treatments. Cows fed CP16+CU consumed 11% less N than in CP18-CU. Cows fed CP18+CU showed the highest excretion of N in urine, and together with CP16+CU, the lowest N excretion in feces. Nitrogen excretion in milk was lower for cows fed CP16+CU. Calculated N balance was not significantly different between diets nor was it significantly different from zero. Efficiency of N capture in milk protein as a function of N intake was higher for animals on CP16+CU. Urinary excretion of purine derivatives was not different between diets, and estimated microbial CP was also similar. Coated urea was not effective at reducing nitrogen excretion by dairy cattle.

Detection and characterization of Pasteuria 16S rRNA gene sequences from nematodes and soils.

Various bacterial species in the genus Pasteuria have great potential as biocontrol agents against plant-parasitic nematodes, although study of this important genus is hampered by the current inability to cultivate Pasteuria species outside their host. To aid in the study of this genus, an extensive 16S rRNA gene sequence phylogeny was constructed and this information was used to develop cultivation-independent methods for detection of Pasteuria in soils and nematodes. Thirty new clones of Pasteuria 16S rRNA genes were obtained directly from nematodes and soil samples. These were sequenced and used to construct an extensive phylogeny of this genus. These sequences were divided into two deeply branching clades within the low-G + C, Gram-positive division; some sequences appear to represent novel species within the genus Pasteuria. In addition, a surprising degree of 16S rRNA gene sequence diversity was observed within what had previously been designated a single strain of Pasteuria penetrans (P-20). PCR primers specific to Pasteuria 16S rRNA for detection of Pasteuria in soils were also designed and evaluated. Detection limits for soil DNA were 100-10,000 Pasteuria endospores (g soil)(-1).

The gamma-aminobutyric acid receptor B, but not the metabotropic glutamate receptor type-1, associates with lipid rafts in the rat cerebellum.

Recent evidence suggests that specialized microdomains, called lipid rafts, exist within plasma membranes. These domains are enriched in cholesterol and sphingolipids and are resistant to non-ionic detergent-extraction at 4 degrees C. They contain specific populations of membrane proteins, and can change their size and composition in response to cellular signals, resulting in activation of signalling cascades. Here, we demonstrate that both the metabotropic gamma-aminobutyric acid receptor B (GABA(B) receptor) and the metabotropic glutamate receptor-1 from rat cerebellum are insoluble in the non-ionic detergent Triton X-100. However, only the GABA(B) receptor associates with raft fractions isolated from rat brain by sucrose gradient centrifugation. Moreover, increasing the stringency of isolation by decreasing the protein : detergent ratio caused an enrichment of the GABA(B) receptor in raft fractions. In contrast, depletion of cholesterol from cerebellar membranes by either saponin or methyl-beta-cyclodextrin treatment, which solubilize known raft markers, also increased the solubility of the GABA(B) receptor. These properties are all consistent with an association of the GABA(B) receptor with lipid raft microdomains.

Protein-protein interactions at G-protein-coupled receptors.

The basic module of signal transduction that involves G-protein-coupled receptors is usually portrayed as comprising a receptor, a heterotrimeric G protein and an effector. It is now well established that regulated interactions between receptors and arrestins, and between G proteins and regulators of G-protein signalling alter the effectiveness and kinetics of information transfer. However, more recent studies have begun to identify a host of other proteins that interact selectively with individual receptors at both the intracellular and extracellular face of the membrane. Although the functional relevance of many of these interactions is only beginning to be understood, current information indicates that these interactions might determine receptor properties, such as cellular compartmentalization or signal selection, and can promote protein scaffolding into complexes that integrate function.

Regulation of gene Expression by 1alpha,25-dihydroxyvitamin D3 and Its analog EB1089 under growth-inhibitory conditions in squamous carcinoma Cells.

