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Biomedical subjects

H F DeLuca

Publications and source records attributed to H F DeLuca.

At least 19 recordsLinked to original sources
PubMed1998

1,25-dihydroxyvitamin D3 is a positive regulator for the two anti-encephalitogenic cytokines TGF-beta 1 and IL-4.

Previously we demonstrated that 1,25-dihydroxyvitamin D3 blocks the progression of relapsing encephalomyelitis. We now propose that 1,25-dihydroxyvitamin D3 blocks these autoimmune symptoms by stimulating the differentiation and/or function of cells that inhibit the encephalitogenic process. To support this belief, we have found that 1,25-dihydroxyvitamin D3 administration to mice increases IL-4 transcripts by 3- to 25-fold and TGF-beta 1 transcripts by 4- to 24-fold. Similarly, IL-4 and TGF-beta 1 transcripts were higher in the central nervous system of 1,25-dihydroxyvitamin D3-treated mice compared with controls. The number of cells recoverable from the lymph nodes of 1,25-dihydroxyvitamin D3-treated mice was only 50% that of controls. Overall, 1,25-dihydroxyvitamin D3 treatment causes a net loss in the total number of lymphocytes while the number of IL-4 and TGF-beta 1 transcripts increased. The systemic and local increase in the expression of these two anti-inflammatory cytokines by 1,25-dihydroxyvitamin D3 may be responsible for the ability of this drug to block encephalomyelitis.

Adjuvants, Immunologic↗
PubMed1998

Identification of a new all-trans-retinol metabolite produced through a new retinol metabolic pathway.

In vitro incubation of all-trans-retinol (atROL) with kidney homogenate from vitamin A-deficient and retinoic acid-supplemented (VAD-RAS) female rats produces a new retinol metabolite. Reverse-phase (RP) and normal-phase (NP) high-performance liquid chromatography (HPLC) analysis showed that this metabolite coelutes with the unknown all-trans-retinol (atROL) metabolite previously found in the day 10 conceptus and kidneys of vitamin A-deficient rats maintained on all-trans-retinoic acid (VAD-RA) and given 2 microg of [3H]atROL. Normal-phase (NP) HPLC purification of the metabolite collected from a RP HPLC column further separated the radiolabeled material into two components. The two isolated compounds have identical or very similar spectroscopic properties. Their nuclear magnetic resonance (1H NMR) and mass spectra (MS) indicated that they are isomers. Spectroscopic studies of the metabolites and their derivatives showed that they are nine-carbon fragments resulting from an oxidative cleavage of the side chain of atROL. The cleavage occurs at C-9, and the product is then oxidized to a keto group. The primary hydroxy group from atROL is preserved in the metabolite. A sulfide bridge is formed between C-11 and C-14, which interrupts the conjugation. The formation of the new metabolites, possessing a 2,5-dihydrothiophene ring, is catalyzed by an enzyme(s) located in the cytosolic fraction of kidneys. The process represents a new retinol metabolic pathway; however, its biological significance is unknown.

Administration, Oral↗
PubMed1998

Parathyroid hormone activation of the 25-hydroxyvitamin D3-1alpha-hydroxylase gene promoter.

The DNA flanking the 5' sequence of the mouse 1alpha-hydroxylase gene has been cloned and sequenced. A TATA box has been located at -30 bp and aCCAAT box has been located at -79 bp. The gene's promoter activity has been demonstrated by using a luciferase reporter gene construct transfected into a modified pig kidney cell line, AOK-B50. Parathyroid hormone stimulates this promoter-directed synthesis of luciferase by 17-fold, whereas forskolin stimulates it by 3-fold. The action of parathyroid hormone is concentration-dependent. 1,25-Dihydroxyvitamin D3 does not suppress basal promoter activity and marginally suppresses parathyroid hormone-driven luciferase reporter activity. The promoter has three potential cAMP-responsive element sites, and two perfect and one imperfect AP-1 sites, while no DR-3 was detected. These results indicate that parathyroid hormone stimulates 25-hydroxyvitamin D3-1alpha-hydroxylase by acting on the promoter of the 1alpha-hydroxylase gene.

25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗
PubMed1998

Cloning and expression of the chicken 25-hydroxyvitamin D3 24-hydroxylase cDNA.

