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Ciesielski F Rochel N Moras D 《The Journal of steroid biochemistry and molecular biology》2007,103(3-5):235-242
The crystal structure of the ligand binding domain (LBD) of the wild-type Vitamin D receptor (VDR) of zebrafish bound to Gemini, a synthetic agonist ligand with two identical side chains branching at carbon 20 reveals a ligand-dependent structural rearrangement of the ligand binding pocket (LBP). The rotation of a Leu side chain opens the access to a channel that can accommodate the second side chain of the ligand. The 25% increase of the LBP's volume does not alter the essential agonist features of VDR. The possibility to adapt the LBP to novel ligands with different chemistry and/or structure opens new perspectives in the design of more specifically targeted ligands. 相似文献
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Atsuko Takeuchi Yuki Ishida Hiroko Sekimoto Sonoko Masuda Toshio Okano Soroku Nishiyama Ichiro Matsuda Tadashi Kobayashi 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1997,691(2):1987
A simplified method for the determination of 25-hydroxy and 1α,25-dihydroxy metabolites of vitamins D2 and D3 in human plasma was developed. Plasma samples were deproteinizated and applied to a Bond Elut C18 OH cartridge to separate 25-hydroxyvitamin D (25-OH-D) and 1α-25-dihydroxyvitamin D [1,25(OH)2D] fractions. The 25-OH-D fraction was purified by a Bond Elut C18 cartridge and 25-OH-D2 and 25-OH-D3 were assayed by HPLC using a Zorbax SIL column. The 1,25(OH)2D fraction obtained above was subsequently applied to HPLC using a Zorbax SIL column to separate 1,25(OH)2D2 and 1,25(OH)2D3 fractions which were determined by a radioreceptor assay (RRA) using calf thymus receptor. The method was applied to nutritional studies. 相似文献
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The active metabolite of vitamin D, 1alpha,25-dihydroxyvitamin D(3), suppresses autoimmune disease in several animal models including experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. The molecular mechanism of this immunosuppression is at present unknown. While 1alpha,25-dihydroxyvitamin D(3) is believed to function through a single vitamin D receptor, there are reports of other vitamin D receptors as well as a "nongenomic" mode of action. We have prepared the EAE model possessing the vitamin D receptor null mutation and determined if 1alpha,25-dihydroxyvitamin D(3) can suppress this disease in the absence of a functional vitamin D receptor. Vitamin D receptor null mice develop EAE although the incidence rate is one-half that of wild-type controls. The administration of 1alpha,25-dihydroxyvitamin D(3) had no significant effect on the incidence of EAE in the vitamin D receptor null mice, while it completely blocked EAE in the wild-type mice. We conclude that 1alpha,25-dihydroxyvitamin D(3) functions to suppress EAE through the well-known VDR and not through an undiscovered receptor or through a "nongenomic" mechanism. 相似文献
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David G. Mottershead Patsie Polly Ruth J. Lyons Robert L. Sutherland Colin K.W. Watts 《Journal of cellular biochemistry》1996,61(3):325-337
The effects of 1α,25(OH)2vitamin D3 on cell growth and differentiation are primarily mediated by the nuclear vitamin D receptor (VDR). In order to study aspects of receptor function and ultimately the structural basis of the VDR-ligand interaction, it is necessary to produce large quantities of purified VDR. To achieve this, we have expressed the human VDR and its ligand binding domain in E. coli as fusion proteins with the maltose binding protein using the expression vector pMal-c2. In this system high level expression of both fusion proteins in a soluble form was achieved, whereas previous attempts to express the VDR in E. coli have resulted in an insoluble product. After affinity purification on amylose resin, the fusion proteins were isolated with yields of 10–20 mg/l of culture. Both forms of the recombinant receptor bound 1α,25(OH)2vitamin D3 with high