Biomedical subjects

H F DeLuca

Publications and source records attributed to H F DeLuca.

At least 217 records · Page 12Linked to original sources

PubMed1986

Modulation of protein biosynthesis during early stages of differentiation in retinoic acid treated F9 teratocarcinoma cells.

Modulation of protein biosynthesis by retinoic acid during induction of differentiation of F9 teratocarcinoma stem cells was investigated by using computerized analysis of double label autoradiography of two-dimensional polyacrylamide gels. As early as 6 h after induction increased synthesis of 5 and decreased synthesis of 2 proteins occur. By 12 h after induction, synthesis of 13 proteins is elevated and by 24 h that of 17. At 24 h the range of stimulation is from two- to fourfold, as demonstrated by a 3H:14C ratio divided by the mode ratio. Examination of the Gaussian distributions of frequency of ratio indicates that many subtle changes in protein synthesis accompany the development of the new phenotype.

Animals↗

PubMed1986

Monoclonal antibodies to the porcine intestinal receptor for 1,25-dihydroxyvitamin D3: interaction with distinct receptor domains.

Monoclonal antibodies to different domains of the porcine intestinal 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] receptor have been produced. A nuclear extract enriched in the 1,25-(OH)2D3 receptor was prepared from small intestinal mucosa of young pigs. The receptor was purified an additional 6600-fold by chromatography on DNA-cellulose, ammonium sulfate precipitation, gel filtration high-performance liquid chromatography, and DEAE-Sepharose chromatography, with an overall yield of 23% and an average purity of 24%. A BALB/c mouse immunized with this material developed serum polyclonal antibodies to the 1,25-(OH)2D3 receptor, as demonstrated by a change in sedimentation of the porcine receptor on sucrose gradients. Spleen cells from this animal were fused with mouse myeloma cells (P3-NSI/1-Ag4-1, SP2/0-Ag14), and 24 hybridomas secreting antibodies to the 1,25-(OH)2D3 receptor were identified by both a radiometric immunosorbent assay and an immunoprecipitation assay. Twenty-one hybridoma lines were cloned by limiting dilution and further characterized as subclass IgG1 antibodies with the exception of one which is an IgA. All but two of the antibodies cross-react with the 1,25-(OH)2D3 receptor from both mammalian (human, monkey, and rat) and avian (chicken) intestine; two antibodies recognize only porcine intestinal receptor. All antibodies are unreactive to the vitamin D serum transport protein. Eight of the antibodies bind denatured receptor on an immunoblot. A solid-phase competition assay was used to identify four groups of antibodies that bind to distinct epitopes on the 1,25-(OH)2D3 receptor. One antibody from each of the four groups was used to examine the effect of antibody binding on the DNA-binding activity of the receptor-hormone complex. One antibody completely inhibited the binding of the 1,25-(OH)2D3 receptor complex to DNA-cellulose, suggesting that the epitope for this antibody may be located in the polynucleotide binding domain of the protein. Antibodies from two additional groups only slightly perturbed DNA binding, while one had no effect, suggesting that these antibodies bind to receptor epitopes distant from the region of the polypeptide directly involved in polynucleotide binding. These antibodies that are directed to several different binding sites on the 1,25-(OH)2D3 receptor provide important new tools to probe the biochemistry and topology of the 1,25-(OH)2D3 receptor and to investigate its role in mediating target tissue response to hormone.

Animals↗

PubMed1986

The action of various vitamin D3 metabolites on calcium and phosphorus metabolism in chick embryo calvariae.

Chick embryos from vitamin D-deficient hens given physiological doses of 1,25-dihydroxyvitamin D3 or 24,25-dihydroxyvitamin D3 or both become severely hypocalcemic, hyperphosphatemic and fail to hatch as compared to those derived from hens given 25-hydroxyvitamin D3 or 24,25-difluoro-25-hydroxyvitamin D3. Calvariae from the former contain less mineral and on incubation in vitro produce significantly lower calcium and higher phosphate concentration in the medium than do the calvariae derived from the embryos of hens supported on 25-hydroxyvitamin D3 or 24,24-difluoro-25-hydroxyvitamin D3.

24,25-Dihydroxyvitamin D 3↗

PubMed1986

Solubilization and reconstitution of chick renal mitochondrial 25-hydroxyvitamin D3 24-hydroxylase.