Using a cDNA probe from the rat 24-hydrovitamin D3 24-hydroxylase, the chicken 25-hydroxyvitamin D3 24-hydroxylase cDNA has been isolated from a chicken kidney lambda gt11 library. The high degree of similarity with the mammalian 24-hydroxylase cDNAs strongly supports the belief that it is the chicken 25-hydroxyvitamin D3 24-hydroxylase cDNA. The deduced amino acid sequences are also very well conserved and 325 of them are identical among the four known 25-hydroxyvitamin D3 24-hydroxylases. This cDNA expressed in E. coli produces 24-hydroxylase activity.

Amino Acid Sequence↗
PubMed1998

Effects of retinoic acid (all-trans and 9-cis) on tumor progression in small-cell lung carcinoma.

The two stereoisomers of retinoic acid (RA), all-trans and 9-cis-RA, are regulators of cell proliferation, differentiation and apoptosis. In this study, the aim was to evaluate the effects of all-trans-and 9-cis-RA on cell growth, proliferation, and on the induction of apoptosis in the human small cell lung carcinoma (SCLC) cell lines NCI-H82 and NCI-H209. The application of various concentrations of all-trans and 9-cis-RA were able to inhibit cell growth and proliferation. Moreover, 3H-thymidine incorporation was inhibited and the number of viable cells decreased, suggesting that all-trans-RA and 9-cis-RA can inhibit cell proliferation in a dose dependent manner. Morphological examinations (light, electron and fluorescence microscopy) demonstrated that both retinoids had profound effects on the induction of apoptosis. Our investigation also showed that, compared to all-trans-RA, 9-cis-RA is a stronger inducer for the inhibition of cell growth and proliferation and that it is more effective in the induction of apoptosis in small cell lung carcinoma cells in culture.

Alitretinoin↗
PubMed1998

A highly sensitive method for large-scale measurements of 1,25-dihydroxyvitamin D.

A quantitative method for measuring 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) was developed utilizing a luciferase reporter gene under the control of the highly inducible 25-hydroxyvitamin D3 24-hydroxylase promoter in a stably transfected cell line. Transient transfections with constructs containing the 24-hydroxylase gene promoter 5' to a luciferase reporter were first performed in cell lines with high levels of vitamin D receptor, i.e., the rat osteosarcoma (ROS 17/2.8) and human breast cancer (T-47D) cell lines. ROS 17/2.8 cells, stably transfected with the plasmid, gave a 60-fold stimulation with 10(-10) M 1,25-(OH)2D3. A standard curve was constructed showing a large range of response to 1,25-(OH)2D3 (1 pg to 1 ng). The assay was adapted to microtiter plates, which permits a large number of samples to be assayed simultaneously. Other metabolites of vitamin D and analogs such as 25-hydroxyvitamin D3, 24,25-dihydroxyvitamin D3, and 1 alpha-hydroxyvitamin D3 have negligible effects on the detection of 1,25-(OH)2D3, thus eliminating the need for purification of sample. The sensitivity of the method permitted the use of 100 microliters of serum with excellent results. Comparison of this method with a commercially available assay demonstrates that it gives higher sensitivity, simpler manipulations, and comparable results.

24,25-Dihydroxyvitamin D 3↗
PubMed1998

1,25-Dihydroxycholecalciferol inhibits the progression of arthritis in murine models of human arthritis.

1,25-Dihydroxycholecalciferol [1,25-(OH)2D3] has been shown to inhibit the progression of experimental autoimmune encephalomyelitis (EAE). Here we tested the possibility that 1, 25-dihydroxycholecalciferol might be therapeutic for another autoimmune disease, arthritis. Two different animal models of arthritis were tested, namely, murine Lyme arthritis and collagen-induced arthritis. Infection of mice with Borrelia burgdorferi (the causative agent of human Lyme arthritis) produced acute arthritic lesions including footpad and ankle swelling. Supplementation with 1,25-dihydroxycholecalciferol of an adequate diet fed to mice infected with B. burgdorferi minimized or prevented these symptoms. Mice immunized with type II collagen also developed arthritis. The symptoms of this disease were also prevented by dietary supplementation with 1,25-dihydroxycholecalciferol. 1, 25-Dihydroxycholecalciferol given to mice with early symptoms of collagen-induced arthritis prevented the progression to severe arthritis compared with untreated controls. These results suggest that 1,25-dihydroxycholecalciferol and/or its analogs may be a valuable treatment approach to this disease.

Animals↗
PubMed1998

Mechanisms and functions of vitamin D.