affinity; estimated Kd values from Scatchard analysis for the purified full-length receptor and the ligand binding domain were 0.16 ± 0.07 nM and 0.04 ± 0.02 nM, respectively. The nonhypercalcemic analogs of vitamin D, MC903 and Δ22-1,25S,26(OH)3vitamin D3, bound the recombinant fusion proteins with a similar affinity to the native ligand, 1α,25(OH)2vitamin D3. In addition, the full-length VDR fusion protein was shown by gel shift analysis to bind weakly to the human osteocalcin gene vitamin D response element, an interaction greatly facilitated by addition of RXRα. These results show that the bacterial expression system detailed here is readily able to produce soluble and functional VDR and its ligand binding domain in high yield. These proteins are easily purified and should be suitable for further structural and functional analysis. © 1996 Wiley-Liss, Inc. 相似文献
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Pseudonocardia autotrophica converted Vitamin D(3) to 25-hydroxyvitamin D(3) and 1alpha,25-dihydroxyvitamin D(3). The hydroxylation of Vitamin D(3) with P. autotrophica was enhanced by the addition of cyclodextrin. In this microbial hydroxylation, a new Vitamin D(3) metabolite was observed in the reaction mixture of P. autotrophica and Vitamin D(3), and was isolated in a pure form by several steps of chromatography. The structure of the new metabolite was determined to be 2alpha,25-dihydroxyvitamin D(3) by UV, NMR and mass spectroscopic analyses. Biological evaluation of the new metabolite was conducted by means of several experiments. 相似文献
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Masaki Ishigai Yoshinori Asoh Kenji Kumaki 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1998,706(2):66
A sensitive and specific liquid chromatographic–mass spectrometric assay has been developed for the determination of 22-oxacalcitriol (OCT), which is a new analog of 1α,25-dihydroxyvitamin D3. The analyte was isolated from serum by two solid-phase extraction steps on a C18 cartridge and NH2 cartridge. The recovery of OCT through two extraction steps was more than 90%. A related substance (ED-94), i.e. OCT with the side-chain shortened by one carbon, was used as an internal standard. Extracts were chromatographed on a C18 reversed-phase column interfaced to the electrospray ionization source. The mass spectrometer was operated in the positive-ion mode of selected reaction monitoring. The chromatographic run-time for one injection was less than 6 min. The intra- and inter-assay coefficients of variation for the lowest concentration examined (30 pg ml−1) were 9.83 and 10.67, respectively. And the analytical recovery of OCT added to serum was quantitative. Assay linearity was obtained in the range of 20–640 pg ml−1. 相似文献
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Rochel N Hourai S Pérez-García X Rumbo A Mourino A Moras D 《Archives of biochemistry and biophysics》2007,460(2):172-176
The crystal structures of vitamin D nuclear receptor (VDR) have revealed that all compounds are anchored by the same residues to the ligand binding pocket (LBP). Based on this observation, a synthetic analog with a locked side chain (21-nor-calcitriol-20(22),23-diyne) has been synthesized in order to gain in entropy energy with a predefined active side chain conformation. The crystal structure of VDR LBD bound to this locked side chain analogue while confirming the docking provides a structural basis for the activity of this compound. 相似文献
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Cristiana Almerighi Anna Sinistro Antonella Cavazza Chiara Ciaprini Giovanni Rocchi Alberto Bergamini 《Cytokine》2009,45(3):190-197
