Isolation of chick intestinal nuclei. Effect of vitamin D3 on nuclear metabolism.
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ABSTRACT:
SUBMITTER: Lawson DE
PROVIDER: S-EPMC1185098 | biostudies-other | 1969 Nov
REPOSITORIES: biostudies-other
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Project description:1. A comparison was made of the nature and intestinal intracellular distribution of the metabolites formed in vitamin D-deficient chicks from [4-(14)C]cholecalciferol and [1-(3)H]cholecalciferol. 2. The simultaneous administration of the two radioactive substances showed the presence in blood, liver, intestine, kidney and bone of cholecalciferol, its ester, 25-hydroxycholecalciferol and a further metabolite of cholecalciferol more polar than 25-hydroxycholecalciferol. The (3)H/(14)C ratios in these four radioactive components were the same as that of the dosed material (4.7:1) with the exception of the most polar material. The (3)H/(14)C ratio was lower in the fourth, most polar, metabolite (0.4:1-1.8:1) in all tissues examined, with the exception of blood. 3. In the chick intestine the polar metabolite accounted for almost 70% of the radioactivity in this tissue after a dose of 0.5mug. of [4-(14)C,1-(3)H]cholecalciferol. This polar metabolite from the intestine also had the lowest (3)H/(14)C ratio of all the tissues. It appears that in the chick intestine the polar metabolite reaches a maximum concentration of 1ng./g. of tissue, above which it cannot be increased irrespective of the dose of the vitamin. 4. The intestinal intracellular organelle with the highest concentration of (14)C radioactivity is the nucleus, and this radioactivity is almost entirely due to the polar metabolite with the lowered (3)H/(14)C ratio, in this case <0.2:1. It appears to be further localized in the chromatin of the nuclei. However, about half of the polar metabolite in the intestine is extranuclear. 5. Double-labelled 25-hydroxycholecalciferol was prepared and after its administration to vitamin D-deficient chicks the polar metabolite with the lowered (3)H/(14)C ratio was detected in liver, kidney, intestine, bone, muscle and heart. 6. None of the polar metabolite with the lowered (3)H/(14)C ratio was detected 16hr. after dosing with either the double-labelled vitamin or the double-labelled 25-hydroxycholecalciferol in blood and adipose tissue of vitamin D-deficient chicks, nor in the intestine, liver and kidney of supplemented birds. 7. The reasons for this loss of (3)H relative to (14)C are discussed in relation to possible chemical structures of this new polar metabolite.
| S-EPMC1185099 | biostudies-other
Project description:1. An attempt has been made to locate the site of action of vitamin D(3) as it affects the translocation of calcium across the intestine. 2. Calcium appears to be pumped out of cells by a process dependent on energy from metabolism. 3. The effects of cold, inhibitors and vitamin D(3) on the translocation of calcium by everted sacs of intestine were studied and compared with results obtained in vivo. 4. A model was proposed to explain the results which suggests that vitamin D(3) inhibits a metabolically operated pump that returns calcium from the mucosal cell to the lumen. 5. Some observations on the effect of sodium lauryl sulphate on the translocation of calcium in vivo and in vitro are reported.
| S-EPMC1207062 | biostudies-other
Project description:RationalePatients with chronic rhinosinusitis with nasal polyps (CRSwNP) have been shown to be vitamin D3 (VD3) deficient, which is associated with more severe disease and increased polyp size. To gain mechanistic insights into these observational studies, we examined the impact of VD3 deficiency on inflammation and VD3 metabolism in an Aspergillus fumigatus (Af) mouse model of chronic rhinosinusitis (Af-CRS).MethodsBalb/c mice were fed control or VD3 deficient diet for 4 weeks. Mice were then sensitized with intraperitoneal Af, and one week later given Af intranasally every three days for four weeks while being maintained on control or VD3 deficient diet. Airway function, sinonasal immune cell infiltrate and sinonasal VD3 metabolism profiles were then examined.ResultsMice with VD3 deficiency had increased Penh and sRaw values as compared to controls as well as exacerbated changes in sRaw when coupled with Af-CRS. As compared to controls, VD3 deficient and Af-CRS mice had reduced sinonasal 1α-hydroxylase and the active VD3 metabolite, 1,25(OH)2D3. Differential analysis of nasal lavage samples showed that VD3 deficiency alone and in combination with Af-CRS profoundly upregulated eosinophil, neutrophil and lymphocyte numbers. VD3 deficiency exacerbated increases in monocyte-derived dendritic cell (DC) associated with Af-CRS. Conversely, T-regulatory cells were decreased in both Af-CRS mice and VD3 deficient mice, though coupling VD3 deficiency with Af-CRS did not exacerbate CD4 or T-regulatory cells numbers. Lastly, VD3 deficiency had a modifying or exacerbating impact on nasal lavage levels of IFN-γ, IL-6, IL-10 and TNF-α, but had no impact on IL-17A.ConclusionsVD3 deficiency causes changes in sinonasal immunity, which in many ways mirrors the changes observed in Af-CRS mice, while selectively exacerbating inflammation. Furthermore, both VD3 deficiency and Af-CRS were associated with altered sinonasal VD3 metabolism causing reductions in local levels of the active VD3 metabolite, 1,25(OH)2D3, even with adequate circulating levels.