Chick kidney mitochondrial 25-hydroxyvitamin D3 24-hydroxylase has been solubilized with sodium cholate and reconstituted with NADPH, beef adrenal ferredoxin, and beef adrenal ferredoxin reductase, each component being essential for maximal 24-hydroxylase activity. The product 24(R),25-dihydroxyvitamin D3 was identified by cochromatography with synthetic compound on straight-phase and reversed-phase high-performance liquid chromatography and by periodate oxidation. The enzyme has an apparent Km for 25-hydroxyvitamin D3 of 0.67 microM. At 1 microM 25-hydroxyvitamin D3, 24,25-dihydroxyvitamin D3 production is linear with time for up to 15 min and with protein concentrations of up to 2 mg/mL. The antioxidant diphenyl-p-phenylenediamine (1.3 X 10(-4) M) has no effect on this reaction. Reconstituted 24-hydroxylase activity is enhanced by the addition of NaCl and KCl up to 100 mM, with higher concentrations having an inhibitory effect. 1 alpha-Hydroxylase is not present in this preparation from vitamin D replete chicks. The similarities of this reconstituted system to the 25-hydroxyvitamin D3 1 alpha-hydroxylase and the adrenal systems suggest that the 25-hydroxyvitamin D3 24-hydroxylase is also a cytochrome P-450 type mixed-function oxidase.

25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗

PubMed1986

A radiometric immunosorbent assay for the detection of anti-hormone-binding protein antibodies.

A radiometric immunosorbent assay (RISA) for the detection of monoclonal antibodies to hormone-binding proteins has been developed. The assay involves incubating hybridoma supernatants in microtiter wells that have been coated with goat anti-mouse IgG antibodies. Any mouse IgG in the test supernatant is thus specifically retained in the wells. Radioactive ligand-binding protein complexes are then incubated in the wells. The presence of anti-binding protein antibodies in the supernatant is indicated by specific retention of radioactive ligand-binding protein complexes in the wells. Crude antigen preparations, such as tissue homogenates, can be used to detect antibodies. The assay is capable of detecting antibody at concentrations 20 ng/ml (approximately 100 pM IgG). The RISA has been used successfully to screen for monoclonal antibodies to the intracellular receptor for 1,25-dihydroxyvitamin D3 and should be useful for the detection of antibodies to ligand-binding proteins in general.

Animals↗

PubMed1986

The relationship between the vitamin D system and cancer.

The classic function of 1,25-dihydroxyvitamin D3, the hormonally active form of vitamin D, is the maintenance of normal levels of calcium and phosphorus in the blood. 1,25-Dihydroxyvitamin D3 binds to a specific receptor protein and exerts its biologic action by a mechanism analogous to that proposed for other steroid hormones, that is, the receptor-ligand complex acts on the chromatin to induce transcription of specific genes. Intracellular receptors that bind 1,25-dihydroxyvitamin D3 with high affinity have been found in a large number of tumor cell lines examined as melanoma, osteosarcoma, and human breast and colonic carcinoma cells. The 1,25-dihydroxyvitamin D3 receptor in these cells has characteristics similar to the receptor in bone and intestine, the known target tissues of the hormone. In fact, 1,25-dihydroxyvitamin D3 inhibits the proliferation of melanoma, osteosarcoma, and breast carcinoma cells. More recently, 1,25-dihydroxyvitamin D3 has been shown to suppress the growth and induce monocytic differentiation of murine and human myeloid leukemia cells in vitro. These results point to a previously unsuspected involvement of vitamin D in cell proliferation and differentiation and suggest that analogs of the vitamin D hormone may be of interest as possible therapeutic agents in the treatment of malignancy.

Calcitriol↗

PubMed1986

The metabolism and functions of vitamin D.

