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Superconducting thin films of Tl–Sr–Ca–Cu–O were prepared on single-crystal MgO(100) substrates by the mist microwave-plasma chemical vapor deposition method, and the dependence of T c-zero of films on the input molar ratio of oxygen O R =F O 2 /(F O 2 +total F A r ) was investigated. In addition, the effect of the Bi addition on Tl–Sr–Ca–Cu–O superconducting thin films was studied. The prepared films were constituted of the 1212 and/or 1223 phases with c-axis orientation perpendicular to the substrates. The maximum T c-zero (zero resistance; 102 K) of as-deposited films was obtained at the input molar ratio of oxygen ( O R ) of 0.30, the microwave power of 300 W and the addition amount (x= Bi/(Tl+Bi)) of 0.30.

The effect of the input molar ratio of oxygen (O R) and supply rate of mist on the preparation of Y–Ba–Cu–O superconducting thin films by the MPCVD method have been investigated. Although the preparation of Y–Ba–Cu–O superconducting thin films was attempted at the input molar ratio of oxygen O R=F O2/(F O2+ total F Ar) from 0.1 to 0.8, the Y–Ba–Cu–O superconducting phase was formed only in the narrow range of O R from 0.1 to 0.3. In addition, the formation of the Y–Ba–Cu–O superconducting phase was limited not only by the input molar ratio of oxygen but by the supply rate of mist.

Superconducting thin films of (Tl, Pb)–Sr–Ca–Cu–O were prepared on single-crystal MgO(100) substrates by the mist microwave-plasma chemical vapor deposition method. The X-ray diffraction patterns showed that the prepared films consisted of the 1223 and 1212 phases with c-axis orientation perpendicular to the substrates. The dependence of the critical temperature of films on the input molar ratio of oxygen was investigated. The maximum critical temperature (100 K) of as-deposited films was obtained at the input molar ratio of oxygen about of 0.3.

Atomic layer epitaxy (ALE) using a GaCl source is investigated by a real-time in situ gravimetric monitoring method. An ALE system with an electrobalance is used as a monitoring system. The direct gravimetric information from the growing surface in the growth system of ALE is monitored during an ALE cycle. It is shown that the surface chemical species are one-monolayer Ga atoms during H2 purge after GaCl supply, and the in situ gravimetric method is a powerful tool for the investigation of the ALE growth mechanism.

Superconducting thin films of (Bi, Pb)-Sr-Ca-Cu-O were prepared on single-crystal MgO(100) substrates by the mist microwave-plasma chemical vapor deposition method. The prepared films constituted of the 2212 and 2201 phases with c-axis orientation perpendicular to the substrates. The dependence of critical temperature of films on the input mole ratio of oxygen OR=FO2 /(FO2 +total FAr) was investigated. The maximum critical temperature (78 K) of as-deposited films was obtained at the input molar ratio of oxygen about of 0.60 and the microwave power of 300 W.

Atomic layer epitaxy (ALE) using GaCl source in an inert carrier gas system is investigated by means of two in situ monitoring methods: gravimetric method and surface photo-absorption (SPA) method. It is shown that the growth of a monomolecular layer unit occurs in the inert carrier gas system as well as in the H2 carrier gas system. In the SPA measurement, the reflected light intensity during GaCl supply and subsequent inert gas or H2 purge is stronger in the inert carrier gas system than that in the H2 carrier gas system. Based on these results, reaction mechanisms are proposed for the two systems.

Oxidative dehydrogenation of (Z)-but-2-ene on iron oxide films deposited on an inactive quartz plate has been studied. The films were composed of tiny maghaemite particles of even size. Since the film thickness was equivalent to the particle size, the films were considered to be in a two-dimensional array over the quartz plate. The catalytic activity of the films was independent of the film thickness. The selectivity of butene into butadiene was however, dependent on the thickness. An enhanced selectivity to butadiene on thin films was attributed to a haematite thin layer formed on the surface of small maghaemite crystallites, constituents of the oxide films.

Iron oxide films consisting of small-sized maghemite particles were formed by spin-coating a solution of iron nitrate dissolved in ethylene glycol onto a quartz plate. These maghemite particles, of a homogeneous size level, were in a two-dimensional array over the quartz plate, so that the film thickness was controlled by the particle size. Heats of adsorption of (Z)-but-2-ene on films of various thicknesses were measured for comparison with those on maghemite powders, leading to the conclusion that films thicker than 3300 Ă behave like the powders, but that films thinner than 3300 A adsorb (Z)-but-2-ene much more strongly than do powders. This is discussed in terms of the variation in saturation magnetization of the films with film thickness.