Analogs of 1alpha,25-dihydroxyvitamin D(3) (1alpha, 25(OH)2D3) inhibit growth in vitro and in vivo of cells derived from a variety of tumors. Here, we examined the effects of 1alpha,25(OH)2D3 and its analog EB1089 on proliferation and target gene regulation of human head and neck squamous cell carcinoma (SCC) lines SCC4, SCC9, SCC15, and SCC25. A range of sensitivities to 1alpha,25(OH)2D3 and EB1089 was observed, from complete G0/G1 arrest of SCC25 cells to only 50% inhibition of SCC9 cell growth. All lines expressed similar levels of vitamin D3 receptor (VDR) mRNA and protein, and no significant variation was observed in 1alpha,25(OH)2D3-dependent induction of the endogenous 24-hydroxylase gene, or of a transiently transfected 1alpha,25(OH)2D3-sensitive reporter gene. The antiproliferative effects of 1alpha,25(OH)2D3 and EB1089 in SCC25 cells were analyzed by screening more than 4,500 genes on two cDNA microarrays, yielding 38 up-regulated targets, including adhesion molecules, growth factors, kinases, and transcription factors. Genes encoding factors implicated in cell cycle regulation were induced, including the growth arrest and DNA damage gene, gadd45alpha, and the serum- and glucocorticoid-inducible kinase gene, sgk. Induction of GADD45alpha protein in EB1089-treated cells was confirmed by Western blotting. Moreover, while expression of proliferating cell nuclear antigen (PCNA) was reduced in EB1089-treated cells, coimmunoprecipitation studies revealed increased association between GADD45alpha and PCNA in treated cells, consistent with the capacity of GADD45alpha to stimulate DNA repair. While 1alpha,25(OH)2D3 and EB1089 modestly induced transcripts encoding the cyclin-dependent kinase inhibitor p21(waf1/cip1), no changes in protein levels were observed, indicating that p21(waf1/cip1) induction does not contribute to the antiproliferative effects of 1alpha,25(OH)2D3 and EB1089 in SCC cells. Finally, in partially resistant SCC9 cells, there was extensive loss of target gene regulation (10 of 10 genes tested), indicating that resistance arises from widespread loss of 1alpha,25(OH)2D3-dependent gene regulation in the presence of normal levels of functional VDRs.

A phenomenological exploration of loneliness in the older adult.

Loneliness is an important phenomenon of concern for psychiatric mental health nursing. Research findings have related loneliness in older adults to mental health problems especially depression. However, little is known about loneliness from the perspective of older adults living in the community. A phenomenological inquiry using Giorgi's research methodology was undertaken to explore and describe the meaning of loneliness for older adults. Twenty participants, most who are over the age of 75, were interviewed and 1385 naïve meaning units were extrapolated from tape-recorded verbatim transcripts. These units were clustered into five major themes describing the loneliness experience. The findings are described and discussed relative to the literature on loneliness. Implications for mental health nursing are presented.

Action of low calcemic 1alpha,25-dihydroxyvitamin D3 analogue EB1089 in head and neck squamous cell carcinoma.

BACKGROUND: 1alpha,25-Dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] and its analogues inhibit growth of various types of cancer cells. Although the therapeutic potential of 1,25(OH)(2)D(3) is limited by its tendency to induce hypercalcemia, analogues such as EB1089 are potent inhibitors of cell growth and exhibit reduced calcemic effects. We analyzed the antiproliferative and calcemic effects of EB1089 in tissue culture and animal models of head and neck squamous cell carcinoma (SCC) to investigate its potential as a chemotherapeutic/chemopreventive agent. METHODS: The effects of 1,25(OH)(2)D(3) and EB1089 on cell growth and expression of p21(WAF1/CIP1) and p27(KIP1), which encode cyclin-dependent kinase inhibitors, and a novel target, gadd45alpha, a growth-arrest and DNA-damage gene, were monitored in cultured murine AT-84 SCC cells. The effects of these agents on AT-84 cell growth in vitro and on growth of AT-84 tumors in syngeneic C3H mice were monitored; treatment started at the time of tumor implantation (early tumor model) or after 12 days (late tumor model). Weight and serum calcium levels were also monitored in these animals. All P values were two-sided. RESULTS: Both 1,25(OH)(2)D(3) and EB1089 arrested proliferation of AT-84 cells in G(0)/G(1) phase, inhibited p21(WAF1/CIP1) expression, and induced expression of p27(KIP1) protein. 1,25(OH)(2)D(3) also enhanced the expression of gadd45alpha, apparently by a p53-independent mechanism. There was a statistically significant decrease in tumor growth for 1,25(OH)(2)D(3)-treated mice (P<.001 for early tumor model) and EB1089-treated mice (P<.001 and P =.001 for early and late tumor models, respectively). Unlike 1,25(OH)(2)D(3), EB1089 did not induce cachexia or hypercalcemia. The effects of 1,25(OH)(2)D(3) and EB1089 on expression of p21(WAF1/CIP1) and GADD45alpha were similar in tumors and in vitro. CONCLUSIONS: EB1089 completely inhibited growth of AT-84 SCC cells at nanomolar concentrations, reduced tumor growth, and did not have calcemic effects. Our results support continued investigation of EB1089 as a chemopreventive/chemotherapeutic agent for head and neck SCC.

Advances in the molecular understanding of GABA(B) receptors.

