Biomedical subjects

N Sawada

Publications and source records attributed to N Sawada.

At least 55 records · Page 3Linked to original sources

PubMed2000

Characteristics of choline transport across the blood-brain barrier in mice: correlation with in vitro data.

PURPOSE: We examined the functional properties of choline transport across the blood-brain barrier (BBB) in mice. We compared the kinetic parameters and transport properties with those found in our in vitro uptake experiments using mouse brain capillary endothelial cells (MBEC4). METHODS: The permeability coefficient-surface area product (PS) values of [3H]choline at the BBB were estimated by means of an in situ brain perfusion technique in mice. RESULTS: [3H]Choline uptake was well described by a two-component model: a saturable component and a nonsaturable linear component. The [3H]choline uptake was independent of pH and Na+, but was significantly decreased by the replacement of Na+ with K+. Various basic drugs, including substrates and inhibitors of the organic cation transporter, significantly inhibited the [3H]choline uptake. These in situ (in vivo) results corresponded well to the in vitro results and suggest that the choline transporter at the BBB is a member of the organic cation transporter (OCT) family. CONCLUSION: The choline transport mechanism at the BBB is retained in MBEC4.

Algorithms↗

PubMed2000

Dual metabolic pathway of 25-hydroxyvitamin D3 catalyzed by human CYP24.

Human 25-hydroxyvitamin D3 (25(OH)D3) 24-hydroxylase (CYP24) cDNA was expressed in Escherichia coli, and its enzymatic and spectral properties were revealed. The reconstituted system containing the membrane fraction prepared from recombinant E. coli cells, adrenodoxin and adrenodoxin reductase was examined for the metabolism of 25(OH)D3, 1alpha,25(OH)2D3 and their related compounds. Human CYP24 demonstrated a remarkable metabolism consisting of both C-23 and C-24 hydroxylation pathways towards both 25(OH)D3 and 1alpha,25(OH)2D3, whereas rat CYP24 showed almost no C-23 hydroxylation pathway [Sakaki, T. Sawada, N. Nonaka, Y. Ohyama, Y. & Inouye, K. (1999) Eur. J. Biochem. 262, 43-48]. HPLC analysis and mass spectrometric analysis revealed that human CYP24 catalyzed all the steps of the C-23 hydroxylation pathway from 25(OH)D3 via 23S, 25(OH)2D3, 23S,25,26(OH)3D3 and 25(OH)D3-26,23-lactol to 25(OH)D3-26, 23-lactone in addition to the C-24 hydroxylation pathway from 25(OH)D3 via 24R,25(OH)2D3, 24-oxo-25(OH)D3, 24-oxo-23S,25(OH)2D3 to 24,25,26,27-tetranor-23(OH)D3. On 1alpha,25(OH)2D3 metabolism, similar results were observed. These results strongly suggest that the single enzyme human CYP24 is greatly responsible for the metabolism of both 25(OH)D3 and 1alpha,25(OH)2D3. We also succeeded in the coexpression of CYP24, adrenodoxin and NADPH-adrenodoxin reductase in E. coli. Addition of 25(OH)D3 to the recombinant E. coli cell culture yielded most of the metabolites in both the C-23 and C-24 hydroxylation pathways. Thus, the E. coli expression system for human CYP24 appears quite useful in predicting the metabolism of vitamin D analogs used as drugs.

Adrenodoxin↗

PubMed2000

Cholestasis with altered structure and function of hepatocyte tight junction and decreased expression of canalicular multispecific organic anion transporter in a rat model of colitis.

