Recent progress in enzymology and molecular biology of enzymes involved in vitamin D metabolism.
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25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗
Biomedical subjects
Y Ohyama
Publications and source records attributed to Y Ohyama.
[Cytokine mRNA expression in the labial glands of patients with Sjögren's syndrome].
It has been found that the pattern of cytokine mRNA expression was closely associated with the initiation and the progression of the disease process. The cytokine mRNA expression in the labial glands of patients with Sjögren's syndrome was examined by a PCR-based method. Th1 cytokines, such as IL-2 and IFN-gamma, were consistently detected in all patients examined, while Th2 cytokines, such as IL-4 and IL-5, were detected in some cases with strong B cell accumulation in the labial glands. These results suggest that Th1 cytokines are essential in the induction and/or maintenance of the disease, while Th2 cytokines are involved in the progression of the disease process. Other studies were also reviewed and a role of cytokines, produced by T cells and the target organ, was discussed.
Cytokines↗
Further oxidation of hydroxycalcidiol by calcidiol 24-hydroxylase. A study with the mature enzyme expressed in Escherichia coli.
The coding region of the cDNA for rat kidney calcidiol 24-hydroxylase (P450cc24), which is involved in calcium homeostasis in animals, was inserted into an expression vector pKK223-3. The recombinant plasmid was formed in a specific manner without deletion or substitution of any parts of the coding region of the cDNA. When the resulting plasmid was introduced into Escherichia coli JM109, the recombinant cells produced a protein which was immunoreactive to an antibody against P450cc24. When the cell-free extract of the transformed cells was incubated with calcidiol together with bovine adrenodoxin and NADPH-adrenodoxin reductase, not only hydroxycalcidiol but also other metabolites such as oxocalcidiol and oxohydroxycalcidiol were produced. Similarly, calcitriol was converted not only to calcitetrol but also to oxocalcitriol and oxohydroxycalcitriol. These results indicate that a single enzyme expressed in the bacteria is responsible for all these successive reactions.
Animals↗
Microdetermination of 2,3-dinor-6-ketoprostaglandin F1 alpha in human urine using gas chromatography-high-resolution selected-ion monitoring.
The microdetermination of 2,3-dinor-6-ketoprostaglandin F1 alpha (I) in human urine is described. Samples to which the [2H4]-analogue was added as an internal standard were extracted by chromatographic sample preparation using a Bond Elut C18 cartridge and a silica gel column. Conversion of the extracted I into the 1-methyl ester-6-methoxime-9,11,15-trisdimethylisopropylsilyl ether derivative was followed by gas chromatography-high-resolution selected-ion monitoring (GC-HR-SIM). Interfering substances from the urine matrix were eliminated during GC-HR-SIM analysis using a DB-1 column. A good linear response over the range 10 pg-100 ng per tube was demonstrated. Compound I could be detected in the range 26-375 pg/ml of human urine. The proposed method can be applied to the determination of I in human urine.
6-Ketoprostaglandin F1 alpha↗
Cloning and characterization of a novel natriuretic peptide in frog (Rana catesbeiana).
C-type natriuretic peptide (CNP) is one of three members of the natriuretic peptide family. In the course of cloning frog CNP precursor cDNA, we found cDNA clones encoding a novel natriuretic peptide precursor. This novel natriuretic peptide precursor is a 118-residue peptide, carrying a 22-residue signal peptide at the N terminus and a 17-residue ring structure characteristic of the natriuretic peptide family at the C terminus. The precursor is highly homologous to mammalian CNP precursors. Its C-terminal Cys codon is directly followed by a termination codon. This feature is characteristic of CNP precursor cDNAs. The cGMP-stimulating activity of the novel natriuretic peptide was at the same level with those of frog CNP and mammalian CNPs, when rat CNP receptor was used in the cGMP assay system. Thus, this novel natriuretic peptide is structurally and functionally a member of the C-type natriuretic peptide. We designated the peptide as frog CNP II, and the previously described CNP as frog CNP I. By Northern blot analysis, expression of this novel CNP mRNA was found in brain, heart, lung, and stomach. On the other hand, frog CNP mRNA was mainly expressed in brain, where mammalian CNP mRNA is exclusively expressed. The distribution of the two frog CNPs suggests a complex system for regulating electrolyte and water balance in frog.
