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Biomedical subjects

H F DeLuca

Publications and source records attributed to H F DeLuca.

At least 91 records · Page 5Linked to original sources
PubMed1995

Cloning of the human 1 alpha,25-dihydroxyvitamin D-3 24-hydroxylase gene promoter and identification of two vitamin D-responsive elements.

A genomic DNA clone for 1 alpha,25-dihydroxyvitamin D-3 (1,25-(OH)2D3) 24-hydroxylase was isolated from a human chromosome 20 library. It spans 2.42 kb, containing the first two exons, the first and part of the second introns, and a 1.26 kb 5'-flanking region. Putative transcription cis-elements were revealed throughout the 5'-flanking region, including TATA box, CAAT box, GC boxes, vitamin D-responsive elements (VDRE), AP1, and AP2 sites. In a CAT reporter gene expression assay, the 24-hydroxylase promoter with its 1.2 kb 5'-flanking sequence elicits a 1,25-(OH)2D3-induced transactivation activity. Gel mobility shift assays of those putative DREs have identified that two different elements can form specific complexes with porcine intestinal nuclear extract (PINE). The specificity of VDRE-PINE complexes was verified by supershift assay with VDR-specific monoclonal antibody VXIE10B6. The proximal element VDREp (-172/-143) consists of three direct repeat half-sites, GAGTCAgcgAGGTGAgcgAGGGCG, in anti-sense orientation. The distal element VDREd (-293/-273) consists of two direct repeat half-sites, GCGTTCaccGGGTGT, also in anti-sense orientation. Both VDREs can direct a reporter gene expression using a heterologous herpes simplex virus thymidine kinase (TK) promoter in a 1,25-(OH)2D3-dependent fashion. Further characterization of these VDREs in various constructs with either a native or TK promoter suggests that both VDREs are required for the optimal induction of 24-hydroxylase expression by 1,25-(OH)2D3.

Base Sequence↗
PubMed1995

A possible role of vitamin D receptors in regulating vitamin D activation in the kidney.

The vitamin D endocrine system is regulated reciprocally by renal 25-hydroxyvitamin D3 1 alpha- and 24-hydroxylases. Previously, we reported that renal proximal convoluted tubules, the major site of 1 alpha, 25-dihydroxyvitamin D3 production, have vitamin D receptors. In the presence of vitamin D receptors, renal proximal convoluted tubules cannot maintain the state of enhanced production of 1 alpha, 25-dihydroxyvitamin D3. To clarify this discrepancy, we proposed a working hypothesis for the reciprocal control of renal 25-hydroxyvitamin D3 1 alpha- and 24-hydroxylase activities. In rat models of enhanced renal production of 1 alpha, 25-dihydroxyvitamin D3, expression of vitamin D receptors and 25-hydroxyvitamin D3 24-hydroxylase mRNAs was strikingly suppressed in renal proximal convoluted tubules but not in the cortical collecting ducts. In vitamin D-deficient rats with up-regulated renal 25-hydroxyvitamin D3 1 alpha-hydroxylase activity, expression of vitamin D receptor mRNA in renal proximal convoluted tubules was also down-regulated, indicating that the down-regulation of vitamin D receptor mRNA is not the result of the enhanced production of 1 alpha, 25-dihydroxyvitamin D3. In Japanese quail models with up-regulated renal 25-hydroxyvitamin D3 1 alpha-hydroxylase activity by sex steroids, expression of vitamin D receptor mRNA was also down-regulated in the kidney but not in the duodenum. These results suggest that the down-regulation of vitamin D receptors plays a critical role in production of 1 alpha, 25-dihydroxyvitamin D3 in renal proximal convoluted tubules.

Animals↗
PubMed1995

Identification of the porcine intestinal accessory factor that enables DNA sequence recognition by vitamin D receptor.

The nuclear accessory protein in porcine intestinal nuclear extracts that activates the binding of the vitamin D receptor to its vitamin D response elements has been highly purified. It contains a protein that binds 9-cis-[3H]retinoic acid, was detected on immunoblots with an anti-retinoid X receptor (RXR) peptide antibody, and supports the binding of retinoic acid receptor gamma to the retinoic acid receptor beta gene response element. Most important, the two specific complexes formed by porcine nuclear extract with the vitamin D response elements from either the osteocalcin gene or the rat 24-hydroxylase gene are shifted to a larger complex by both an anti-vitamin D receptor antibody and an anti-RXR antibody, leaving no doubt that in vivo the nuclear accessory factor for the vitamin D receptor in the intestine is an RXR protein.