Cholestasis is frequently associated with inflammatory bowel disease. Because some cholestasis is resulted from altered hepatocyte tight junctions (TJs) or the canalicular multispecific organic anion transporter, we have investigated the following topics in a rat model of inflammatory bowel disease: (1) alterations in hepatocyte TJs and in the canalicular multispecific organic anion transporter, (2) etiologic factors for cholestasis, and (3) effects of antibiotics on cholestasis. Rats with trinitrobenzene sulfonic acid-induced colitis were studied 24 hours after treatment. Hepatocyte TJs and the canalicular multispecific organic anion transporter were evaluated by immunostaining for TJ-associated proteins, 7H6 and ZO-1, and multidrug resistance protein 2 (mrp2). To investigate etiologic factors causing cholestasis, portal endotoxin and proinflammatory cytokines were examined. The effects of polymyxin B, penicillin G, or metronidazole on immunostaining for 7H6, ZO-1, mrp2, and cholestasis were investigated. (1) Immunostaining for 7H6 and ZO-1 colocalized outlining the bile canaliculi and immunostaining for mrp2 localized on the canalicular membrane in controls. Treatment with trinitrobenzene sulfonic acid induced significant cholestasis and caused translocation of immunostaining for 7H6, but not that for ZO-1, to the cytoplasm and diminished immunostaining for mrp2 on the canaliculus membrane. (2) The levels of portal endotoxin, but not proinflammatory cytokines, was increased. (3) Polymyxin B, but not the other antibiotics, prevented alterations in immunostaining for both 7H6 and mrp2, and cholestasis. We described that both hepatocyte TJs and the canalicular multispecific organic anion transporter were altered and that gut-derived endotoxin levels in the portal blood were increased in this rat colitis model.

Animals↗

PubMed2000

Oxidative stress augments secretion of endothelium-derived relaxing peptides, C-type natriuretic peptide and adrenomedullin.

OBJECTIVE: Excess oxidative stress is one of the major metabolic abnormalities on vascular walls in hypertension and atherosclerosis. In order to further elucidate the endothelial function under oxidative stress, the effect of hydrogen peroxide (H2O2) on expression of two novel endothelium-derived vasorelaxing peptides, C-type natriuretic peptide (CNP) and adrenomedullin (AM) from bovine carotid artery endothelial cells (BCAECs) was examined. METHODS: BCAECs were treated with H2O2 (0.1-1.0 mmol/ l) and/or an antioxidant, N-acetylcysteine (NAC) (5-10 mmol/l), and incubated for 48 h. The concentrations of CNP and AM were measured with the specific radioimmuno assays that we originally developed. CNP and AM mRNA expressions were also examined by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Treatment of BCAECs with 0.5 and 1 mmol/l H2O2 induced 9-and 10-fold increases of CNP concentration in the media. Addition of 10 mmol/l NAC significantly suppressed the effect of H2O2 by 52%. RT-PCR analysis showed that CNP mRNA expression in BCAECs was also rapidly augmented within 1 h with H2O2 (1 mmol/l) treatment, and reached a peak at 3 h to show a 10-fold increase. AM secretion from BCAECs also increased to two-fold with exposure to 0.5 mmol/l H2O2, accompanied with the augmented level of AM mRNA. NAC 10 mmol/l completely suppressed the effect of H2O2 on AM secretion. CONCLUSIONS: In this study, it has been demonstrated that H2O2 augments endothelial secretion of the two endothelium-derived relaxing peptides, CNP and AM. Our findings suggest the increased secretion of CNP and AM from endothelium under oxidative stress may function to compensate the impaired nitric oxide-dependent vasorelaxation in hypertension and atherosclerosis.

Acetylcysteine↗

PubMed2000

Altered gene expression of uncoupling protein-2 and -3 in stroke-prone spontaneously hypertensive rats.