Amino Acid Sequence↗
Identification of a vitamin D-responsive element in the 5'-flanking region of the rat 25-hydroxyvitamin D3 24-hydroxylase gene.
The 5'-flanking region of the rat vitamin D3 24-hydroxylase (P450cc24) gene was examined and a vitamin D-responsive element (VDRE) responsible for the 1 alpha,25-dihydroxyvitamin D3 (1,25-(OH)2D3) enhancement was identified. Unidirectional deletion analyses of the 5'-flanking region indicated that the region [-167/-102] is involved in vitamin D responsiveness. Further functional analyses showed that the segment [-204/-129] conferred the hormone responsiveness in an orientation-independent manner when it was placed upstream to the heterologous thymidine kinase promoter or the rabbit beta-globin promoter. The segment [-204/-129] contained two direct repeat motifs homologous to other VDREs found in the osteocalcin and osteopontin genes. Synthetic oligonucleotides containing the putative VDRE were used for functional analyses and gel mobility shift assays. The proximal [-151/-137], but not the distal [-169/-155] direct repeat activated the transcription in response to 1,25-(OH)2D3 through the beta-globin promoter. Furthermore, the proximal direct repeat formed a complex with the vitamin D receptor and a nuclear accessory factor(s) from COS cells (or retinoid X receptor) in the presence of 1,25-(OH)2D3. These results indicate that a direct repeat motif, AGGTGAgt-gAGGGCG, located at -151 base pairs upstream in the antisense strand binds to a heterologous dimer consisting of the VDR occupied with 1,25-(OH)2D3 and the nuclear accessory factor and that it plays a critical role in mediating the vitamin D enhancement of the rat P450cc24 gene expression.
Animals↗
Isolation and identification of midkine and pleiotrophin in bovine follicular fluid.
Ovarian factors that promote growth of vascular smooth muscle cells were investigated. Two distinct heparin-binding polypeptides were isolated from bovine follicular fluid by successive chromatographies. N-Terminal and tryptic peptide fragment analysis of these polypeptides revealed that they are identical to midkine (MK) and pleiotrophin (PTN), respectively, which form a new family of heparin-binding growth/differentiation factors. Two N-terminally distinct forms of midkine were also identified in the bovine follicular fluid. The concentrations of MK and PTN in the bovine follicular fluid were estimated to be 125 micrograms/l and 400 micrograms/l, respectively. The present findings that MK and PTN are quite rich in the follicular fluid will provide a new insight into so far unclarified functions of MK and PTN, especially their roles in the maturation of ovarian follicles.
Amino Acid Sequence↗
Congenital euthyroid goitre with impaired thyroglobulin transport.
A case of congenital goitre with defective thyroglobulin (Tg) synthesis was studied from the clinical, biochemical and morphological perspectives. The patient, 5.5-year-old boy, who was clinically euthyroid, showed a positive perchlorate discharge test (37.2%). However, the iodination system seemed to be normal since radioiodine uptake into the thyroid was very high, and inspection of the H2O2-generating system using thyroid slices and an assay for peroxidase activity in microsomes showed no abnormalities. On the other hand, virtually no Tg was detected in the serum, and the amount of Tg in thyroid tissue, estimated with gel electrophoresis, was below 10% of the normal value, the quality of Tg being unchanged. Morphological observations demonstrated the presence of Tg in the markedly distended rough endoplasmic reticulum of the cytoplasm of follicular cells and a lack of Tg in the colloid of the follicular lumen. These results suggest that the thyroid is defective in Tg synthesis, probably associated with impaired transport of Tg from the cells to the lumen.
Child, Preschool↗
Felodipine therapy may not alter glucose and lipid metabolism in hypertensives. Felodipine Multicenter Prospective Study Group in Japan.