| S-EPMC5646812 | biostudies-literature
Project description:Undegraded polyribosome preparations may be obtained from chick intestinal mucosa if ribonuclease activity is strictly controlled. This is best achieved by homogenization of the mucosa directly in rat liver cell-sap. 2. The extent of amino acid incorporation by chick intestinal polyribosomes is greatly influenced by the source of the cell-sap. Sephadex-treated intestinal cell-sap caused impaired incorporation and release of completed polypeptide chains, whereas Sephadex-treated rat liver cell-sap promoted the polymerization of up to 90 amino acids per ribosome. Under optimum conditions 30-35% of the nascent polypeptide chains are completed and released. 3. The preparation of an antiserum against the calcium-binding protein formed in response to vitamin D is described. It is shown that the antiserum is highly specific for calcium-binding protein. 4. This antiserum was used to investigate the ability of chick intestinal polyribosomes to synthesize calciumbinding protein. Only polyribosomes from chicks receiving vitamin D have the ability to synthesize calcium-binding protein. Moreover, the product formed in vitro has the same electrophoretic mobility as calcium-binding protein synthesized in vivo. 5. It is concluded that one of the main functions of vitamin D in the small intestine is to induce the synthesis de novo of calcium-binding protein.
| S-EPMC1167995 | biostudies-other
Project description:CYP27A1 is a mitochondrial cytochrome P450 which can hydroxylate vitamin D3 and cholesterol at carbons 25 and 26, respectively. The product of vitamin D3 metabolism, 25-hydroxyvitamin D3, is the precursor to the biologically active hormone, 1?,25-dihydroxyvitamin D3. CYP27A1 is attached to the inner mitochondrial membrane and substrates appear to reach the active site through the membrane phase. We have therefore examined the ability of bacterially expressed and purified CYP27A1 to metabolize substrates incorporated into phospholipid vesicles which resemble the inner mitochondrial membrane. We also examined the ability of CYP27A1 to metabolize 20-hydroxyvitamin D3 (20(OH)D3), a novel non-calcemic form of vitamin D derived from CYP11A1 action on vitamin D3 which has anti-proliferative activity on keratinocytes, leukemic and myeloid cells. CYP27A1 displayed high catalytic activity towards cholesterol with a turnover number (k(cat)) of 9.8 min(-1) and K(m) of 0.49 mol/mol phospholipid (510 ?M phospholipid). The K(m) value of vitamin D3 was similar for that of cholesterol, but the k(cat) was 4.5-fold lower. 20(OH)D3 was metabolized by CYP27A1 to two major products with a k(cat)/K(m) that was 2.5-fold higher than that for vitamin D3, suggesting that 20(OH)D3 could effectively compete with vitamin D3 for catalysis. NMR and mass spectrometric analyses revealed that the two major products were 20,25-dihydroxyvitamin D3 and 20,26-dihydroxyvitamin D3, in almost equal proportions. Thus, the presence of the 20-hydroxyl group on the vitamin D3 side chain enables it to be metabolized more efficiently than vitamin D3, with carbon 26 in addition to carbon 25 becoming a major site of hydroxylation. Our study reports the highest k(cat) for the 25-hydroxylation of vitamin D3 by any human cytochrome P450 suggesting that CYP27A1 might be an important contributor to the synthesis of 25-hydroxyvitamin D3, particularly in tissues where it is highly expressed.