After our previous report that osteoclast-like multinucleated cells (MNCs) were formed in response to 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3] in cocultures of mouse spleen cells and osteoblast-rich populations freshly isolated from fetal mouse calvariae, we examined whether such primary osteoblast-like cells can be replaced by established cell lines in inducing osteoclast-like cell formation. We first used two clonal cell lines simultaneously established from newborn mouse calvariae. One was the osteoblastic cell line MC3T3-E1, and the other was the preadipose cell line MC3T3-G2/PA6. Tartrate-resistant acid phosphatase (TRACP; a marker enzyme of osteoclasts)-positive mononuclear cells and MNCs were formed in the cocultures of spleen cells and MC3T3-G2/PA6 cells in the presence of 1 alpha,25-(OH)2D3. Dexamethasone greatly potentiated TRACP-positive MNC formation induced by 1 alpha,25-(OH)2D3, whereas the glucocorticoid alone had no effect on it. In contrast, osteoblastic MC3T3-E1 cells failed to induce TRACP-positive cells in the cocultures. Another bone marrow-derived stromal cell line ST2 also induced TRACP-positive MNC formation in the cocultures in response to 1 alpha,25-(OH)2D3 and dexamethasone. Salmon calcitonin enhanced cAMP production in the cocultures only when TRACP-positive cells were formed. Autoradiographic studies demonstrated that [125I]calcitonin specifically bound to TRACP-positive cells formed in the cocultures. When spleen cells and either MC3T3-G2/PA6 or ST2 cells were cocultured on sperm whale dentine slices in the presence of 1 alpha,25-(OH)2D3 and dexamethasone, numerous resorption lacunae were formed. These results show that the two bone marrow-derived stromal cell lines can support osteoclast-like cell differentiation in cocultures with spleen cells.

We have developed a mouse marrow culture system, in which multinucleated cells (MNCs) are formed within 6-8 days. These MNCs showed several characteristics of osteoclasts, including tartrate-resistant acid phosphatase (TRACP) and the ability to resorb calcified dentine. lα,25-Dihydroxyvitamin D3 [lα,25(OH)2D3] stimulated the formation of TRACPpositive MNCs, and salmon calcitonin (CT) inhibited it. In this study, we examined whether the TRACP-positive MNCs formed from mouse marrow cells possess CT receptors, another typical characteristic of osteoclasts. Mouse marrow cells cultured for 8 days with 10 nM lα,25(OH)2D3 and freshly isolated authentic mouse osteoclasts were incubated with [125I]-salmon CT in the presence or absence of excess amounts of unlabeled CT, stained for TRACP, and processed for autoradiography. The [125I]-CT exclusively bound to TRACP-positive mononuclear cells and MNCs formed in the lα,25(OH)2D3-treated cultures and also to isolated mouse osteoclasts. Both [125I]-CT binding and TRACP a...

We have developed a mouse marrow culture system in which multinucleated osteoclast (OC) -like cells are formed within 8 days. Using this culture system, we examined the effect of prostaglandins (PGs), potent bone-resorting agents, on OC-like cell formation. Four PGs (PGE1 and PGE2 at 10-8-10-5 M, 6-keto-PGF1αat 10-5M, and PGF2αat 10-6-10-5M) significantly stimulated the formation of OC-like cells. The potency of the PGs in inducing OC-like cell forma-tion was well correlated with the potency in increasing the production of cyclic adenosine 3', 5'-monophosphate (cAMP) in bone marrow cells. Addition of dibutyryl-CAMP also induced OC-like cell formation. Moreover, isobutylmethylxanthine (IBMX), a potent inhibitor of phosphodiesterase, potentiated the OC-like cell formation induced by PGE2. Calcitonin induced cAMP production in cultures treated with PGE2, but not in cultures with vehicle. When bone marrow mononuclear cells were cultured on dentine slices in the presence of PGE2, multinucleated OC-like cells were similarly formed and they resorbed calcified tissues.These results suggest that PGs stimulate resorption of calcified tissues by promoting osteoclast formation. The activity of PGs in inducing OC-like cell formation is considered mediated by a mechanism involving cAMP.

The common marmoset, a New World monkey, requires a large amount of vitamin D3 to maintain its normal growth. This monkey is reported to have an end-organ resistance to 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3). In this study, the bone morphology of marmosets fed a high vitamin D3 diet (intake of vitamin D3, 110 IU/day/100 g of body weight) was compared by X-ray and histological examinations with that of rhesus monkeys (Old World monkey) fed a normal diet (intake of vitamin D3, 5 IU/day/100 g of body weight). Three of 20 marmosets were found by X-ray examination to have osteomalacic changes in their bones despite the high daily intake of vitamin D3, whereas none of the 5 rhesus monkeys showed any signs of osteomalacia. Osteomalacic marmosets had distinct increases in osteoid surface, relative osteoid volume, and active osteoclastic bone resorption, whereas non-osteomalacic marmosets had no increase in osteoid tissues in their bones. None of the marmosets, either osteomalacic or non-osteomalacic, was hypercalcemic despite the extremely high circulating levels of 1 alpha,25(OH)2D3. However, the serum 25-hydroxyvitamin D3 (25OHD3) and 24R,25-dihydroxyvitamin D3 (24R,25(OH)2D3) levels were significantly lower in the osteomalacic than in the non-osteomalacic marmosets. These results suggest that the marmoset is likely to exhibit osteomalacic bone changes despite the high daily intake of vitamin D3. These changes resemble those in vitamin D-dependent rickets, type II.