The effects of long-term monotherapy with felodipine, a calcium antagonist, on blood pressure, glucose tolerance, and serum lipid profiles were prospectively investigated in 51 hypertensive patients: 13 with normal glucose tolerance and 38 with glucose intolerance. The levels of plasma glucose, serum lipids, and glycosylated hemoglobin A1c were determined before and during long-term (7.5 +/- 0.5 months; range, 6 to 9 months) therapy with felodipine. A 75-g oral glucose tolerance test was performed before and during long-term felodipine therapy. Significant decreases in both systolic and diastolic blood pressures in both patient groups were maintained during the therapy. Neither fasting nor post-glucose load venous plasma glucose levels were altered in either group of patients, and no patients with normal glucose tolerance developed diabetes mellitus during the study. Serum lipid levels did not change significantly in either group of patients except for significant decreases in high-density lipoprotein cholesterol and apolipoprotein A-I in the group with normal glucose tolerance tests, but those changes remained within the normal range. Furthermore, neither serum lipid nor apolipoprotein levels were altered, even in patients with hypercholesterolemia (total cholesterol levels, > 5.69 mmol/L = 220 mg/dL). These results suggest that long-term therapy with felodipine may not alter glucose and lipid metabolism in hypertensive patients, and felodipine appears to be useful as an antihypertensive agent for hypertensive patients with either dyslipidemia or impaired glucose metabolism.
Analysis of Variance↗
Regulation of messenger ribonucleic acid expression of 1 alpha,25-dihydroxyvitamin D3-24-hydroxylase in rat osteoblasts.
We have reported that PTH inhibits 25-hydroxyvitamin D3-24-hydroxylase messenger RNA (mRNA) expression induced by 1 alpha,25-dihydroxyvitamin D3 [1 alpha, 25-(OH)2D3] in rat kidney but not intestine. In the present study, we examined whether the suppression of 24-hydroxylase mRNA expression by PTH occurs commonly in tissues and cells which have PTH receptors. Administration of 1 alpha, 25-(OH)2D3 into rats fed a synthetic vitamin D-repleted diet containing adequate calcium greatly increased serum levels of calcium and 1 alpha, 25-(OH)2D3. Also, there was a 4-fold increase in bone 24-hydroxylase activity in response to 1 alpha, 25-(OH)2D3 administration. In rats fed a low calcium diet, renal 24-hydroxylase activity was suppressed probably due to secondary hyperparathyroidism. In contrast, the low calcium feeding did not suppress bone 24-hydroxylase activity. The expression of 24-hydroxylase mRNA in rat osteoblastic C-26 and C-11 cells was similar and attained maximal levels 24 h after cells were incubated with 10(-8) M 1 alpha, 25-(OH)2D3. Induction of 24-hydroxylase mRNA expression by 1 alpha, 25-(OH)2D3 was much greater and earlier in immature C-26 cells than mature C-11 cells. Simultaneous addition of PTH, prostaglandin E2, or cAMP together with 1 alpha, 25-(OH)2D3 did not down-regulate mRNA expression of 24-hydroxylase induced by the vitamin in both C-26 and C-11 cells. Of the three osteoblastic cells (C-26, C-20, and C-11) examined, C-26 cells showed the least mRNA expression of vitamin D receptors, in spite of the highest expression of 24-hydroxylase mRNA. These results suggest that unlike in the kidney, bone 24-hydroxylase mRNA expression is not down-regulated by PTH despite of the presence of PTH receptors. They also suggest that the degree of the induction of 24-hydroxylase mRNA by 1 alpha, 25-(OH)2D3 is not explained simply by the vitamin D receptors content.
Adenylyl Cyclases↗
Short-term starvation increases calcidiol-24-hydroxylase activity and mRNA level in rat kidney.
The renal mitochondrial calcidiol-24-hydroxylase activity and the corresponding cytochrome P-450 mRNA level were measured in rats subjected to short-term starvation alone or in combination with calcitriol treatment. Short-term starvation of 24 and 48 h increased the mRNA level by five- and six-fold, respectively. The 24-hydroxylase activity increased by five- and threefold, respectively. Treatment with calcitriol markedly increased the enzyme activity about 20-fold and the mRNA level about six-fold. In rats subjected to calcitriol treatment combined with 24 h of starvation, a significant further increase in enzyme activity was observed. The mRNA levels increased but the difference was not significant statistically. The results indicate that the mechanism by which starvation stimulates the enzymes is different, at least in part, from that behind the stimulatory effect of calcitriol.
Animals↗
Clinical utility of insulin-like growth factor binding protein-3 in the evaluation and treatment of short children with suspected growth hormone deficiency.