The vitamin D hormone, 1,25-dihydroxyvitamin D3, functions by way of a nuclear receptor (vitamin D receptor [VDR]) in a manner analogous to the other members of the steroid-thyroid hormone superfamily. Although the vitamin D receptor has been cloned, its three-dimensional structure remains unknown. The VDR binds to the direct repeat response elements called DR-3 in the promoter region of target genes to stimulate or suppress transcription of those genes encoding for proteins that carry out a wide variety of functions. The binding of the VDR to a DR-3 requires the presence of its ligand and a companion protein, namely the RXR group of retinoid receptors. The RXR binds to the 5' arm of the response element while the VDR binds to the 3' arm. In addition, the transcription factor TFIIB has been shown to bind VDR but there is currently no evidence that a co-repressor or co-activator of VDR is also involved. Phosphorylation of VDR in the transcription complex occurs as does bending of the DNA prior to the initiation or suppression of transcription. As VDR has been detected in cells not previously thought to be target organs, scientists continue to discover new functions of vitamin D. Among these new functions are those noted in the immune system. Experiments in mice have illustrated that the autoimmune diseases of multiple sclerosis and rheumatoid arthritis can be successfully treated with the vitamin D hormone and its analogs. New experiments illustrating the use of the vitamin D hormone and its analogs in suppressing transplant rejection indicate that these compounds may be superior to cyclosporin and may not have the side effects attributed to the cyclosporin immunosuppression therapies.

Animals↗
PubMed1997

Cloning and expression of rat 25-hydroxyvitamin D3-1alpha-hydroxylase cDNA.

A full-length cDNA for the rat kidney mitochondrial cytochrome P450 mixed function oxidase, 25-hydroxyvitamin D3-1alpha-hydroxylase (P4501alpha), was cloned from a vitamin D-deficient rat kidney cDNA library and subcloned into the mammalian expression vector pcDNA 3.1(+). When P4501alpha cDNA was transfected into COS-7 transformed monkey kidney cells, they expressed 25-hydroxyvitamin D3-1alpha-hydroxylase activity. The sequence analysis showed that P4501alpha was of 2,469 bp long and contained an ORF encoding 501 amino acids. The deduced amino acid sequence showed a 53% similarity and 44% identity to the vitamin D3-25-hydroxylase (CYP27), whereas it has 42.6% similarity and 34% identity with the 25-hydroxyvitamin D3-24-hydroxylase (CYP24). Thus, it composes a new subfamily of the CYP27 family. Further, it is more closely related to the CYP27 than to the CYP24. The expression of P4501alpha mRNA was greatly increased in the kidney of vitamin D-deficient rats. In rats with the enhanced renal production of 1alpha,25-dihydroxyvitamin D3 (rats fed a low Ca diet), P4501alpha mRNA was greatly increased in the renal proximal convoluted tubules.

Amino Acid Sequence↗
PubMed1997

Cloning and characterization of the mouse vitamin D receptor promoter.

The gene encoding the mouse vitamin D receptor has been cloned. A new exon 1 has been found that changes the numbering established for the human VDR gene. Exons 2 and 3 in the human VDR gene (coding for the zinc fingers 1 and 2, respectively) are named exons 3 and 4 in the mouse vitamin D receptor. The 1.5-kb 5'-flanking region of the new exon 1 was analyzed and revealed the presence of putative cis-acting elements. Despite the absence of a TATA box, this 5'-flanking region contains several characteristics of a GC-rich promoter including four Sp1 sites present in tandem and two CCAAT boxes. Interestingly, the Sp1 site that is the most proximal to the new exon 1 overlaps a perfect site for Krox-20/24. Krox-20 is a transcription factor involved in brain development, and also in bone remodeling. In luciferase reporter gene expression assays, we showed that sequences from this 5'-flanking region elicit high transactivation activity. Furthermore, in the NIH 3T3 cell line, a 3- to 5-fold increase in response to forskolin treatment (an activator of adenylate cyclase and in turn of protein kinase A pathway) was observed.

3T3 Cells↗
PubMed1997

Salt concentration determines 1,25-dihydroxyvitamin D3 dependency of vitamin D receptor-retinoid X receptor--vitamin D-responsive element complex formation.