Osteoclast-like multinucleated cells were formed from mouse bone marrow mononuclear cells, and their morphology on coverslips and on calcified dentine slices was compared by means of transmission electron microscopy. Addition of 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3] to bone marrow cells cultured on coverslips greatly stimulated the formation of multinucleated cells within 8 days. These multinucleated cells had the cytological features of osteoclasts (abundant pleomorphic mitochondria, a large number of vacuoles and lysosomes, many stacks of Golgi membranes, and an extensive canalicular system), but they developed neither ruffled borders nor clear zones. The multinucleated cells appeared to result from direct fusion of mononuclear progenitor cells, whose structural features were similar to those of multinucleated cells. Like isolated osteoclasts, both multinucleated cells and their precursors exhibited an intense reaction for tartrate-resistant acid phosphatase (TRACP) in the cisterns of endoplasmic reticulum and lysosomes. Multinucleated cells formed from alveolar macrophages in the presence of 1 alpha,25(OH)2D3 were totally negative for TRACP reaction. When marrow cells were cultured on dentine slices in the presence of 1 alpha,25(OH)2D3, some of the multinucleated cells were located in the shallow resorption lacunae of dentine surfaces, and they developed the characteristic ruffled borders and clear zones. The narrow extracellular spaces of the ruffled borders, the adjacent pale endocytotic vacuoles, and the dark lysosomes located in the perinuclear cytoplasm of the multinucleated cells contained numerous apatite crystals delete in resorption lacunae. These results indicate that 1) the multinucleated cells formed on coverslips from mouse marrow cells treated with 1 alpha,25(OH)2D3 exhibit non-functional basic features of osteoclast morphology, and 2) differentiation of the multinucleated cells into functional osteoclasts requires some components of calcified dentine.

We measured endogenous angiotensins (ANGs) I, II&III using a system of extraction by Sep-Pak column followed by high performance liquid chromatography (HPLC) combined with radioimmunoassay (RIA). An excellent separation of ANGs was obtained by HPLC. The recovery of ANGs I, II&III was 80–84%, when these authentic peptides were added to 6 ml of plasma. The coefficient of variation of the ANGs was 0.04–0.09 for intra-assay and 0.08–0.13 for inter-assay, thereby indicating a good reproducibility. Plasma ANGs I, II&III measured by this method in 5 normal volunteers were 51,4.5 and 1.2 pg/ml. In the presence of captopril, ANGs II&III decreased by 84% and 77%, respectively, while ANG I increased 5.1 times. This method is therefore useful to assess the precise levels of plasma ANGs.

We developed a mouse bone marrow culture system to examine the process of osteoclast-like multinucleated cell formation from its progenitors. When mouse marrow cells were cultured for 8 days with 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3, 10(-10) to 10(-7) M] or human PTH (1-34) (25-100 ng/ml), tartrate-resistant acid phosphatase (TRACP)-positive multinucleated cells formed. No TRACP-positive multinucleated cells appeared in the absence of these hormones. 1 alpha,25-(OH)2D3 and PTH also increased the number of the clusters of TRACP-positive mononuclear cells. Time course studies showed that these TRACP-positive mononuclear cell clusters appeared before the formation of TRACP-positive multinucleated cells, suggesting that the TRACP-positive mononuclear cells are precursors of the multinucleated cells. Salmon calcitonin markedly inhibited the formation of TRACP-positive multinucleated cells but not TRACP-positive mononuclear cell clusters induced by 1 alpha,25-(OH)2D3 or PTH. TRACP-positive mononuclear cells and multinucleated cells were rarely stained for nonspecific esterase, but some mononuclear cells were positively stained for both nonspecific esterase and TRACP. More that 90% of the TRACP-positive mononuclear cell clusters and multinucleated cells were found near colonies of alkaline phosphatase-positive mononuclear cells (possibly osteoblasts). When marrow mononuclear cells were cultured on sperm whale dentine slices in the presence of 1 alpha,25-(OH)2D3 or PTH, numerous resorption lacunae were formed. These results suggest that 1) TRACP-positive multinucleated cells formed in response to osteotropic hormones in mouse marrow cultures satisfy most of the criteria of osteoclasts, and 2) osteoblasts may play an important role in osteoclast formation.

Bone is a highly specialized form of connective tissue, composed of bone forming osteoblasts and bone-resorbing osteoclasts, several types of matrix proteins and proteoglycans, and bone salts of calcium and phosphorus. It not only forms the skeleton or framework of the bodies, but serves as a calcium reservoir in most vertebrates. The newest knowledge on bone cell biology has allowed us to consider the mechanisms of bone formation and resorption and their coupling system. A great number of such systemic hormones as 1 α, 25-dihydroxyvitamin D3, parathyroid hormone and calcitonin and local factors including prostaglandins, interleukins, transforming growth factors, insulin-like growth factors and bone morphogenetic protein have been shown to be involved in the regulation of bone formation and resorption.In this review, we will summarize the newest knowledge on bone cell biology to understand the pathogenesis of several metabolic bone diseases including osteoporosis, rheumatoid arthritis and inflammatory periodontal diseases.