BACKGROUND: Uncoupling proteins (UCPs) are the inner mitochondrial membrane-associated proteins, which dissipate the proton gradient and generate heat instead of ATP. The involvement of UCPs in energy expenditure and glucose metabolism has been suggested. Recently, we succeeded in cloning of rat UCP2 and UCP3. OBJECTIVE: The aim of this study was to elucidate the pathophysiological role of UCP2 and UCP3 in hypertension associated with hyperglycemia in stroke-prone spontaneously hypertensive rats (SHR-SP). METHODS: UCP2 and UCP3 mRNA levels of cardiac and gastrocnemius muscles in SHR-SP and Wistar-Kyoto (WKY) rats were determined at 6 weeks (prehypertensive stage) and at 15 weeks (hypertensive stage). RESULTS: UCP2 and UCP3 mRNA levels in the heart of SHR-SP at 6 weeks were significantly higher than those of WKY rats (1.6-fold, 3.6-fold, respectively). These tendencies did not change in the heart at 15 weeks. UCP2 and UCP3 mRNA levels in the skeletal muscle of SHR-SP at 6 weeks were significantly higher than those of WKY rats (1.4-fold, 2.4-fold, respectively). In contrast, at 15 weeks, UCP2 and UCP3 mRNA levels in the skeletal muscle of SHR-SP were significantly lower than those of WKY rats (70 and 36% of WKY rats, respectively). Therefore, the decrease of UCP2 and UCP3 in the skeletal muscle was observed with the concomitant development of hypertension in SHR-SP. UCP2 mRNA levels in the epididymal fat of SHR-SP at 15 weeks were similar to that of WKY rats. CONCLUSIONS: Altered gene expression of UCP2 and UCP3 might be related to some pathophysiological aspects in hypertension and glucose metabolism in SHR-SP.

Adipose Tissue↗

PubMed2000

Expression of GFRalpha-1, receptor for GDNF, in rat brain capillary during postnatal development of the BBB.

It is well known that the blood-brain barrier (BBB) matures at approximately 2 wk after birth in the rat. Recently, we showed that glial cell line-derived neurotrophic factor (GDNF) enhances the barrier function of porcine endothelial cells forming the BBB in culture. In the present study, we examined the relation between permeability of the BBB, using Evans blue as a tracer, and expression of the GDNF family receptor (GFRalpha-1) during postnatal development of the BBB. Morphometric analysis showed that exudation of Evans blue from capillaries of the cerebral cortex progressively decreased until postnatal day 21. Inversely, immunohistochemical examinations showed expression of GFRalpha-1 in the capillaries at postnatal day 3 and expression that reached the same levels as observed in adult rats by postnatal day 10. However, c-ret, which is thought to mediate a signal evoked by binding of GDNF to GFRalpha-1, was not expressed in the capillaries of the brain cortex in 3-mo-old rats. On the other hand, the tight junction proteins occludin and ZO-1 appeared to be fully expressed at birth. The reciprocal relation between GFRalpha-1 expression and the permeability of the BBB strongly suggests active participation of GDNF in postnatal development of the BBB, although the mechanism(s) involved is still veiled.

Animals↗

PubMed2000

Expression of receptors for glial cell line-derived neurotrophic factor (GDNF) and neurturin in the inner blood-retinal barrier of rats.

The retina is protected from somatic circulation by the blood-retinal barrrier (BRB) composed of tight junctions between retinal vascular endothelial cells (the inner BRB) and those between retinal pigment epithelial cells (the outer BRB). Our recent studies showed that glial cell line-derived neurotrophic factor (GDNF) secreted from astrocytes regulates the permeability of the BBB. In the present study, we immunohistochemically examined the expression of GDNF, neurturin (NTN) and their receptors, GFRalpha1 for GDNF and GFRalpha2 for NTN, because the capillaries of the inner BRB show specialization very similar to the blood-brain barrier (BBB). GDNF and NTN were detected in glial fibrillary acidic protein (GFAP)-positive cells, including Müller cells. GFRalpha1 and GFRalpha2 were localized in von Willebrand factor-positive cells. GDNF and NTN enhanced the barrier function of endothelial cells derived from porcine brain cortex. These results strongly suggest that the barrier function of the BRB is regulated by GDNF and NTN secreted from glial cells, like the BBB.

Animals↗

PubMed1999

Enhanced binding of the neural siglecs, myelin-associated glycoprotein and Schwann cell myelin protein, to Chol-1 (alpha-series) gangliosides and novel sulfated Chol-1 analogs.