We have shown previously that serum insulin-like growth factor binding protein-3 (IGFBP-3) levels have good predictive value for complete, but not partial, growth hormone deficiency (GHD). In this study, we compare IGFBP-3 levels in short children previously divided into groups on the basis of their post-stimulation GH levels. Complete GHD (N = 59) included those children with peak post-stimulation GH < 5 micrograms/l. The partial GHD group (N = 49) had post-stimulation GH peaks of > 5 micrograms/l but < 10 micrograms/l. The normal children with short stature (N = 103) had post-stimulation GH peaks > 10 micrograms/l. Partial GHD and normal children with short stature also were divided into either low IGF-I or normal IGF-I subgroups. The clinical sensitivity of IGFBP-3 for complete GHD was 92%, whereas its sensitivity for partial GHD was 39%. For partial GHD, among those with low IGF-I (N = 19) 68% were also low for IGFBP-3, while 80% of those with normal IGF-I (N = 30) were also normal for IGFBP-3. The clinical specificity of IGFBP-3 for normal children with short stature was 69%. For these groups, among those with low IGF-I (N = 22) 73% also were low for IGFBP-3, while 80% of those with normal IGF-I (N = 81) also were normal for IGFBP-3. In addition, we tested whether IGFBP-3 can predict the response to GH treatment in prepubertal children by comparing pretreatment IGFBP-3 with the height gain achieved by 1 year of GH treatment.(ABSTRACT TRUNCATED AT 250 WORDS)
Adolescent↗
[CT & MRI findings of refractory vasculitis].
We described CT and MRI findings of various refractory vasculitis. Refractory vasculitis must be divided into two categories including one group with major vascular involvement and another one with small or microvascular involvement. In cases with major vessel involvement such as Takayasu's arteritis, Kawasaki disease or vasculo-Behçet disease, CT and MRI depicted direct findings of vessels itself including stenosis, obstruction, aneurysm, and wall thickening. On the other hand, CT and MRI showed various extravascular findings in cases with small or microvascular involvement, such as CNS involvement in collagen vascular disease, pulmonary lesions in Wegener's granulomatosis and Churg-Strauss syndrome, and peritoneal lesion in SLE. Our results suggest that CT and MRI findings reflect various vascular condition of refractory vasculitis and correspond to various pathology.
Humans↗
Structural characterization of the gene encoding rat 25-hydroxyvitamin D3 24-hydroxylase.
The structural gene encoding 25-hydroxyvitamin D3 24-hydroxylase (P-450cc24) was isolated from the rat genomic DNA. It spans approximately 15 kb, is composed of 12 exons, and was demonstrated by Southern blot analysis to be present as a single copy. One major T residue was identified at the cap site, a putative TATA (ATAAATA) box was located at position -30, and a putative CCAAT box was at -58. Four possible vitamin D responsive elements that may be involved in regulation of 24-hydroxylase expression were found in the 5'-flanking region. Alignment with mitochondrial P-450 proteins showed that 7 out of 11 intron insertion sites of P-450cc24 gene occupied positions identical with those in the CYP11 family (P-450scc, P-450(11 beta)). The structure of the gene is discussed in relation to present knowledge about the mechanism of regulation of the 25-hydroxyvitamin D3 24-hydroxylase and calcium homeostasis.
Amino Acid Sequence↗
HS-142-1, a novel antagonist for natriuretic peptides, has no effect on the third member of membrane bound guanylate cyclases (GC-C) in T84 cells.
HS-142-1, a novel non-peptide antagonist for natriuretic peptides, exerts antagonistic actions almost equally on two similar guanylate cyclase-linked natriuretic peptide receptors (GC-A and GC-B), but has little or no effect on the binding of natriuretic peptides to a membrane protein, the so-called "clearance receptor", which binds all natriuretic peptides. The third mammalian form of membrane bound guanylate cyclases (GC-C) was identified not as a natriuretic peptide receptor, but as a receptor for heat-stable enterotoxins (STa). In this study, we examined effects of HS-142-1 on GC-C (STaR) in T84 cells and showed that HS-142-1 exerts neither agonistic nor antagonistic activity for GC-C, indicating that HS-142-1 is not a common antagonist for a family of membrane bound guanylate cyclase receptors, but a specific antagonist for the guanylate cyclase-linked natriuretic peptide receptors.