| S-EPMC3303980 | biostudies-literature
Project description:Cortisol was reported to downregulate body-fluid Ca(2+) levels in mammals but was proposed to show hypercalcemic effects in teleostean fish. Fish, unlike terrestrial vertebrates, obtain Ca(2+) from the environment mainly via the gills and skin rather than by dietary means, and have to regulate the Ca(2+) uptake functions to cope with fluctuating Ca(2+) levels in aquatic environments. Cortisol was previously found to regulate Ca(2+) uptake in fish; however, the molecular mechanism behind this is largely unclear. Zebrafish were used as a model to explore this issue. Acclimation to low-Ca(2+) fresh water stimulated Ca(2+) influx and expression of epithelial calcium channel (ecac), 11?-hydroxylase and the glucocorticoid receptor (gr). Exogenous cortisol increased Ca(2+) influx and the expressions of ecac and hydroxysteroid 11-beta dehydrogenase 2 (hsd11b2), but downregulated 11?-hydroxylase and the gr with no effects on other Ca(2+) transporters or the mineralocorticoid receptor (mr). Morpholino knockdown of the GR, but not the MR, was found to impair zebrafish Ca(2+) uptake function by inhibiting the ecac expression. To further explore the regulatory mechanism of cortisol in Ca(2+) uptake, the involvement of vitamin D(3) was analyzed. Cortisol stimulated expressions of vitamin D-25hydroxylase (cyp27a1), cyp27a1 like (cyp27a1l), 1?-OHase (cyp27b1) at 3 dpf through GR, the first time to demonstrate the relationship between cortisol and vitamin D(3) in fish. In conclusion, cortisol stimulates ecac expression to enhance Ca(2+) uptake functions, and this control pathway is suggested to be mediated by the GR. Lastly, cortisol also could mediate vitamin D(3) signaling to stimulate Ca(2+) uptake in zebrafish.
| S-EPMC3161063 | biostudies-literature
Project description:PurposeTo study the in vitro effect of vitamin D3 on the healing response of human Tenon's fibroblasts (HTF) and its possible role in preventing excessive postoperative subconjunctival fibrosis.MethodsEffect of vitamin D3 on cytotoxicity and cell survival of primary cultured HTF was measured by lactate dehydrogenase and PrestoBlue assays, respectively. Proliferation and migration of vitamin D3-treated HTF (D3-HTF) was determined by CyQUANT proliferation and scratch assay, respectively. The mRNA expression profiles of control-HTF and D3-HTF from six subjects (three with glaucoma and long-term use of topical medications, three with primary pterygium) were assessed by RNA sequencing analyses to identify potential biomarkers for the inhibitory effect on HTF by vitamin D3. Validation of these biomarkers and their potential pathways were performed by quantitative real-time polymerase chain reaction (qRT-PCR) detection.ResultsPure monolayers of HTF from controls (retinal detachment or squint surgeries), pterygium, and glaucoma subjects were successfully prepared and passaged. Proliferation and migration of pterygium and glaucoma HTF were inhibited by vitamin D3 in a dose-dependent manner, and without cytotoxicity or decrease in cellular viability with concentrations up to 10 µM. The qRT-PCR results were consistent with the transcriptome analyses, vitamin D3 appears to enhance CYP24A1, SHE, KRT16 but suppresses CILP expression in HTF.ConclusionsVitamin D3 can inhibit the in vitro activity of HTF without compromising cellular survivability at concentration up to 10 µM. This has potential clinical application for improving the outcome of pterygium and filtering surgeries.Translational relevanceVitamin D3 can suppress the in vitro proliferation, migration, and transdifferentiation of human Tenon's fibroblasts, without the cytotoxicity of mitomycin-C, the current standard antifibrotic agent in clinical use.