A metabolite of 25-hydroxycholecalciferol has been isolated in pure form from chicken kidney homogenates. It has been identified as 25-hydroxy-24-oxocholecalciferol by means of ultraviolet absorption spectrophotometry, mass spectrometry, infrared spectrometry, nuclear magnetic resonance spectrometry, and specific chemical reactions.

MgOを0, 1, 5, 10, 15および20 mo1%添加したTiO焼結体の物理的性質を測定し次の結果を得た.生成相はすべてNaCl型構造のTiOで,MgOはTiO相に約15%まで固溶した.MgO添加によりTiO焼結体の粒成長は抑制され,添加量の増加とともに平均粒径は減少した.MgO添加量の増加に従って焼結性は低下し,相対密度は減少した.一般に焼結体の硬さの増加に対するMgO添加の効果は大きくなく, 15%添加により無添加の最大値よりわずかに大きいビッカース硬度1350が得られた.焼結体の電気抵抗はMgO量の増加とともに増加した.これはMgOの添加による粒成長の抑制と気孔率の増加を原因とする., Some physical properties of sintered TiO doped with 0, 1, 5, 10, 15 and 20 mol% MgO were measured. All of the Ti phases obtained were identified to be NaCl type structure and solid solubility of MgO in TiO phase was assumed to be about 15%. The grain growth of TiO was suppressed by the doping of Mg0 and as the addition of MgO increased, the grain size decreased. Moreover, as the addition of MgO increased, the sinterability of TiO decreased and the relative density decreased. The effect of the addition of MgO on the hardness of TiO ceramics was not so significant. 0nly when 15 mo1% MgO was doped, the microvicker's hardness was about 1350, slightly larger than that of the maximum value of TiO ceramics without any additives. The electrical resistivity of TiO ceramics increased with an increase of the addition of MgO. This is assumed to be by reasons of the suppression of grain growth and the increase of the porosity of TiO ceramics.

We have reported that the duodenal ornithine decarboxylase activity and the tissue content of putrescine increase markedly after a single intravenous injection of 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3) into vitamin D-deficient chicks (Shinki, T., Takahashi, N., Miyaura, C., Samejima, K., Nishii, Y., and Suda, T. (1981) Biochem. J. 195, 685-690). In the present study, we examined in the same experimental system the effect of 1 alpha,25(OH)2D3 on the activity of duodenal spermidine N1-acetyltransferase, a rate-limiting enzyme catalyzing the conversion from spermidine to putrescine. The duodenal spermidine N1-acetyltransferase activity began to increase 30 min after a single intravenous injection of 625 ng of 1 alpha,25(OH)2D3 and attained a maximum in 2 h. As little as 1.25 ng of 1 alpha,25(OH)2D3 induced a small but significant increase in the spermidine N1-acetyltransferase activity, and the maximal response was obtained by 125 ng of the vitamin. The dose levels of 1 alpha,25(OH)2D3 required to induce duodenal spermidine N1-acetyltransferase activity were only one-tenth as much as those required to induce ornithine decarboxylase activity. The spermidine N1-acetyltransferase activity was induced commonly in the target tissues of vitamin D, whereas ornithine decarboxylase activity occurred only in intestine. The 1 alpha,25(OH)2D3-induced spermidine N1-acetyltransferase activity was greatly inhibited by prior administration of actinomycin D or cycloheximide. When 30,000 X g intestinal supernatants were incubated with [14C]spermidine, [14C]putrescine was formed. These results clearly indicate that the induction of spermidine N1-acetyltransferase activity is the 1 alpha,25(OH)2D3-induced earliest de novo synthesis in several proteins induced by the vitamin reported to date and that the 1 alpha,25(OH)2D3-induced duodenal synthesis of putrescine occurs by the pathways from both ornithine and spermidine.