Extended glycoconjugate binding specificities of three sialic acid-dependent immunoglobulin-like family member lectins (siglecs), myelin-associated glycoprotein (MAG), Schwann cell myelin protein (SMP), and sialoadhesin, were compared by measuring siglec-mediated cell adhesion to immobilized gangliosides. Synthetic gangliosides bearing the alpha-series determinant (NeuAc alpha2,6-linked to GalNAc on a gangliotetraose core) were tested, including GD1alpha (IV(3)NeuAc, III(6)NeuAc-Gg(4)OseCer), GD1alpha with modified sialic acid residues at the III(6)-position, and the "Chol-1" gangliosides GT1aalpha (IV(3)NeuAc, III(6)NeuAc, II(3)NeuAc-Gg(4)OseCer) and GQ1balpha (IV(3)NeuAc, III(6)NeuAc, II(3)(NeuAc)(2)-Gg(4)OseCer). The alpha-series gangliosides displayed enhanced potency for MAG- and SMP-mediated cell adhesion (GQ1balpha > GT1aalpha, GD1alpha > GT1b, GD1a >> GM1 (nonbinding)), whereas sialoadhesin-mediated adhesion was comparable with alpha-series and non-alpha-series gangliosides. GD1alpha derivatives with modified sialic acids (7-, 8-, or 9-deoxy) or sulfate (instead of sialic acid) at the III(6)-position supported adhesion comparable with that of GD1alpha. Notably, a novel GT1aalpha analog with sulfates at two internal sites of sialylation (NeuAcalpha2,3Galbeta1,4GalNAc-6-sulfatebeta1, 4Gal3-sulfatebeta1,4Glcbeta1,1'ceramide) was the most potent siglec-binding structure tested to date (10-fold more potent than GT1aalpha in supporting MAG and SMP binding). Together with prior studies, these data indicate that MAG and SMP display an extended structural specificity with a requirement for a terminal alpha2, 3-linked NeuAc and great enhancement by nearby precisely spaced anionic charges.

Animals↗

PubMed1999

Induction of tight junctions in human connexin 32 (hCx32)-transfected mouse hepatocytes: connexin 32 interacts with occludin.

Small gap junction plaques are associated with tight junction strands in some cell types including hepatocytes and it is thought that they may be closely related to tight junctions and the establishment of cell polarity. In order to examine roles of gap junctions in regulating expression and structure of tight junctions, we transfected human Cx32 cDNA into immortalized mouse hepatocytes (CHST8 cells) which lack endogenous Cx32 and Cx26. Immunocytochemistry revealed that endogenous integral tight junction protein occludin was strongly localized and was colocalized with Cx32 at cell borders in transfectants, whereas neither was detected in parental cells. In Northern blots, mRNAs encoding occludin and the other integral tight junction proteins, claudin-1 and -2, were induced in the transfectants compared to parental cells. In Western blots, occludin protein was increased in the transfectants compared to parental cells, and binding of occludin to Cx32 protein was demonstrated by immunoprecipitation. In freeze fracture of the transfectants, tight junction strands were more numerous and complex compared to parental cells, and small gap junction plaques appeared within induced tight junction strands. Nevertheless, no change in barrier function of tight junctions was observed. These results indicate that in hepatocytes, gap junction, and tight junction expression are closely coordinated, and that Cx32 may play a role in regulating occludin expression.

Animals↗

PubMed1999

Induction of thymidine phosphorylase expression and enhancement of efficacy of capecitabine or 5'-deoxy-5-fluorouridine by cyclophosphamide in mammary tumor models.

Thymidine phosphorylase (dThdPase) is an essential enzyme for the activation of the oral cytostatic drugs capecitabine (N(4)-pentyloxycarbonyl-5'-deoxy-5-fluorocytidine, Xeloda(trade mark)) and its intermediate metabolite doxifluridine [5'-deoxy-5-fluorouridine (5'-dFUrd, Furtulon((R)))] to 5-fluorouracil (5-FUra) in tumors. In a previous study, we found that several cytostatics were able to up-regulate tumor levels of dThdPase in a human colon cancer xenograft model. In the present study, we confirmed that the administration of cytostatics used for breast cancer treatment, such as taxanes and cyclophosphamide (CPA), up-regulated the tumor level of dThdPase in mammary tumor models as well. Because the dThdPase up-regulation was observed even when CPA was given orally, we investigated further the usefulness of combination therapy with the 2 oral drugs, 5'-dFUrd/capecitabine and CPA in mammary tumor models. Daily oral administration of CPA up-regulated human dThdPase levels in the tumor tissue of mice bearing a human mammary tumor xenograft, MX-1, whereas in the small intestine and liver, it did not affect levels of pyrimidine nucleoside phosphorylases (PyNPase) including dThdPase and uridine phosphorylase. The preferential up-regulation of PyNPase activity in the tumor by CPA administration was also confirmed in mice bearing a syngeneic murine mammary adenocarcinoma, A755. In both models, combination therapy of 5'-dFUrd/capecitabine with CPA showed synergistic antitumor activity, without significant potentiation of toxicity. In contrast, treatment with CPA and either 5-FUra or UFT (a mixture of tegafur and uracil) in combination showed only additive activity. Our results suggest that CPA and capecitabine/5'-dFUrd, both available for oral administration, would be good partners, and that clinical trials with this drug combination against breast cancer are warranted.