CD40 ligand (CD40L) stimulation induces proinflammatory and immunomodulatory activity in monocytes. Here, we report on the effects of the steroid hormone 1α,25-dihydroxyvitamin D3 (1,25D3) on human blood monocytes that have been stimulated with the CD40L ligand. Co-treatment of CD40L-stimulated monocytes with 1,25D3 resulted in reduced production and secretion of tumor necrosis factor (TNF)-α and interleukin (IL)-1β, as well as in reduced expression of the surface co-stimulatory molecules CD80 and CD86. In addition, costimulation of CD4+ T lymphocytes by monocytes co-treated with CD40L and 1,25D3 resulted in reduced cell proliferation and diminished interferon (IFN)-γ but enhanced IL-10 production by CD4+ T cells. Finally, 1,25D3 interfered with the ability of CD40L to rescue monocytes from apoptosis induced by serum withdrawal. These findings suggest that 1,25D3 may regulate the interaction of monocytes with T cells or other cell types that express CD40L, thus influencing the outcome of the immune or inflammatory response. 相似文献
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1,25-dihydroxyvitamin D3 receptor is upregulated in aortic smooth muscle cells during hypervitaminosis D 总被引:2,自引:0,他引:2
Several studies have demonstrated that excess of vitamin D3 is toxic particularly to vascular tissues. A notable pathological feature is arterial calcification. The nature of the toxic metabolite in hypervitaminosis D and the pathogenesis of arterial calcification are not clearly understood. The present study was undertaken to explore whether arterial calcification is a sequel of increased calcium uptake by arterial smooth muscle mediated by up regulation of vitamin D receptor in the cells in response to elevated circulating levels of vitamin D3 in serum. The experimental study was performed in 20 New Zealand white female rabbits aged 6 months. Animals in the test group were injected 10,000 IU of cholecalciferol intramuscularly twice a week for one month. Six control animals were given intra-muscular injections of plain cottonseed oil. Animals were sacrificed and aortas were examined for pathological lesions, 1,25-dihyroxyvitamin D3 (1,25(OH)2 D3) receptor levels and 45Ca uptake in smooth muscle cells. Serum samples collected at intervals were assayed for levels of 25-OH-D3 and calcium. The results showed that in animals given injections of cholecalciferol, serum levels of 25-OH-D3 were elevated. In four of these animals calcification and aneurysmal changes were seen in the aorta. Histological lesions comprised of fragmentation of elastic fibers as well as extensive loss of elastic layers. 1,25(OH)2 D3 receptor levels were up regulated and 45Ca uptake enhanced in aortas of animals which were given excessive vitamin D3. The evidences gathered suggest that excess vitamin D is arteriotoxic and that the vitamin induces arterial calcification through up regulation of 1,25(OH)2D3 receptor and increased calcium uptake in smooth muscle cells of the arteries. 相似文献
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Carlos Gregorio Silvina Eduardo Luis C. Rodrigues María A. Regueira Ramn Fraga Ricardo Riveiros Miguel Maestro Antonio Mourio 《The Journal of steroid biochemistry and molecular biology》2007,103(3-5):227
Hapten derivatives of 25-hydroxyvitamin D3 and 1α,25-dihydroxyvitamin D3 were synthesized using the Wittig–Horner approach. Both haptens bearing a carboxylic group at the side chain that can be linked to a protein for raising antibodies of potential utility for the determination of 25-hydroxyvitamin D3, 1α,25-dihydroxyvitamin D3 and 1α-hydroxylated vitamin D3 analogues. 相似文献
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Susumi Hatakeyama Satoshi Nagashima Naoko Imai Keisuke Takahashi Jun Ishihara Atsuko Sugita Takeshi Nihei Hitoshi Saito Fumiaki Takahashi Noboru Kubodera 《The Journal of steroid biochemistry and molecular biology》2007,103(3-5):222