The molecular nature of the metabotropic GABA(B) receptor was for some time a mystery, however it was recently discovered that two related G-protein-coupled receptors have to heterodimerize to form the functional GABA(B) receptor at the cell surface. This review discusses the most recent findings in the rapidly expanding field of GABA(B) receptor research, and includes a summary of all splice variants of both receptor subunits identified to date. It also evaluates emerging evidence that certain splice variants might play a role in determining pharmacologically distinguishable receptors, and reviews receptor localization at the sub-cellular level and involvement in neuronal development.

Amphiregulin is a vitamin D3 target gene in squamous cell and breast carcinoma.

1alpha,25-Dihydroxyvitamin D(3) [1,25(OH)2D3] inhibits growth of cells derived from a variety of tumors in vitro and in vivo. Proliferation in vitro of human SCC25 cells, derived from a primary squamous cell carcinoma (SCC) of the tongue, was blocked by 1,25(OH)2D3 and its analog EB1089. A similar effect was observed with 13-cis retinoic acid (RA), which has been used in chemoprevention of SCC. We identified amphiregulin, a member of the epidermal growth factor family, as a 1,25(OH)2D3 target gene in SCC25 cells. Induction of amphiregulin mRNA by 1,25(OH)2D3 was rapid and sustained over 48 h, and was unaffected by cycloheximide. 1,25(OH)2D3 also induced amphiregulin mRNA in estrogen receptor-positive and -negative human breast cancer cell lines, but not in LNCaP human prostate cancer cells. RAR- or RXR-specific retinoids did not affect amphiregulin mRNA levels in SCC25 cells; however, 13-cis RA partially blocked the response to 1,25(OH)2D3. Amphiregulin partially inhibited growth of SCC25 cells in culture. Our data show that amphiregulin is a 1,25(OH)2D3 target gene, and suggest that its induction may contribute to the growth inhibitory effects of 1,25(OH)2D3.

Analysis of the P3 promoter of the human parathyroid hormone (PTH)/PTH-related peptide receptor gene in pseudohypoparathyroidism type 1b.

Hypocalcemia and hyperphosphatemia caused by PTH resistance are the only discernible abnormalities in pseudohypoparathyroidism type 1b (PHP-1b). Because of the selective resistance toward PTH, inactivating mutations in its receptor, the PTH/PTH-related peptide receptor (PTHR1), were thought to be responsible for PHP-1b. However, gene abnormalities responsible for PHP-1b have not been identified in the coding region and well conserved promoters (P1 and P2) of the PTHR1 gene. The purpose of the present study was to analyze the structure of the P3 promoter, the main promoter of the human PTHR1 gene in kidney, in patients with PHP-1b. Southern analysis of genomic DNA from lymphoblastoid cell lines of eight nonfamilial patients with PHP-1b revealed neither gross rearrangements nor methylation abnormalities in the P3 promoter region of the PTHR1 gene. Sequencing revealed no abnormalities in the P3 promoter region, although one patient was homozygous for an (AAAG)n polymorphic variant. In conclusion, despite the selective resistance toward PTH in the kidney, which mainly uses the PTHR1 P3 promoter, PHP-1b in eight cases is not associated with structural abnormalities in this promoter. This study also indicates that inactivation of the P3 promoter is not achieved by methylation as tested in patients' genomic DNA from lymphoblastoid cell lines. The influence of alterations in the polymorphic A-rich repeat sequence on promoter activity warrants further study.

The unique tryptophan residue of the vitamin D receptor is critical for ligand binding and transcriptional activation.

The human vitamin D receptor (hVDR) is a member of the nuclear receptor superfamily of transcriptional regulators. Here we show that tryptophan 286 of the hVDR is critical for ligand binding and transactivation of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] target genes. Two mutants of the hVDR were produced, W286A and W286F, in which the tryptophan was replaced with an alanine or a phenylalanine, respectively. The W286A mutant did not bind 1,25(OH)2D3, interact with steroid receptor coactivator 1 (SRC-1) in vitro, or activate transcription. Moreover, the W286A receptor did not heterodimerize in a ligand-dependent manner with the human retinoid X receptor alpha (hRXRalpha). Although the W286F receptor heterodimerized with hRXRalpha, interacted with SRC-1, and bound 1,25(OH)2D3, its capacity to transactivate was attenuated severely. Thus, tryptophan 286 of hVDR plays an important role in specific 1,25(OH)2D3 ligand interaction and subsequently in hVDR/RXR interaction, SRC-1 binding, and ligand-dependent transactivation of 1,25(OH)2D3 target genes. These results identify the first amino acid that is absolutely required for ligand binding in the VDR and further define the structure-function relationship of 1,25(OH)2D3 interaction with its receptor.