Animals↗

PubMed1999

Elevation of metallothionein level in preneoplastic lesions during chemical hepatocarcinogenesis of the Fischer 344 rat.

Metallothionein (MT) is inducible by various stimuli such as metals and physiological stress. Although elevated MT expression in certain type of tumors has been reported, there are few data available on the roles of MT in tumor formation. In this study, we immunohistochemically examined MT expression in gluthathione S-transferase placental type p (GST-P)-positive preneoplastic liver lesions induced by a chemical carcinogen and further examined the relationship between MT expression and the proliferative activity of the preneoplastic cells, using bromodeoxyuridine (BrdU) labeling indices. Eleven male Fischer rats (F344/DuCrj, 9 weeks old) were subjected to the Solt-Farber protocol. Thereafter, the livers were removed, frozen and sectioned serially for immunohistochemical staining of MT and GST-P. Eight rats were given six injections of BrdU before sacrifice. In 92.8% of GST-P positive lesions, high MT staining intensity was demonstrated. On the other hand, there was no correlation between the intensity of MT staining and the BrdU labeling indices of the preneoplastic lesions. Our results indicate that MT is a useful positive marker for preneoplastic liver lesions, though the reason why MT is expressed in the preneoplastic and neoplastic lesions remains to be elucidated.

2-Acetylaminofluorene↗

PubMed1999

Glial cell line-derived neurotrophic factor induces barrier function of endothelial cells forming the blood-brain barrier.

Since a deep involvement of astrocytes, a kind of glial cells, in differentiation of the blood-brain barrier (BBB) has been suggested, we examined the relation of glial cell line-derived neurotrophic factor (GDNF) to the BBB. First, immunohistochemical examination of the cerebral cortex of rats revealed that glial cell line-derived neurotrophic factor receptor (GFRalpha1) was preferentially expressed on the cell membranes of capillary endothelial cells. Second, to elucidate the effects of GDNF on the BBB, capillary endothelial cells isolated from the porcine cerebral cortex were cultured and then changes in tight junction function of the endothelial cells were examined after addition of GDNF, in terms of transendothelial electrical resistance (TER) and permeability. GDNF at concentrations of 0.1 and 1 ng/ml significantly activated the barrier function of the endothelial cells in the presence of cAMP. Since GDNF is secreted from astrocytes sheathing capillary endothelial cells in the brain cortex, our results strongly suggest that GDNF enhances the barrier function of tight junctions of the BBB on the one hand, and also supports the survival of neurons on the other hand.

Animals↗

PubMed1999

Ganglioside GD1 alpha analogues as high-affinity ligands for myelin-associated glycoprotein (MAG).

The first systematic synthesis of ganglioside GD1 alpha analogues carrying N-acetyldeoxyneuraminic acids linked to C-6 of the GalNAc residue was accomplished. The suitably protected GM1b pentasaccharide derivative was regioselectively glycosylated with the phenyl 2-thioglycosides of 7-deoxy, 8-deoxy, and 9-deoxy-N-acetylneuraminic acid promoted by N-iodosuccinimide (NIS)-trifluoromethanesulfonic acid (TfOH) in acetonitrile, and the resulting hexasaccharides were converted to the target GD1 alpha analogues. All of the analogues retained excellent efficiency in supporting the adhesion to myelin-associated glycoprotein (MAG), raising the possibility that the internal sialic acid linked to the GalNAc residue may be replaced by other anionic substituents, in contrast to the terminal sialic acid, which is essential for MAG binding.