The distribution of enzymes involved in polyamine synthesis and that of the polyamine levels were investigated in the duodenal mucosa of vitamin D-deficient chicks and those supplemented with 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3]. Duodenal epithelial cells were isolated sequentially from the villus tip to the crypt region using a nonenzymatic method. In vitamin D-deficient chicks, the activities of ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase were markedly higher in the crypt cells than in the villus cells. Similar gradients were observed in the distribution of putrescine and spermidine. Six hours after iv administration of 625 ng 1 alpha,25-(OH)2D3, ODC activity and putrescine levels markedly increased both in the villus and crypt region. The rate of the increase in ODC activity and putrescine levels, however, was much higher in the villus than in the crypt region. Neither S-adenosylmethionine decarboxylase activity nor spermidine levels were affected by the treatment with 1 alpha,25-(OH)2D3. To investigate differential uptake of 1 alpha,25-(OH)2D3 in the duodenal mucosa, 0.1 nmol 1 alpha,25-(OH)2[3H]D3 with or without a 200-fold excess of unlabeled 1 alpha,25-(OH)2D3 was injected into rachitic chicks, and epithelial cells were sequentially isolated 2 h later. The radioactivity specifically incorporated in the nuclear fraction was distributed uniformly from the villus tip to crypt cells. The cytoplasmic receptor for 1 alpha,25-(OH)2D3 was similarly distributed in the crypt and villus cells. These results suggest that 1 alpha,25-(OH)2D3 plays a physiological role in cellular activities not only in the villus but also in the crypt region through polyamine biosynthesis.

Objective Polarized osteoclasts form sealing zones, (also called clear zones) detectable as actin rings, and ruffled borders to resorb bone. They secrete protons and catabolic enzymes, including tartrate-resistant acid phosphatase (TRAP), through the ruffled borders. We previously reported that polarized osteoclasts develop areas of TRAP activity (TRAP-marks) when cultured on dentin slices [11]. In this study, we examined how osteoclasts recognize dental implant materials. Methods Osteoclasts obtained from murine co-cultures were cultured on implant materials such as titanium (Ti), alumina, zirconia, and sintered hydroxyapatite (sHA), in addition to dentin. Osteoclasts were also treated with reveromycin A (RM-A), which specifically acts on polarized osteoclasts and induces apoptosis. Polarization of osteoclasts cultured on implant materials was evaluated by measuring actin rings, TRAP-marks, and reveromycin A-induced apoptosis. Results Osteoclasts formed actin rings on all substrates examined. The formation of actin rings on Ti by osteoclasts was inhibited by the GRGDS peptide, but not by the GRGES peptide, suggesting an integrin-mediated polarization of osteoclasts on Ti. Calcitonin, an inhibitory hormone of osteoclast function, disrupted the actin rings that were preformed on Ti and sHA. Osteoclasts put TRAP-marks on sHA and dentin and formed resorption pits on dentin, but failed to form resorption pits on sHA. RM-A induced apoptosis in osteoclasts cultured on Ti and sHA; this was suppressed by calcitonin. Conclusions These results demonstrate that osteoclasts are able to polarize on dental implant materials similar to the polarization observed on bone.

There are few long-term reports of patients with frequent PVCs in the absence of ischemic heart disease. In 86 patients without ischemic heart disease, who had 1000 or more PVCs in 24-hour Holter ECG, the number of PVCs during 24-hours Holter ECG and echocardiographic parameters were followed at least 1 year (66.5 ± 39.7 months). PVC was significantly reduced in the patients with or without underlying diseases (UD). The reduction rate in the number of PVCs was prominent in patients with UD. PVC was significantly reduced in patients under medication, but not in patients without medication. In the comparison between the initial and follow up observation using Wilcoxon's rank test, the number of PVC was significantly reduced (P < 0.05), and EF was also improved (P < 0.05) in angiotensin converting enzyme inhibitor (ACEI) or angiotensin receptor blocker (ARB) group, and in β-blocker group. In Ca-antagonist group and antiarrhythmic drug group, the number of PVCs was also significantly reduced (P < 0.05). Multivariate analysis revealed significantly higher incidence (60% or more with PVC reduction) in ACEI/ARB group. These results suggest that the administration of ACEI/ARB may contribute to the reduction of PVC in non-ischemic heart disease cases with multiple PVC.

Expression of p60c-src, the normal cellular counterpart of the transforming protein of Rous sarcoma virus (RSV), p60c-src, was examined in mouse and rat authentic osteoclasts and mouse osteoclast-like multinucleated cells (MNCs) formed in vitro. In co-cultures of mouse osteoblastic cells and spleen cells, the expression of p60c-src strikingly increased on day 5 in parallel with the appearance of MNCs in the presence of 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3). Immunohistochemical examination confirmed the high level expression of p60c-src in both mouse authentic osteoclasts and MNCs. Electron microscopic examination revealed that p60c-src was primarily localized on ruffled border membranes and vacuoles, but not on the clear zone in rat authentic osteoclasts. These results suggest that p60c-src is important in osteoclastic bone resorption.

Vitamin D3 is metabolized first in the liver to 25-hydroxyvitamin D3 [25(OH)D3] and then in the kidney mainly to lα, 25-dihydroxyvitamin D3 [1α, 25(OH)2D3] and 24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3].1 The 1, α25(OH)2D3 is now regarded as the hormonal form of the vitamin in promoting intestinal calcium transport and bone mineral mobilization.1 The renal lα-hydroxylation of 25(OH)D3 is the rate-limiting step in the production of lα, 25(OH)2D3 and it is strictly regulated by the plasma levels of phosphorus, parathyroid hormone (PTH), calcitonin, estrogen, lα, 25(OH)2D3, and other steroid and peptide hormones.2 Since the discovery of lα, 25(OH)2D3, many lines of evidence have indicated that the hormone is incorporated by receptor mediation into the nuclear fraction of target cells before exerting its biological functions.3 This suggests that the lα, 25(OH)2D3 acts in a way similar to that of other steroid hormones.