| S-EPMC8287040 | biostudies-literature
Project description:We show that cytochrome P450scc (CYP11A1) in either a reconstituted system or in isolated adrenal mitochondria can metabolize vitamin D3. The major products of the reaction with reconstituted enzyme were 20-hydroxycholecalciferol and 20,22-dihydroxycholecalciferol, with yields of 16 and 4%, respectively, of the original vitamin D3 substrate. Trihydroxycholecalciferol was a minor product, likely arising from further metabolism of dihydroxycholecalciferol. Based on NMR analysis and known properties of P450scc we propose that hydroxylation of vitamin D3 by P450scc occurs sequentially and stereospecifically with initial formation of 20(S)-hydroxyvitamin D3. P450scc did not metabolize 25-hydroxyvitamin D3, indicating that modification of C25 protected it against P450scc action. Adrenal mitochondria also metabolized vitamin D3 yielding 10 hydroxyderivatives, with UV spectra typical of vitamin D triene chromophores. Aminogluthimide inhibition showed that the three major metabolites, but not the others, resulted from P450scc action. It therefore appears that non-P450scc enzymes present in the adrenal cortex to some extent contribute to metabolism of vitamin D3. We conclude that purified P450scc in a reconstituted system or P450scc in adrenal mitochondria can add one hydroxyl group to vitamin D3 with subsequent hydroxylation being observed for reconstituted enzyme but not for adrenal mitochondria. Additional vitamin D3 metabolites arise from the action of other enzymes in adrenal mitochondria. These findings appear to define novel metabolic pathways involving vitamin D3 that remain to be characterized.
| S-EPMC2234577 | biostudies-literature
Project description:Cytochrome P450scc (CYP11A1) can hydroxylate vitamin D3 to produce 20-hydroxyvitamin D3 and other poorly characterized hydroxylated products. The present study aimed to identify all the products of vitamin D3 metabolism by P450scc, as well as the pathways leading to their formation. Besides 20-hydroxyvitamin D3, other major metabolites of vitamin D3 were a dihydroxyvitamin D3 and a trihydroxyvitamin D3 product. The dihydroxyvitamin D3 was clearly identified as 20,23-dihydroxyvitamin D3 by NMR, in contrast to previous reports that postulated hydroxyl groups in positions 20 and 22. NMR of the trihydroxy product identified it as 17alpha,20,23-trihydroxyvitamin D3. This product could be directly produced by P450scc acting on 20,23-dihydroxyvitamin D3, confirming that hydroxyl groups are present at positions 20 and 23. Three minor products of D3 metabolism by P450scc were identified by MS and by examining their subsequent metabolism by P450scc. These products were 23-hydroxyvitamin D3, 17alpha-hydroxyvitamin D3 and 17alpha,20-dihydroxyvitamin D3 and arise from the three P450scc-catalysed hydroxylations occurring in a different order. We conclude that the major pathway of vitamin D3 metabolism by P450scc is: vitamin D3 --> 20-hydroxyvitamin D3 --> 20,23-dihydroxyvitamin D3 --> 17alpha,20,23-trihydroxyvitamin D3. The major products dissociate from the P450scc active site and accumulate at a concentration well above the P450scc concentration. Our new identification of the major dihydroxyvitamin D3 product as 20,23-dihydroxyvitamin D3, rather than 20,22-dihydroxyvitamin D3, explains why there is no cleavage of the vitamin D3 side chain, unlike the metabolism of cholesterol by P450scc.
| S-EPMC2562691 | biostudies-literature
Project description:Gut microbiota is a complex aggregation of microbial organisms, which offers diverse protective benefits to the host. Dysbiosis of intestinal microbiota is frequently associated with many diseases. Vitamin D3 (VD), which was originally associated with bone health, also possesses antimicrobial activities and can act through antimicrobial peptide. Cathelicidin is a type of antimicrobial peptide in host to maintain the balance of gut microbiome. Our current study sought to evaluate the protective effect of VD and cathelicidin in mice intestines by administration of VD or mCRAMP-encoding L. lactis. We herein provided a comprehensive profile of the impact of VD and mCRAMP on gut microbiota using 16S rRNA sequencing, followed by bioinformatics and statistical analysis. Our results revealed an increased richness of bacterial community in mice intestines due to VD administration. Moreover, we showed a beneficial effect of VD and mCRAMP by enhancing the colonization of bacterial taxa that are associated with protective effects to the host but repressing the propagation of bacterial taxa that are associated with harmful effects to the host. Various metabolic pathways related to amino acid and lipid metabolism were affected in this process. We further established a bacterial panel as a reliable biomarker to evaluate the efficacy of remodeling the mice gut microbiota by VD and mCRAMP administration. The uncovered effects will deepen the comprehension about the antibacterial mechanisms of VD and mCRAMP and provide new insights for therapeutic implication of them.
| S-EPMC8732771 | biostudies-literature