Animals↗

PubMed1999

Down regulation of peroxisome proliferator-activated receptorgamma expression by inflammatory cytokines and its reversal by thiazolidinediones.

AIMS/HYPOTHESIS: Previous studies show that inflammatory cytokines play a part in the development of insulin resistance. Thiazolidinediones were developed as insulin-sensitizing drugs and are ligands for the peroxisome proliferator-activated receptory (PPARgamma). We hypothesized that the anti-diabetic mechanism of thiazolidinediones depends on the quantity of PPARgamma in the insulin resistant state in which inflammatory cytokines play a part. METHODS: We isolated rat PPARgamma1 and gamma2 cDNAs and examined effects of various cytokines and thiazolidinediones on PPARgamma mRNA expression in rat mature adipocytes. RESULTS: Various inflammatory cytokines, such as tumour necrosis factor-alpha (TNF-alpha), interleukin-1alpha (IL-1alpha), IL-1beta, IL-6 and leukaemia inhibitory factor decreased PPARgamma mRNA expression. In addition, hydrogen peroxide, lysophosphatidylcholine or phorbol 12-myristate 13-acetate also decreased the expression of PPARgamma. The suppression of PPARgamma mRNA expression caused by 10 nmol/l of TNF-alpha was reversed 60% and 55% by treatment with 10(-4) mol/l of troglitazone and 10(-4) mol/l of pioglitazone, respectively. The suppression of glucose transporter 4 mRNA expression caused by TNF-alpha was also reversed by thiazolidinediones. Associated with the change of PPARgamma mRNA expression, troglitazone improved glucose uptake suppressed by TNF-alpha. CONCLUSION/INTERPRETATION: Our study suggests that inflammatory cytokines could be factors that regulate PPARgamma expression for possible modulation of insulin resistance. In addition, we speculate that the regulation of PPARgamma mRNA expression may contribute to the anti-diabetic mechanism of thiazolidinediones.

Adipocytes↗

PubMed1999

Little contribution of GB virus C to the development of primary hepatocellular carcinoma in Japan.

RNA of GB virus C was searched for in sera from 109 patients with primary hepatocellular carcinoma. It was detected in 11 patients (10%), and was more frequent than in 3 of 342 blood donors (0.9%) (P < 0.001). The 11 patients included 4 of 29 patients (14%) with hepatitis B surface antigen and 7 of 74 patients (9%) with antibody to hepatitis C virus. RNA of GB virus C was not detected in any of 4 patients without hepatitis B surface antigen or antibody to hepatitis C virus. These results suggest that GB virus C may contribute little to the development of primary hepatocellular carcinoma in Japan.

Carcinoma, Hepatocellular↗

PubMed1999

Enzymatic properties of mouse 25-hydroxyvitamin D3 1 alpha-hydroxylase expressed in Escherichia coli.

Renal 25-hydroxyvitamin D3 1 alpha-hydroxylase cDNA cloned from the kidneys of mice lacking the vitamin D receptor was expressed in Escherichia coli JM109. As expected, the bacterially-expressed enzyme catalyzes the 1 alpha-hydroxylation of 25-hydroxyvitamin D3 with a Michaelis constant, K(m), value of 2.7 microM. Unexpectedly, the enzyme also hydroxylates the 1 alpha-position of 24,25-dihydroxyvitamin D3 with a K(m) of 1.3 microM, and a fourfold higher Vmax/K(m) compared with the 25-hydroxyvitamin D3 hydroxylase activity, suggesting that 24,25-dihydroxyvitamin D3 is a better substrate than 25-hydroxyvitamin D3 for 1 alpha-hydroxylase. In addition, the enzyme showed 1 alpha-hydroxylase activity toward 24-oxo-25-hydroxyvitamin D3. However, it showed only slight activity towards 23,25-dihydroxyvitamin D3 and 24-oxo-23,25-dihydroxyvitamin D3, and no detectable activity towards vitamin D3 and 24,25,26,27-tetranor-23-hydroxyvitamin D3. These results suggest that the 25-hydroxyl group of vitamin D3 is essential for the 1 alpha-hydroxylase activity and the 24-hydroxyl group enhances the activity, but the 23-hydroxyl group greatly reduced the activity. Another remarkable finding is that living recombinant E. coli cells can convert the substrates into the 1 alpha-hydroxylated products, suggesting the presence of a redox partner of 1 alpha-hydroxylase in E. coli cells.