Animals↗
PubMed1995

Two vitamin D response elements function in the rat 1,25-dihydroxyvitamin D 24-hydroxylase promoter.

The interaction between the two vitamin D response elements (DRE) located at -154 to -134 base pairs (bp) and -262 to -238 bp from the transcription initiation site has been studied using reporter gene assays and binding assays by electrophoretic gel shift measurements. 3 half-sites separated by 3 bp were found necessary for transactivation by the -154 to -125 DRE, while 2 half-sites separated by 3 bp were needed for the DRE at -262 to -238 to function. However, the two DREs together provided maximal activity. The 93-bp fragment separating the two DREs was not required and could be deleted. The most effective binding by receptor was found with the two complete DREs (dissociation constant (Kd) = 13.7 pM), although each DRE bound to the receptor and nuclear accessory factor with about 5 nM Kd. The two DREs (a total of 5 half-sites) apparently account for most if not all of the transactivation of the rat 24-hydroxylase by 1,25-dihydroxyvitamin D3. This system represents the most powerful of the DREs reported to date.

Animals↗
PubMed1995

The vitamin D3 hydroxylase-associated protein is a propionamide-metabolizing amidase enzyme.

Previously we isolated a novel protein that coimmunoprecipitates with the 1,25-dihydroxyvitamin D3-24R-hydroxylase and 25-hydroxyvitamin D3-1 alpha-hydroxylase. This kidney-specific protein found in the inner membrane of mitochondria is named the vitamin D3 hydroxylase-associated protein (VDHAP). To determine a putative function for this protein, an extensive computer search of the deduced amino acid sequence of VDHAP was performed. A BLAST homology search identified amino acid residues 133 through 321 in acetamidase from Aspergillus nidulans that exhibit 38% amino acid identify and 65% amino acid similarity to VDHAP. A protein consensus sequence dictionary, MOTIFS, identified an amidase consensus sequence in VDHAP. This sequence, G-G-S-S-G-G-E-G-A-L-I-A-G-G-G-S-L-L-G-I-G-S-D-V-A-G-S-I-R-L-P-S, in VDHAP is located between amino acids 223 and 254. Propionamide, acetamide, and acrylamide were identified as substrates for an amidase activity in soluble chicken kidney mitochondria. Propionamide is the best substrate with a Vmax of 16.7 nmol NH4+/min/mg protein and an apparent Km of 7.9 mM in soluble chicken kidney mitochondria. A VDHAP monoclonal antibody, IVC2G8, immunoprecipitates 78% of the total propionamidase activity in soluble chicken kidney mitochondria. These results suggest that VDHAP is a propionamidase enzyme in soluble chicken kidney mitochondria and a member of the amidase signature gene family.

Amides↗
PubMed1995

Vitamin D receptor gene polymorphism, bone mass, body size, and vitamin D receptor density.

We determined vitamin D receptor (VDR) gene alleles (based on the BsmI restriction site polymorphism), duodenal mucosal receptor density, bone mass at spine and total body, and body size in 32 healthy premenopausal females. While we found no relationship between allele and receptor density in duodenal mucosa, bone mineral content (BMC) at both spine and total body was significantly associated with VDR gene alleles. BMC was highest for the bb allele, lowest for BB, and intermediate for Bb. A similar association was noted between allele and body size variables, particularly weight. When BMC was adjusted for body weight, the association with VDR polymorphism disappeared. The VDR gene polymorphism may be affecting bone mass not through classical nutritional mechanisms (e.g., intestinal calcium absorption), but through an influence on body size.

Adult↗
PubMed1995

1 alpha-Hydroxylated cholecalciferol compounds act additively with microbial phytase to improve phosphorus, zinc and manganese utilization in chicks fed soy-based diets.

Seven experiments were conducted to determine the efficacy of 1,25-dihydroxycholecalciferol [1,25-(OH)2D3] and microbial phytase in improving P, Zn and Mn utilization of chicks fed P, Zn- and Mn-deficient soy protein diets containing surfeit levels of cholecalciferol. Efficacy of 1 alpha-hydroxycholecalciferol (1 alpha-OH D3) was also studied. A dose titration study indicated that supplemental phytase at 1200 units/kg diet would increase bone ash by at least 65% when added to a corn-soybean meal diet containing 0.43 g P/100 g (0.1 g nonphytate P/100 g). These responses were similar to those obtained from supplemental P (0.1 g/100 g) as KH2PO4 or from added 1,25-(OH)2D3 (10 micrograms/kg). Dietary addition of both 1200 units phytase and 10 micrograms/kg 1,25-(OH)2D3 elicited bone ash responses that were near 100%. When chicks were fed a Zn-deficient soy-concentrate diet (13 mg Zn/kg), diet supplementation with 1,25-(OH)2D3 or phytase increased growth rate by 40% and tibia Zn content by > 100%; adding 1,25-(OH)2D3 together with phytase increased tibia Zn content by 160%. Utilization of both Zn and Mn contained in the corn-soybean meal diet also was markedly enhanced by supplemental phytase, 1,25-(OH)2D3, or the combination. The cholecalciferol analog 1 alpha-OH D3 was found to improve dietary P utilization maximally (70% bone ash response) at a dose of 20 micrograms/kg diet, and effects were additive when 1 alpha-OH D3 was fed in the presence of phytase.(ABSTRACT TRUNCATED AT 250 WORDS)