Vitamin D functions by stimulating intestinal calcium and phosphorus absorption, by stimulating bone calcium mobilization, and by increasing renal reabsorption of calcium in the distal tubule. These functions on bone and possibly kidney, but not intestine, require the parathyroid hormone. As a result of these functions, serum calcium and phosphorus concentrations are elevated to supersaturating levels required for the mineralization of bone to prevent rickets, osteomalacia, and hypocalcemic tetany. Recent experiments demonstrate that maintaining serum calcium and phosphorus levels in vitamin D-deficient rats in the normal range results in normal bone growth and mineralization. However, increased calcification results because bone resorption by osteoclasts is a vitamin D-dependent process. Thus, bone resorption, modeling and remodeling must be considered vitamin D-dependent processes. Vitamin D must be metabolized to 25-hydroxyvitamin D3 by the liver and subsequently by the kidney to 1,25-dihydroxyvitamin D3 before function. 1,25-Dihydroxyvitamin D3 is metabolized to a C-23 carboxylic acid (calcitroic acid) but the pathway is unknown. Although 25-hydroxyvitamin D3 is metabolized to 24R,25-dihydroxyvitamin D3, 25,26-dihydroxyvitamin D3 and 25-hydroxyvitamin D3-26,23-lactone, these pathways play no role in the function of vitamin D as shown by appropriate fluoro analogs of 25-hydroxyvitamin D3. 1,25-Dihydroxyvitamin D3 binds to a specific receptor in the intestinal nuclei to elicit a stimulation of calcium transport. 1,25-Dihydroxyvitamin D3 plus the receptor causes transcription of specific genes that code for calcium and phosphorus transport proteins. Only one protein, the calcium binding protein, has been identified as being vitamin D dependent. Two others have been described, but no clear description of the molecular mechanism of action of 1,25-dihydroxyvitamin D3 is yet available.

Animals↗

PubMed1986

The adrenal: a new target organ of the calciotropic hormone 1,25-dihydroxyvitamin D3.

Target cells for 1,25-dihydroxyvitamin D3 were demonstrated in the adrenal medulla by frozen-section autoradiography. The appearance of these target cells was age-dependent in neonatal mice. Immunocytochemical staining for phenylethanolamine-N-methyltransferase revealed that both epinephrine and non-epinephrine cells concentrate 1,25-dihydroxyvitamin D3 in their nuclei. In contrast, immunocytochemical staining for "vitamin D-dependent calcium-binding protein" (D-CaBP) demonstrated that D-CaBP immunoreactivity is localized in only a small percentage of adrenomedullary cells, in mice and rats. Comparison of PNMT and D-CaBP immunoreactivities in sequential sections showed that epinephrine-producing cells do not contain D-CaBP. These results indicate that adrenal medullary cells have receptors for 1,25-dihydroxyvitamin D3 and that 1,25-dihydroxyvitamin D3 may directly affect certain functions of these endocrine cells.

Adrenal Medulla↗

PubMed1986

Purification of human serum vitamin D-binding protein by 25-hydroxyvitamin D3-Sepharose chromatography.

25-Hydroxyvitamin D3-Sepharose was prepared by coupling 25-hydroxyvitamin D3-3 beta-(1,2-epoxypropyl)-ether to thio-activated Sepharose CL-6B, forming a protease-resistant linkage between the sterol and the matrix. Vitamin D-binding protein from human plasma was obtained 85-92% pure after ligand affinity chromatography. Subsequent hydroxylapatite chromatography provided homogeneous protein. The purified vitamin D-binding protein was fully active in regard to 25-hydroxyvitamin D3 and actin binding capabilities.

Calcifediol↗

PubMed1986

Biosynthesis of retinoyl-beta-glucuronide, a biologically active metabolite of all-trans-retinoic acid.

All-trans-retinoic acid is metabolized in vitro to a biologically active metabolite, retinoyl-beta-glucuronide. We have studied the synthesis of this metabolite in vitro. The identity of the product was established by cochromatography on reverse-phase high-performance liquid chromatography, beta-D-glucuronidase hydrolysis, and fast atom bombardment and collisionally activated decomposition/fast atom bombardment mass spectrometry. The formation of retinoyl-beta-glucuronide is catalyzed by a UDP-glucuronosyltransferase with apparent Km's of 54.7 microM for all-trans-retinoic acid and 2.4 mM for UDP-glucuronic acid. The reaction requires enzyme, UDP-glucuronate, and no other factor. It is strongly inhibited by millimolar concentrations of coenzyme A. The specific activity of UDP-glucuronosyltransferase is greatest in the liver and least in the kidney of those tissues examined. The specific activity of the enzyme is increased by vitamin A deficiency. The increased specific activity observed in the vitamin A-deficient rat liver is uncharacteristic of retinoic acid inactivation enzymes; therefore, retinoyl-beta-glucuronide may be of functional importance.