Atrial Natriuretic Factor↗
Isolation and characterization of high molecular weight melanogenic inhibitors naturally occurring in melanoma cells.
Intrinsic melanogenic inhibitors with high molecular weights have been isolated from Greene's amelanotic hamster melanoma by DEAE ion-exchange and gel filtration chromatographies. The native molecular weights of two partially purified inhibitors have been determined to be 15 kDa (beta-type) and 67 kDa (gamma-type), respectively, using HPLC gel filtration. Both types of inhibitors, despite their inability to directly inhibit isolated tyrosinase, have been shown to markedly inhibit melanin formation in cultured B16 cells. In contrast to the beta-type inhibitor, the gamma-type inhibitor can induce depigmentation in B16 cells without abolishing their internal tyrosinase activity. Further, it has been determined that both inhibitors contain various amounts of unsaturated fatty acids, C15:1, C18:1, C18:2, C18:3, C20:3, and C20:4, which exhibit depigmentary activities on cultured B16 cells. C15:1 is low in the beta-type, but high in the gamma-type whereas C18:3 is high in the beta-type but low in the gamma-type. These results suggest that the differential action of these inhibitors is most likely due to the composition of the unsaturated fatty acids.
Animals↗
Comparison between insulin-like growth factor-I (IGF-I) and IGF binding protein-3 (IGFBP-3) measurement in the diagnosis of growth hormone deficiency.
To analyze the utility of insulin-like growth factor-I (IGF-I) and insulin-like growth factor binding protein-3 (IGFBP-3) radioimmunoassay in the diagnosis of growth hormone deficiency (GHD) we measured IGF-I and IGFBP-3 in sera from normal children (n = 309), short children (n = 99) and patients with GHD (n = 73). In 80% and 93% of classical GHD (cGHD), IGF-I and IGFBP-3 levels, respectively, were below the age-related cutoff levels (lower limit). In 81% and 88% of normal short children (NS), IGF-I and IGFBP-3 levels, respectively, were above the age-related cutoff levels. Thus, both IGF-I and IGFBP-3 were good parameters for screening GHD. In contrast, in more than half of partial GHD (pGHD), either IGF-I or IGFBP-3 was above the age-related cutoff levels. The poor discrimination between patients with pGHD and NS by using these two parameters may be the result of their relatively similar GH levels, as compared to cGHD, or due to the limitations of GH stimulation tests. In about 80-90% of NS, IGF-I and IGFBP-3 were above the age-related cutoff levels at all ages. A hundred percent of cGHD under 10 years old had IGFBP-3 below the age-related cutoff levels, whereas 79% of cGHD under 10 years old had IGF-I below the age-related cutoff levels. Thus in the younger age groups, IGFBP-3 may be more sensitive than IGF-I. It may be because IGFBP-3 levels are relatively higher than those of IGF-I in younger subjects.(ABSTRACT TRUNCATED AT 250 WORDS)
Adolescent↗
Expression of mRNA coding for pituitary hormones and pituitary-specific transcription factor in the pituitary gland of the rdw rat with hereditary dwarfism.
The rdw rat (gene symbol: rdw) with hereditary dwarfism has been shown immunohistochemically to have subnormal numbers not only of GH- but also of prolactin- and thyrotrophin-positive cells. To characterize the dwarfism of this strain, the expression of pituitary hormone mRNAs was examined by Northern hybridization. The pituitary gland in the rdw rat expressed 30-100 times less GH and prolactin mRNAs than normal controls, whereas mRNAs for pro-opiomelanocortin and the alpha subunit of rat glycoprotein hormone revealed a significant increase. There was a non-significant difference in rat LH-beta subunit and FSH-beta subunit between normal and rdw rats. The suppressed expression of a pituitary-specific transcription factor, Pit-1, is considered to cause hereditary dwarfism in mouse strains Snell and Jackson, whose phenotypes resemble those of the rdw rat. In this study, however, no difference in mRNA expression for Pit-1 was found between rdw rats and controls. This work indicates that the rdw rat may not have the same genotype as the phenotypically similar dwarf mice, Snell, Jackson and Ames.
Animals↗