6-Phytase↗
PubMed1995

Retinol in addition to retinoic acid is required for successful gestation in vitamin A-deficient rats.

Vitamin A-deficient, pregnant rats resorb their fetuses around Day 15 of gestation even when given retinoic acid (RA) daily. This resorption is prevented by the administration of retinol. Retinol was found to be required no later than Day 10 of gestation, and up to 500 micrograms of retinol administered on Day 11 or later was not able to change the course of fetal resorption in these animals. Vitamin A-deficient pregnant rats supported on 40 micrograms all-trans-RA daily that are given 2 micrograms of retinol on Day 10 and then placed on a retinol-sufficient diet at 24 h after administration of the retinol dose gave birth to normal pups. When a single 2-micrograms dose of retinol was administered on Day 10 and the vitamin A-deficient, RA-supported animals continued on the deficient diet supplemented with RA, the fetal resorption did not occur and the fetuses appeared normal when examined on Day 20 of gestation. Experiments using radiolabeled retinol tracer indicated that while there was some accumulation of radioactivity through Day 14 in the fetus, the administered retinol was mostly eliminated from the animal by Day 16. Thus, retinol is clearly required by Day 10 for normal gestation.

Animals↗
PubMed1995

An investigation of sources of variation in calcium absorption efficiency.

To examine putative sources of interindividual variation in calcium absorption efficiency, we studied 41 healthy premenopausal women (mean age, 36.4 yr). About half were randomized to pretreatment with supplemental 25-hydroxyvitamin D (25OHD; 20 micrograms/day [corrected] for approximately 34 days) before testing. We measured dietary factors, humoral regulators, intestinal motility, mucosal histology, mucosal vitamin D receptor levels, and calcium absorption efficiency. In winter tests, but not in summer tests, calcium absorption fraction was significantly higher in the pretreated group (mean, 0.465 vs. 0.387). Serum 25OHD, intestinal transit, and urinary calcium to creatinine ratio were all significantly and positively correlated to calcium absorption efficiency. However, neither the level of 1,25-dihydroxyvitamin D receptors in duodenal mucosa nor circulating 1,25-dihydroxyvitamin D was related to calcium absorption efficiency. These findings, which are consistent with other published human data, suggest that 25OHD plays a more prominent role in the regulation of calcium absorption than is generally believed. In a multiple regression model, serum 25OHD, mouth to cecum transit time, and fasting urinary calcium/creatinine ratio explained 44% of the observed variation in calcium absorption efficiency.

Absorption↗
PubMed1994

The avian vitamin D receptors: primary structures and their origins.

cDNA clones encoding Japanese quail chorioallantoic membrane and chicken kidney 1,25-dihydroxyvitamin D3 receptors were isolated and the total 448-amino acid (aa) sequence was deduced. The sequences of the chicken and quail receptors are identical. The nucleotide and deduced amino acid sequences of the avian receptors are similar but not identical to the reported rat or human receptor sequences. There is a 78% similarity in the nucleotide sequences and 98.5% and 87.5% similarities in the amino acid sequences of the DNA-binding and ligand-binding domains, respectively. Two avian receptor proteins (58 and 60 kDa) arise from a single mRNA transcript by alternate initiation of translation. The avian 1,25-dihydroxyvitamin D3 receptors were produced using a bacterial expression system. Form A receptor was expressed from a cloned cDNA that contains the first translation signal (ATG) and corresponds with the 60-kDa avian receptor protein, and form B receptor was initiated from the third ATG on the same mRNA transcript to give rise to the 58-kDa protein. The cysteine-rich DNA-binding domain is almost conserved among human, rat, and avian receptors. The position of the nine cysteines was conserved in all three sequences. The avian receptor differs in the second zinc finger domain, where a methionine replaces a leucine, a serine replaces an asparagine, and a lysine replaces an arginine at aa 77, 83, and 87, respectively, of the avian sequence. The increased length of the avian receptor results from a 20-aa extension of the N-terminal region. RNA hybridization indicates there is a single mRNA species of approximately 2700 bp for both the chicken and quail receptors compared to 4400 bp for the rat transcript. Surprisingly, the translated avian sequence is larger (448 aa) than the 423-aa rat receptor protein. Therefore, our results confirm that despite the difference in molecular mass between different receptor proteins, there is a similarity in gene organization such that the DNA-binding and hormone-binding domains are positionally conserved from the C terminus.