Animals↗

PubMed1986

1,25-Dihydroxyvitamin D3-stimulated mRNAs in rat small intestine.

The technique of differential hybridization has been employed to study gene expression associated with vitamin D action on the mammalian intestine. A cDNA library consisting of 10(6) independent recombinants was constructed from poly(A)+ RNA extracted from vitamin D-deficient rats given 1,25-dihydroxyvitamin D3. A survey of 20,000 clones resulted in identification of four distinct cDNAs whose corresponding mRNAs are significantly increased 12 h after an intrajugular dose of 1,25-dihydroxyvitamin D3 given to vitamin D-deficient rats. DNA sequence analysis identified these mRNAs as mitochondrial ATP synthetase, vitamin D-dependent calcium binding protein, cytochrome oxidase subunit I, and cytochrome oxidase subunit III. The time course of response of three of these mRNAs was similar, with maximum values at 12 h after dosing, while that of cytochrome oxidase subunit I showed two peaks at 6 and 18 h following a single dose of 1,25-dihydroxyvitamin D3. The levels of all four mRNAs were elevated in rats supplied with vitamin D when hypocalcemia was produced by dietary calcium restriction.

Animals↗

PubMed1986

Studies on the relation of DNA synthesis to retinoic acid-induced differentiation of F9 teratocarcinoma cells.

Inhibition of DNA synthesis in F9 embryonal carcinoma cells with high thymidine induces differentiation similar to that induced with retinoic acid (RA). The presence of differentiated cells is evident after 15 h of treatment with 2 mM thymidine, during which period DNA synthesis is inhibited 99%. The addition of RA during the period of high thymidine treatment does not increase the amount of differentiation seen at the end of the 15-h treatment, but does increase the amount seen after thymidine is removed. The inhibition of proliferation by low serum concentration does not induce differentiation in the absence of RA. In partially synchronized cultures of F9 cells, the addition of RA alters the pattern of DNA replication during the first third of S phase. If RA is present during this part of S phase, differentiation is evident both morphologically and biochemically during the following cell cycle. Addition of RA during the second half of S phase does not lead to obvious differentiation until after the next cell cycle. These results suggest that particular events during the early replication period of F9 cells are targets for RA action in induction of differentiation of F9 cells.

Animals↗

PubMed1986

Photoactivable analogs for labeling 25-hydroxyvitamin D3 serum binding protein and for 1,25-dihydroxyvitamin D3 intestinal receptor protein.

3-Azidobenzoates and 3-azidonitrobenzoates of 25-hydroxyvitamin D3 as well as 3-deoxy-3-azido-25-hydroxyvitamin D3 and 3-deoxy-3-azido-1,25-dihydroxyvitamin D3 were prepared as photoaffinity labels for vitamin D serum binding protein and 1,25-dihydroxyvitamin D3 intestinal receptor protein. The compounds prepared were easily activated by short- or long-wavelength uv light, as monitored by uv and ir spectrometry. The efficacy of the compounds to compete with 25-hydroxyvitamin D3 or 1,25-dihydroxyvitamin D3 for the binding site of serum binding protein and receptor, respectively, was studied to evaluate the vitamin D label with the highest affinity for the protein. The presence of an azidobenzoate or azidonitrobenzoate substituent at the C-3 position of 25-OH-D3 significantly decreased (10(4)- to 10(6)-fold) the binding activity. However, the labels containing the azido substituent attached directly to the vitamin D skeleton at the C-3 position showed a high affinity, only 20- to 150-fold lower than that of the parent compounds with their respective proteins. Therefore, 3-deoxy-3-azidovitamins present potential ligands for photolabeling of vitamin D proteins and for studying the structures of the protein active sites.

Azides↗

PubMed1986

Vitamin D metabolism during pregnancy.