The electrophoretic mobility shift assay was used to determine in vitro formation of the vitamin D receptor-retinoid X receptor beta (VDR-RXR beta) heterodimer complex on vitamin D-response elements (VDREs) from rat osteocalcin, mouse osteopontin, rat 25-hydroxyvitamin D3 24-hydroxylase, and human parathyroid hormone (PTH) genes. Baculovirus-expressed rat VDR was used as VDR and the binding reactions were performed at salt concentrations ranging from 50 to 170 mM KCI. Without ligand, optimum complex formation was observed at 50 mM KCI and markedly decreased with increasing KCI for all VDREs. In the presence of 1,25-dihydroxyvitamin D3, optimum complex formation occurred between 110 and 130 mM KCI for positive (enhancer) VDREs. At low salt concentrations (50-70 mM KCI), 1,25-dihydroxyvitamin D3 did not increase complex formation and actually caused a slight decrease. However, above 90 mM KCI, 1,25-dihydroxy-vitamin D3 markedly increased complex formation and at 150-170 mM KCI, a concentration that presumably mimics physiologic nuclear levels, 1,25-dihydroxy-vitamin D3 appeared to be required for complex formation. With the suppressive cis-acting sequence, i.e., PTH-VDRE, optimum detection of VDR complexes in the presence of 1,25-dihydroxyvitamin D3 occurred at a lower salt concentration (90-110 mM KCI). Moreover, no specific complexes were formed at high salt concentrations, even when 1,25-dihydroxyvitamin D3 was added. Thus, when analyzing an effect of ligand on VDR-RXR-VDRE complex formation, it is essential that the reaction be carried out with a range of salt concentrations. Further, 1,25-dihydroxyvitamin D3 appears to be required for formation of the VDR-RXR beta-VDRE complex at salt concentrations approaching physiological.

Animals↗
PubMed1997

Cloning and origin of the two forms of chicken vitamin D receptor.

The cDNA for the chicken vitamin D receptor (VDR) has been cloned in full length by screening cDNA libraries from chicken intestine and chicken kidney. The chicken kidney cDNA library constructed by both oligo(dT) and random primer methods yielded several full-length clones. These contained a 66-bp additional coding sequence at the 5' end in comparison to the cDNAs of human, rat, and mouse VDR. The Japanese quail VDR cDNA was also cloned in full length. We found that the 5' coding sequence reported previously had been artifactually inverted. Both chicken and quail cDNAs have three conserved ATG sites for translation initiation with the 3' one corresponding to the only ATG site found in the mammalian cDNAs. Northern blot analysis showed that the VDR gene is expressed as a single 2.69-kb transcript in chicken intestine and kidney. An analysis of the avian multiple ATG sites revealed that the first ATG is in a suboptimal context of TCCATGT, while the second ATG is in AGC-ATGG, matching the optimal context: R-3NNATGG+4. As a result, the two forms of chicken VDR (cVDR) (form A, 60.3 kDa; form B, 58.6 kDa) likely arise from the first and second ATG sites of a single mRNA during translation. Mutational analysis confirmed this belief. The wild-type construct starting from the first ATG site with its original context was subcloned into pCMV5 expression vector and expressed in COS-1 cells. Two receptor proteins, exactly comigrating with the form A and form B cVDRs isolated from chicken intestine, were detected by immunoblotting. Point mutations optimizing the first ATG context led to exclusive production of form A, while knocking out the first ATG site resulted in the sole generation of form B. Form A translated from the first ATG site has 451 amino acids, and form B from the second ATG has 437 amino acids. Comparison between avian and mammalian VDRs indicated that VDR is essentially conserved in DNA binding and ligand binding domains. The significant difference especially at the N-terminus demonstrates divergence of this receptor during evolution of these species.

Amino Acid Sequence↗
PubMed1997

Effect of vitamin D receptor genotypes on calcium absorption, duodenal vitamin D receptor concentration, and serum 1,25 dihydroxyvitamin D levels in normal women.

It is well established that bone mineral density is under strong genetic control. Recently it was reported that the Bsm I restriction fragment length polymorphism of the vitamin D receptor (VDR) gene could account for up to 75% of the genetic variance in bone mineral density. However, the physiological basis for such an effect has not been established. The VDR gene codes for the vitamin D receptor protein which regulates intestinal calcium absorption. In order to assess the biochemical basis we studied the effect of common allelic variation of the VDR gene on intestinal VDR protein concentration, calcium absorption, and serum 1,25 dihydroxyvitamin D (1,25(OH)2D). Ninety-two Caucasian women were genotyped for Bsm I and Taq I polymorphism at the VDR gene locus. From these we compared 49 young women aged 25-35 years and 43 elderly women aged 65-83 years, who had all three measurements performed. There were no significant differences in intestinal VDR protein concentration, serum 1, 25(OH)2D, or radioactive calcium absorption among VDR genotype groups. Therefore, the small intestine does not seem to be a target for VDR gene polymorphism.

Absorption↗