Amino Acid Sequence↗
PubMed1994

Synthesis and biological activity of 2-hydroxy and 2-alkoxy analogs of 1 alpha,25-dihydroxy-19-norvitamin D3.

1 alpha, 2 alpha,25-Trihydroxy-19-norvitamin D3, 1 alpha, 2 beta,25-trihydroxy-19-norvitamin D3, and their alkoxy analogs were efficiently prepared in a convergent synthesis, starting with (-)-quinic acid and a Windaus--Grundmann type ketone. Configurations of the A-ring fragment substituents were determined by 1H,1H COSY 2D spectra and 1H NOE difference spectroscopy. The new analogs exhibited selective activity in stimulating intestinal calcium transport while having little or no activity in mobilizing bone calcium. They also showed HL-60-differentiating activity equal to or 10 times lower than that of 1 alpha,25-dihydroxyvitamin D3.

Animals↗
PubMed1994

26,27-Dihomo-1 alpha-hydroxy- and 26,27-dihomo-24-epi-1 alpha,25-dihydroxyvitamin D2 analogs that differ markedly in biological activity in vivo.

26,27-Dihomo-1 alpha-hydroxyvitamin D2, 26,27-dihomo-24-epi-1 alpha-hydroxyvitamin D2, and 26,27-dihomo-24-epi-1 alpha,25-dihydroxyvitamin D2 have been synthesized. In contrast to 1 alpha-hydroxyvitamin D2 and 24-epi-1 alpha-hydroxyvitamin D2, 26,27-dihomo-1 alpha-hydroxyvitamin D2 (1) and the 24-epi analog (2) have no activity in intestinal calcium transport, bone calcium mobilization, or skeleton mineralization. On the other hand, 26,27-dihomo-24-epi-1 alpha,25-dihydroxyvitamin D2 is equal to 1,25-dihydroxyvitamin D3 in biological activity. Vitamin D 25-hydroxylase readily converts 1 alpha-hydroxyvitamin D2 to 1,25-dihydroxyvitamin D2. In contrast, the same preparations fail to hydroxylate 26,27-dihomo-1 alpha-hydroxyvitamin D2 and 26,27-dihomo-24-epi-1 alpha-hydroxyvitamin D2 on carbon 25. Thus, homologation of carbons 26 and 27 of the vitamin D compound likely sterically hinders vitamin D 25-hydroxylase.

Animals↗
PubMed1994

Isolation and characterization of a novel cDNA from HL-60 cells treated with 1,25-dihydroxyvitamin D-3.

A novel cDNA clone (VDUP1) has been isolated from a cDNA library constructed from mRNA obtained from HL-60 cells stimulated by 1,25-dihydroxyvitamin D-3. Northern blot analysis showed that VDUP1 cDNA hybridizes to a 2.9 kb mRNA species which is up-regulated in HL-60 cells by 1,25-dihydroxyvitamin D-3 (1,25-(OH)2D3) treatment. In vitro expression of VDUP1 cDNA produced a 46 kDa protein. A search of the GenBank database revealed that the 3' untranslated region of VDUP1 is homologous to a sequence expressed in brain. A detailed time course study showed that the VDUP1 mRNA starts to increase at 6 h after 1,25-dihydroxyvitamin D-3 treatment, reaches a plateau at around 18 h and stays elevated for 24 h. The VDUP1 mRNA is not regulated by phorbol 12-myristate 13-acetate (PMA) in HL-60 cells. Inhibition of protein synthesis by cycloheximide does not prevent the induction of VDUP1 mRNA by 1,25-dihydroxyvitamin D-3. Cycloheximide itself increases VDUP1 mRNA levels. These results suggest that the degradation of VDUP1 mRNA is either translation-dependent or regulated by a labile protein.

Amino Acid Sequence↗
PubMed1994

The role of vitamin D in chorioallantoic membrane calcium transport.