1α,25-Dihydroxy-2β-(3-hydroxypropoxy)vitamin D3 (ED-71), an analog of active vitamin D3, 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3], possesses a hydroxypropoxy substituent at the 2β-position of 1,25(OH)2D3. ED-71 has potent biological effects on bone and is currently under phase III clinical studies for bone fracture prevention. It is well-known that the synthesis and secretion of parathyroid hormone (PTH) is regulated by 1,25(OH)2D3. Interestingly, during clinical development of ED-71, serum intact PTH in osteoporotic patients did not change significantly upon treatment with ED-71. The reason remains unclear, however. Brown et al. reported that 3-epi-1,25(OH)2D3, an epimer of 1,25(OH)2D3 at the 3-position, shows equipotent and prolonged activity compared to 1,25(OH)2D3 at suppressing PTH secretion. Since ED-71 has a bulky hydroxypropoxy substituent at the 2-position, epimerization at the adjacent and sterically hindered 3-position might be prevented, which may account for its weak potency in PTH suppression observed in clinical studies. We have significant interest in ED-71 epimerization at the 3-position and the biological potency of 3-epi-ED-71 in suppressing PTH secretion. In the present studies, synthesis of 3-epi-ED-71 and investigations of in vitro suppression of PTH using bovine parathyroid cells are described. The inhibitory potency of vitamin D3 analogs were found to be 1,25(OH)2D3 > ED-71 ≥ 3-epi-1,25(OH)2D3 3-epi-ED-71. ED-71 and 3-epi-ED-71 showed weak activity towards PTH suppression in our assays. 相似文献
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de Lyra EC da Silva IA Katayama ML Brentani MM Nonogaki S Góes JC Folgueira MA 《The Journal of steroid biochemistry and molecular biology》2006,100(4-5):184-192
1,25(OH)2D3 is an antiproliferative agent that may inhibit proliferation of breast cancer (BC) cells in vitro and BC development in animals. Epidemiological studies have shown a high incidence of BC in people less exposed to solar rays. To unravel the role of Vitamin D3 in BC patients, we have investigated serum levels of 25(OH)D3 and its active form 1,25(OH)2D3 as well as tissue expression of 1alpha-hydroxylase, 24-hydroxylase, and Vitamin D-receptor (VDR), determined by semiquantitative RT-PCR, in 88 Brazilian BC patients and 35 women without cancer (submitted to mammoplasties or resection of benign lesions). Median age of women with and without cancer was 51 and 46 years, respectively, and the majority of BC patients were classified as clinical stage II (67%). Although no differences in 25(OH)D3 serum concentration were found, 1,25(OH)2D3 (40+/-21 pg/ml) levels in BC patients were lower than in women without cancer (53+/-23). Our results indicate that 24-hydroxylase, VDR and 1alpha-hydroxylase mRNA tissue expression is similar in both groups and no correlation between 24-hydroxylase, 1alpha-hydroxylase, and VDR expression in breast tumors was found. A low 1,25(OH)2D3 serum concentration seems to be associated to breast cancer, however, the mechanism involved in this regulation is still unclear. 相似文献
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Wang Z Senn T Kalhorn T Zheng XE Zheng S Davis CL Hebert MF Lin YS Thummel KE 《Analytical biochemistry》2011,418(1):126-133
Simultaneous and accurate measurement of circulating vitamin D metabolites is critical to studies of the metabolic regulation of vitamin D and its impact on health and disease. To that end, we have developed a specific liquid chromatography–tandem mass spectrometry (LC–MS/MS) method that permits the quantification of major circulating vitamin D3 metabolites in human plasma. Plasma samples were subjected to a protein precipitation, liquid–liquid extraction, and Diels–Alder derivatization procedure prior to LC–MS/MS analysis. Importantly, in all human plasma samples tested, we identified a significant dihydroxyvitamin D3 peak that could potentially interfere with the determination of 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] concentrations. This interfering metabolite has been identified as 4β,25-dihydroxyvitamin D3 [4β,25(OH)2D3] and was found at concentrations comparable to 1α,25(OH)2D3. Quantification of 1α,25(OH)2D3 in plasma required complete chromatographic separation of 1α,25(OH)2D3 from 4β,25(OH)2D3. An assay incorporating this feature was used to simultaneously determine the plasma concentrations of 25OHD3, 24R,25(OH)2D3, 1α,25(OH)2D3, and 4β,25(OH)2D3 in healthy individuals. The LC–MS/MS method developed and described here could result in considerable improvement in quantifying 1α,25(OH)2D3 as well as monitoring the newly identified circulating metabolite, 4β,25(OH)2D3. 相似文献