Vitamin D metabolism is altered in the pregnant animal, presumably in response to fetal demands for calcium. Circulating levels of 1,25-dihydroxyvitamin D are elevated in the pregnant animal. The stimulus of this increase and the hydroxylase(s) (placental or renal) responsible are unknown. Maternal plasma 25-hydroxyvitamin D levels have been reported to be both unchanged and decreased during pregnancy but very much dependent upon exposure to ultraviolet light and vitamin D supplementation. The major vitamin D metabolites (25-hydroxyvitamin D, 24,25-dihydroxyvitamin D, and 1,25-dihydroxyvitamin D) circulate in fetal plasma but generally at lower concentrations than in the mother (exception is the sheep). All of these metabolites are able to cross the placenta. The fetal kidney and placenta both have 25-hydroxyvitamin D1 alpha- and 24-hydroxylase activity. However, the relative contribution of mother, fetus, and placenta to fetal vitamin D metabolism has yet to be fully determined.

Animals↗

PubMed1986

Decreased c-myc expression is an early event in retinoic acid-induced differentiation of F9 teratocarcinoma cells.

Retinoic acid-induced differentiation of mouse F9 teratocarcinoma cells is known to be accompanied by changes in gene expression. We examined the expression of the c-myc gene during retinoic acid-induced differentiation of F9 cells. Retinoic acid caused a 50% reduction in the level of c-myc mRNA after 3 hr of treatment and a 90% reduction after 12 hr. We have also examined several features of c-myc genomic structure in F9 cells, PYS2 (differentiated parietal yolk sac) cells, and liver--namely, methylation, amplification, and gross organization. Comparison of Hpa II and Msp I digests of DNAs from F9, PYS2, and liver showed that the c-myc gene in F9 cells is hypomethylated relative to that in PYS2 cells and in liver. The Hpa II sites that undergo methylation during differentiation were found to be in the second intron. Gross structural changes of the c-myc gene have not occurred in F9 or PYS2, and the c-myc gene is not amplified.

Animals↗

PubMed1986

Synthesis of coenzyme A ester of retinoic acid: intermediate in vitamin A metabolism.

Coenzyme A esters of all-trans- and 13-cis-retinoic acid were synthesized for use in studying vitamin A metabolism. The esters were obtained by two different synthetic methods starting from retinoic acids, which were converted to activated succinimidyl esters or anhydrides. These in turn were coupled with coenzyme A to form their respective thioesters. The retinoyl coenzyme A esters were purified by reverse-phase high performance liquid chromatography.

Coenzyme A↗

PubMed1986

Excessive cholecalciferol in a layers diet: decline in some aspects of reproductive performance and increased bone mineralisation of progeny.

Feeding hens a diet containing 5,000 micrograms (200,000 ICU)/kg of cholecalciferol for four 28-d periods had no adverse effect on hen-day egg production or hatchability. Egg weight, shell quality, food consumption and fertility were significantly decreased in hens fed 5,000 micrograms/kg. of cholecalciferol compared with those fed 24 micrograms (960 ICU) cholecalciferol/kg diet. Plasma calcium increased significantly as the concentration of cholecalciferol was increased in the diet. However, no histologically detectable changes in the viscera or changes in the proportion of bone ash were observed with any concentration of the vitamin. Chicks hatched from dams receiving excessive doses of cholecalciferol (5,000 micrograms/kg) and maintained on a rachitogenic diet for 4 weeks had a significantly higher proportion of tibial ash but there was no effect on either body weight or tibial calcium.

Animals↗

PubMed1986

Calcitriol corrects bone loss induced by oophorectomy in rats.

The effects of calcitriol on histomorphometric parameters of bone structure, formation, and resorption were evaluated in 21 normal or oophorectomized rats. Twelve rats were oophorectomized; six of these received daily subcutaneous injections of 135 pmol of calcitriol for 14 wk starting 38 wk after oophorectomy. Nine rats were sham operated; five of these received the same treatment with calcitriol. There were no differences in serum calcium and phosphorus between sham-operated and oophorectomized animals and rats with or without calcitriol. Calcitriol treatment of sham-operated rats produced an increase in cancellous and cortical bone mass. Oophorectomy resulted in decreased cancellous and cortical bone mass and in a decreased ratio between mineralized and nonmineralized bone. There was no difference in bone-osteoclast interface and osteoid seam width among all groups of rats. Calcitriol corrected, at least in part, the bone loss in oophorectomized animals, normalized the ratio between mineralized and nonmineralized bone, and restored mean growth plate width to normal.

Animals↗