24,25-Dihydroxyvitamin D 3↗

PubMed1999

Metabolic studies using recombinant escherichia coli cells producing rat mitochondrial CYP24 CYP24 can convert 1alpha,25-dihydroxyvitamin D3 to calcitroic acid.

Previously we expressed rat 25-hydroxyvitamin D3 24-hydroxylase (CYP24) cDNA in Escherichia coli JM109 and showed that CYP24 catalyses three-step monooxygenation towards 25-hydroxyvitamin D3 and 1alpha,25-dihydroxyvitamin D3 [Akiyoshi-Shibata, M., Sakaki, T., Ohyama, Y., Noshiro, M., Okuda, K. & Yabusaki, Y. (1994) Eur. J. Biochem. 224, 335-343]. In this study, we demonstrate further oxidation by CYP24 including four- and six-step monooxygenation towards 25-hydroxyvitamin D3 and 1alpha,25-dihydroxyvitamin D3, respectively. When the substrate 25-hydroxyvitamin D3 was added to a culture of recombinant E. coli, four metabolites, 24, 25-dihydroxyvitamin D3, 24-oxo-25-hydroxyvitamin D3, 24-oxo-23, 25-dihydroxyvitamin D3 and 24,25,26,27-tetranor-23-hydroxyvitamin D3 were observed. These results indicate that CYP24 catalyses at least four-step monooxygenation toward 25-hydroxyvitamin D3. Furthermore, in-vivo and in-vitro metabolic studies on 1alpha,25-dihydroxyvitamin D3 clearly indicated that CYP24 catalyses six-step monooxygenation to convert 1alpha,25-dihydroxyvitamin D3 into calcitroic acid which is known as a final metabolite of 1alpha,25-dihydroxyvitamin D3 for excretion in bile. These results strongly suggest that CYP24 is largely responsible for the metabolism of both 25-hydroxyvitamin D3 and 1alpha,25-dihydroxyvitamin D3.

Animals↗

PubMed1999

Enzymatic properties of human 25-hydroxyvitamin D3 1alpha-hydroxylase coexpression with adrenodoxin and NADPH-adrenodoxin reductase in Escherichia coli.

We have cloned human 25-hydroxyvitamin D3 1alpha-hydroxylase cDNAs from normal subjects and patients with pseudovitamin D-deficient rickets (PDDR), and expressed the cDNAs in Escherichia coli JM109 cells. Kinetic analysis of normal 1alpha-hydroxylase in the reconstituted system revealed that Km values for 25(OH)D3 and (24R), 25(OH)2D3 were 2.7 and 1.1 microM, respectively. The lower Km value and higher Vmax/Km value for (24R),25(OH)2D3 indicated that it is a better substrate than 25(OH)D3 for 1alpha-hydroxylase. These results are quite similar to those of mouse 1alpha-hydroxylase. To establish a highly sensitive in vivo system, 1alpha-hydroxylase, adrenodoxin and NADPH-adrenodoxin reductase were coexpressed in E. coli cells. The recombinant E. coli cells showed remarkably high 1alpha-hydroxylase activity, suggesting that the electrons were efficiently transferred from NADPH-adrenodoxin reductase through adrenodoxin to 1alpha-hydroxylase in E. coli cells. Using this system, the activities of four mutants of 1alpha-hydroxylase, R107H, G125E, R335P and P382S, derived from patients with PDDR were examined. Although no significant reduction in expression of these mutants was observed, none showed detectable activity. These results strongly suggest that the mutations found in the patients with PDDR completely abolished 1alpha-hydroxylase activity by replacement of one amino acid residue.

25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