The time course of change in plasma calcium levels and renal metabolism of 25-hydroxyvitamin D3 [25(OH)D3] was investigated in chicks maintained on a vitamin D-deficient diet for 4 weeks. Plasma calcium concentrations dropped sharply between the 7th and 14th day of the feeding period. Renal 25(OH)D3-1 alpha-hydroxylase activity was reciprocally enhanced concurrently with the decrease in plasma calcium levels. The elevated activity of 1 alpha-hydroxylase had declined significantly by the 21st and 28th days in spite of the more severe hypocalcemia. When graded amounts of vitamin D3 were administered to the chicks maintained on this diet for 14 or 28 days, there were considerable differences in the change of plasma calcium levels and 25(OH)D3 metabolism induced by vitamin D3 administration between the 14-day and 28-day birds. The minimal dose levels of vitamin D3 to completely suppress renal 1 alpha-hydroxylase activity were 25 micrograms in the 14-day, and 2.5 mg in the 28-day birds. These differences were not observed between the 14-day and 28-day birds when 1 alpha-hydroxyvitamin D3 [1 alpha(OH)D3] was administered. Renal adenylate cyclase activity induced by parathyroid hormone (PTH) was much lower in the 28-day chicks than that in 1-day-old and the 14-day birds. These results are explained by the hypersecretion of PTH and the subsequent refractoriness of the target organs in severe vitamin D deficiency. Plasma calcium levels per se did not appear to be a major factor in the regulation of 25(OH)D3 metabolism.

The relationship between the appearance of vitamin D-dependent calcium-binding protein (CaBP) and calcium absorption was studied in sequentially isolated duodenal mucosal preparations from vitamin D-deficient chicks and those supplemented with vitamin D3 or 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3]. The duodenal calcium absorption activity was significantly increased 6 h after a single iv injection of 0.625 microgram of 1 alpha,25-(OH)2D3, attained a maximum at 12 h, and declined gradually thereafter. On the other hand, only small amounts of CaBP appeared in the crypt and lower villus regions 6 h after 1 alpha,25-(OH)2D3 administration. At 12 h, the CaBP was found in the entire villus, but its content was still much higher in the crypt and lower villus. At 24 h and 48 h, the distribution of CaBP showed the opposite gradient, higher in the villus and lower in the crypt. At 72 h, CaBP was found only in the upper villus. The life time of duodenal mucosal cells was calculated to be 108 h as indicated by the cells labeled with [3H]thymidine. Thus, the movement of CaBP from the crypt to the villus tip was considered to be much faster than the cell migration, suggesting that both crypt and villus cells are capable of producing CaBP. Daily administration of vitamin D3 or 1 alpha,25-(OH)2D3 for 2 weeks resulted in a marked increase in CaBP levels mainly in the mid- and upper villus regions. The higher the intestinal calcium transport activity was, the higher the duodenal CaBP content. These results suggest that CaBP is not necessary in initiating intestinal calcium transport, but it plays an important role in maintaining the enhanced transport mechanism.

A sensitive and rapid in vitro assay of 25-hydroxyvitamin D3 [25-(OH)D3]-1 alpha- and 24-hydroxylase activities was developed using rat kidney homogenates. A potent inhibitor of the enzymes in rat plasma was removed by thoroughly perfusing rats with saline. Kidney homogenates prepared from vitamin D-deficient rats preferentially produced tritiated 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3] from 25(OH) [3H]D3. Addition of 10 microliter or more of rat plasma to 3 ml of 10% kidney homogenates suppressed 1 alpha-hydroxylase activity dose-dependently. Thyroparathyroidectomy (TPTX) of vitamin D-deficient rats greatly abolished 1 alpha-hydroxylase activity. Administration of parathyroid hormone to the TPTX rats increased 1 alpha-hydroxylase activity and that of 1 alpha,25(OH)2D3 enhanced 24-hydroxylase markedly. Since this assay is technically simple, rapid and sensitive, it will be useful in studying the regulatory mechanism in the renal metabolism of 25(OH)D3 in mammals.