1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) is essential for the transport of eggshell calcium to the embryo across the chorioallantoic membrane (CAM). CAM contains the vitamin D receptor that increases following 1,25-(OH)2D3 injection into embryos at day 10 of incubation. Further, a single injection of 100 ng of 1,25-(OH)2D3 into vitamin D-deficient quail eggs at day 10 of incubation resulted in a significant increase in both body and yolk calcium. This is accompanied by an increase in carbonic anhydrase from low levels in deficiency to normal levels. Acetazolamide (AZ), a specific carbonic anhydrase inhibitor injected into the quail embryos, caused hypocalcemia and hyperphosphatemia. This is similar to the hypocalcemia and hyperphosphatemia found in vitamin D-deficient embryos. These results suggest that one mechanism of action of vitamin D in the mobilization of eggshell calcium is the activation of carbonic anhydrase that acidifies the calcium carbonate shell.

Acetazolamide↗
PubMed1994

Immunoaffinity purification of active rat recombinant 1,25-dihydroxyvitamin D3 receptor.

We have developed a large-scale immunoaffinity purification procedure for the recombinant vitamin D receptor. The purified receptor is homogeneous, and is bound by 1,25-dihydroxyvitamin D3 with a Kd of 5 x 10(-10) M. The isolated receptor binds to the osteocalcin vitamin D response element in the presence of porcine intestinal nuclear extract stripped of endogenous vitamin D receptor as well. However, the binding of D3 and the vitamin D3 response element does not completely assure a native conformation of the protein. The availability of large quantities of highly purified active vitamin D receptor makes possible detailed structural analysis.

Animals↗
PubMed1994

Identification of a vitamin D-response element in the rat calcidiol (25-hydroxyvitamin D3) 24-hydroxylase gene.

The calcidiol (25-hydroxyvitamin D3) 24-hydroxylase is one of the key enzymes in the metabolism of vitamin D. This enzyme acts on both calcidiol and calcitriol (1,25-dihydroxyvitamin D3) to initiate degradation of these potent vitamin D metabolites and is tightly regulated. Calcitriol itself induces this enzyme and acts at the transcriptional level. Transcriptional regulation of genes by calcitriol has been shown to occur via the vitamin D-receptor binding to a vitamin D-response element located upstream of the transcription start site. We now report a vitamin D-response element located between nt -262 and nt -238 of the rat calcidiol 24-hydroxylase gene. This sequence binds the calcitriol receptor and confers vitamin D-dependent transactivation of transcription to its own, as well as heterologous, promoter.

Animals↗
PubMed1994

A new member to the astacin family of metalloendopeptidases: a novel 1,25-dihydroxyvitamin D-3-stimulated mRNA from chorioallantoic membrane of quail.

1,25-Dihydroxyvitamin D-3 is essential for the utilization of eggshell calcium by avian embryo through the chorioallantoic membrane (CAM). A cDNA library was constructed from poly(A)+ RNA extracted from vitamin D-deficient CAMs given 1,25-dihydroxyvitamin D-3. Screening this library by differential hybridization yielded a full-length (approximately 1.8 kb) cDNA, whose corresponding mRNA is increased 3-fold 2.5 h after a single injection of 1,25-(OH)2D3. The complete nucleotide sequence for the full-length cDNA has been determined. An open-reading frame, corresponding to a 310 amino acid, 41 kDa protein was found. Searching protein sequence data bases revealed a strong similarity to the following proteases: astacin, a crayfish digestive protease, Oryzias latipes hatching enzyme constituent protease (Orz), Xenopus laevis developmentally regulated UVS.2 protein secreted by the hatching gland of embryos, the NH2-terminal domain of human bone morphogenetic protein (BMP-1) and Drosophila dorsal-ventral patterning tolloid. The cDNA has approximately 36% overall identity with astacin and BMP-1, and is more than 60% identical to either Orz or UVS.2. Moreover, multiple alignment analysis indicates that 37 residues, including 3 cysteine residues, are strictly conserved in the complete 200-amino acid astacin sequence. All 6 proteins contain a zinc-binding motif (HEXXH), found at the active site of most metalloendopeptidases. This motif is found within an extended sequence of HEXXHXXGFXHE that is unique to this subgroup of metalloendopeptidases. In addition, the 6 proteins have 50% identity (including the present cDNA) and 79% are conserved in 4 of these proteins in a 24-amino acid sequence that includes the putative active site. The level of mRNA for the new protein reaches a maximum at day 12 of embryonic life and declines thereafter. It is suggested that this clone corresponds to an mRNA encoding for a protease that may play a role in the degradation of eggshell matrix.

Allantois↗