The effect of vitamin D3 on medullary bone formation was investigated in egg-laying Japanese quail and in immature male chicks treated with sex hormones. When laying quail were fed a vitamin D-deficient diet for 16 days, their eggshell weights and egg production rate were markedly reduced in a time-dependent manner with a significant decrease in plasma calcium and 25-hydroxyvitamin D3 levels. The calcium content of the medullary bone of femurs decreased markedly with the progress of vitamin D deficiency, whereas that of the cortical bone remained unchanged. Quantitative histological examination also showed that the area of the mineralized portion of medullary bone in quail that were fed the vitamin D-deficient diet markedly decreased compared with that in the control laying quail, whereas the total area of the mineralized and unmineralized portions of medullary bone in the bone marrow cavity increased moderately. Daily administration of vitamin D3 (0.75 microgram/day) to the vitamin D-deficient quail increased the mineralization of medullary bone as early as day 4. Daily administration of both estradiol (0.3 mg/day) and testosterone (0.9 mg/day) for 3 weeks to immature male chicks induced an apparent hypercalcemia and matrix formation of medullary bone, regardless of the vitamin D status of the chicks. Mineralization of medullary bone was observed only when vitamin D3 was administered together with the sex hormones. These results suggest that vitamin D3 is directly involved in the mineralization of medullary bone in birds.

Metabolism of 25-hydroxyvitamin D3 (25-OH-D3) in pregnancy was investigated invitro in New Zealand White rabbits fed a rabbit chow. Kidney homogenates from pregnant mothers and fetuses were separately incubated with [3H]-25-OH-D3. The homogenates from fetuses produced significant amounts of [3H]-1α,25-dihydroxyvitamin D3 [1α,25-(OH)2-D3] from its precursor, while those from mothers predominantly produced [3H]-24,25-dihydroxyvitamin D3 [24,25-(OH)2-D3]. The identity of the radioactive metabolites produced from [3H]-25-OH-D3 was established by periodate cleavage and comigration with synthetic 1α,25-(OH)2-D3 or 24,25-(OH)2-D3 on high pressure liquid chromatography. These results clearly indicate that the fetal kidney is at least one of the sites of 1α,25-(OH)2-D3 synthesis in pregnancy.

We have developed a mouse bone marrow culture system in which multinucleated osteoclast (OC)-like cells are formed within 8 days. Using this culture system, we examined the effect of prostaglandins (PGs), potent bone-resorbing agents, on OC-like cell formation. Four PGs (PGE1 and PGE2 at 10(-8)-10(-5) M, 6-keto-PGF1 alpha at 10(-5) M, and PGF2 alpha at 10(-6)-10(-5) M) significantly stimulated the formation of OC-like cells. The potency of the PGs in inducing OC-like cell formation was the highest in PGE1 and PGE2, followed by PGF2 alpha and 6-keto-PGF1 alpha in that order, and the order was highly correlated with the order of the potency in increasing the production of cyclic adenosine 3',5'-monophosphate (cAMP) in bone marrow cells. Addition of dibutyryl-cAMP also induced OC-like cell formation. Moreover, isobutylmethylxanthine (IBMX), a potent inhibitor of phosphodiesterase, potentiated the OC-like cell formation induced by PGE2, whereas salmon calcitonin greatly inhibited it. Calcitonin induced cAMP production in cultures treated with PGE2, but not in cultures with vehicle. When bone marrow mononuclear cells were cultured on dentine slices in the presence of PGE2, multinucleated OC-like cells were similarly formed and they resorbed calcified dentine, resulting in so-called Howship's lacunae. These results suggest that PGs stimulate resorption of calcified tissues by promoting osteoclast formation. The activity of PGs in inducing OC-like cell formation is considered mediated mainly by a mechanism involving cAMP.

We developed a co-culture system with mouse spleen cells and osteoblastic cells to examine the role of osteoblasts in osteoclast formation. When mouse spleen cells and osteoblastic cells isolated from fetal mouse calvariae were co-cultured in the presence of 10 nM 1 alpha, 25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3], numerous tartrate-resistant acid phosphate (TRACP)-positive mononuclear and multinucleated cells were formed within 8 days. Neither the same co-cultures without the vitamin nor separate cultures of either spleen cells or osteoblastic cells with the vitamin produced TRACP-positive cells. Salmon calcitonin (CT) markedly increased cAMP production in the co-cultures treated with 1 alpha,25(OH)2D3. Autoradiographic studies clearly demonstrated that [125I]-CT specifically bound to the TRACP-positive cells formed in the co-cultures with the vitamin. When spleen cells and osteoblastic cells were co-cultured on dentine slices in the presence of 1 alpha,25(OH)2D3, numerous resorption lacunae were formed on the slices. Neither co-cultures of alveolar macrophages and osteoblastic cells nor those of spleen cells and mouse skin-derived fibroblasts induced TRACP-positive cells even in the presence of 1 alpha,25(OH)2D3. When spleen cells and osteoblastic cells were cultured separately from each other by a membrane filter (0.45 micron), no TRACP-positive cells were formed. These results indicate that osteoblastic cells are required for the differentiation of osteoclast progenitors in splenic tissues into multinucleated osteoclasts.

Induction of osteoclast-like multinucleated cells (MNCs) by various fragments of PTH-related protein (PTHrP) was examined in mouse marrow cultures. Osteoclast-like MNCs were defined as tartrate-resistant acid phosphatase (TRACP)-positive MNCs with calcitonin receptors. In all experimental protocols examined, PTHrP-(1-34) induced TRACP-positive MNCs at almost the same rate as PTH-(1-34). PTHrP-(1-29) was less potent than PTHrP-(1-34). PTHrP-(1-25) and PTHrP-(1-14) had no effect. PTHrP-(1-34) was more potent than PTH-(1-34) in increasing the accumulation of cAMP, but the former appeared to lose its activity more rapidly than the latter. Isobutylmethylxanthine increased the effect of PTHrP-(1-34) and PTH-(1-34) in inducing TRACP-positive MNCs. Furthermore, the calcium ionophore A23187 significantly increased the formation of TRACP-positive MNCs. The effect of PTH-(1-34) and PTHrP-(1-34) in inducing TRACP-positive MNCs was potentiated by adding A23187 but suppressed by adding verapamil simultaneously. The inhibition by verapamil was overcome by adding A23187. [Nle8,18,Tyr34]PTH-(3-34)amide inhibited the effect of not only PTH-(1-34) but also PTHrP-(1-34) in inducing both the accumulation of cAMP and the TRACP-positive MNC formation. These results show that PTHrP is a potent stimulator of osteoclast-like MNC formation to almost the same extent as PTH. It increases the number of osteoclast-like MNCs by a mechanism involving cAMP and calcium ions, and is most likely mediated through the same receptor. The controversial results of the bone-resorbing activity of PTH and PTHrP reported so far may be explained by the differences in the relative potencies of the respective hormones in increasing the intracellular cAMP and calcium ions and by the shorter half-life of PTHrP in culture medium.

The role of vitamin D in the maturation of epiphyseal chondrocytes was investigated in the developing chick embryo. Cartilage tissues were divided into two parts: resting cartilage and growth cartilage. A cytosol component to which 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3) is specifically bound first appeared in the growth cartilage on day 15, rapidly increased, and attained a maximum on day 19. The calcium content of the growth cartilage also began to increase on day 15 and continued to increase in parallel with the 1 alpha,25(OH)2D3 receptor levels. Glycosaminoglycan (GAG) synthesis by the growth cartilage cells increased from day 11-17 and rapidly declined thereafter reciprocally with the increase in calcium and receptor levels. In the resting cartilage, no cytosol receptor for 1 alpha,25(OH)2D3 was detected up to hatching time. The calcium content and GAG synthesis in the resting cartilage were very low and did not change appreciably throughout development. No receptor-like macromolecule for 24R,25-dihydroxyvitamin D3 (24R,25(OH)2D3) was recognized in either the resting or growth cartilage. 1 alpha,25(OH)2D3 added to the culture of chondrocytes from the epiphyseal growth cartilage inhibited GAG synthesis and stimulated its release from the cell layer into the medium in a dose-dependent manner. These in vitro effects of 1 alpha,25(OH)2D3 were not observed in chondrocytes obtained from 13-day-old growth cartilage and 19-day-old resting cartilage. 25-Hydroxyvitamin D3 and 24R,25(OH)2D3 had no effect on chondrocytes in any of the preparations. These results suggest that 1 alpha, 25 (OH)2D3 is directly involved in the maturation of chondrocytes and possibly in the calcification of growth cartilage.

Osteoprotegerin (OPG) is a decoy receptor for receptor activator of NF-κB ligand (RANKL). We previously reported that OPG deficiency elevated the circulating level of RANKL in mice. Using OPG−/− mice, we investigated whether OPG is involved in the shedding of RANKL by cells expressing RANKL. Osteoblasts and activated T cells in culture released a large amount of RANKL in the absence of OPG. OPG or a soluble form of receptor activator of NF-κB (the receptor of RANKL) suppressed the release of RANKL from those cells. OPG- and T cell-double-deficient mice showed an elevated serum RANKL level equivalent to that of OPG−/− mice, indicating that circulating RANKL is mainly derived from bone. The serum level of RANKL in OPG−/− mice was increased by ovariectomy or administration of 1α,25-dihydroxyvitamin D3. Expression of RANKL mRNA in bone, but not thymus or spleen, was increased in wild-type and OPG−/− mice by 1α,25-dihydroxyvitamin D3. These results suggest that OPG suppresses the shedding of RANKL from osteoblasts and that the serum RANKL in OPG−/− mice exactly reflects the state of bone resorption.

Thin magnetic films consisting of aggregated γ-Fe2O3(maghemite) fine particles were prepared by spin-coating a gel solution of iron (III) nitrate dissolved in ethylene glycol on glass substrates. Film thickness was controlled by adjusting the viscosity and stirring time of the get solution. As-coated films were calcined in air at various temperatures up to 600°C. The calcined films were confirmed by transmission electron microscope observation to consist of fine iron oxide particles. The small coercive force (≦250 Oe) of the films is well explained in terms of the shape and size of the iron oxide particles, and the variation of magnetization with calcination temperature is closely related to the crystallographic transition of maghemite phase to hematite phase.