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ABSTRACTThe collective migration of vascular endothelial cells plays important roles in homeostasis and angiogenesis. Oxygen tension in vivois a key factor affecting the cellular dynamics. We previously reported hypoxic conditions promote the internalization of vascular endothelial (VE)-cadherin and increase the collective migration of vascular endothelial cells. However, the mechanism through which cells regulate collective migration as affected by oxygen tension is not fully understood. Here, we investigated oxygen-dependent collective migration, focusing on intracellular protein p21-activated kinase (PAK) and hypoxia-inducing factor (HIF)-1α. The results indicate that the oxygen-dependent variation of the migration speed of vascular endothelial cells is mediated by the regulation of VE-cadherin through the PAK pathway, as well as other mechanisms via HIF-1α, especially under extreme hypoxic conditions.

Resonant acoustic® mixing (RAM) technology is a system that performs high-speed mixing by vibration through the control of acceleration and frequency. In recent years, real-time process monitoring and prediction has become of increasing interest, and process analytical technology (PAT) systems will be increasingly introduced into actual manufacturing processes. This study examined the application of PAT with the combination of RAM. near-infrared spectroscopy, and chemometric technology as a set of PAT tools for introduction into actual pharmaceutical powder blending processes. Content uniformity was based on a robust partial least squares regression (PLSR) model constructed to manage the RAM configuration parameters and the changing concentration of the components. As a result, real-time monitoring may be possible and could be successfully demonstrated for in-line real-time prediction of active pharmaceutical ingredients and other additives using chemometric technology. This system is expected to be applicable to the RAM method for the risk management of quality.

AbstractTo effectively utilize a photovoltaic (PV) system, reactive-power control methods for solar inverters have been considered. Among the various methods, the constant-voltage control outputs less reactive power compared with the other methods. We have developed a constant-voltage control to reduce the reactive-power output. However, the developed constant-voltage control still outputs unnecessary reactive power because the control parameter is constant in every waveform of the PV output. To reduce the reactive-power output, we propose a dynamic reactive-power control method with a PV output prediction. In the proposed method, the control parameter is varied according to the properties of the predicted PV waveform. In this study, we performed numerical simulations using a distribution system model, and we confirmed that the proposed method reduces the reactive-power output within the voltage constraint.

Compensation of a voltage within the appropriate range becomes difficult when a large number of photovoltaic (PV) systems are installed. As a solution to this problem, the installation of a low-voltage regulator (LVR) has been studied. In this paper, we propose a method for instantly and accurately determining the line drop compensator (LDC) method parameters as a part of a voltage management scheme, which consists of prediction, operation, and control. In the proposed method, the solution candidates of the proper LDC parameters are narrowed by using a random forest that learns the relationship between the power-series data and the properness of the LDC parameters, thereby reducing the computational cost. We performed numerical simulations to verify the validity of the proposed method. From the results, the LDC parameters can be rapidly and accurately determined. Additionally, the desirable voltage control performance is verified.

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.

Tetracyclines such as doxycycline and minocycline are used to suppress bacterial growth in patients with inflammatory diseases. Tetracyclines have been shown to prevent bone loss, but the underlying mechanisms are unknown. Osteoclasts and dendritic cells (DCs) are derived from common progenitors such as bone marrow-derived macrophages (BMMs). Here, we showed that minocycline converts the differentiation pathway, which results in DC-like cells and not osteoclasts. Minocycline inhibited the receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis of BMMs but had no effects on cell growth and phagocytic activity. It influenced neither the proliferation nor differentiation of bone-forming osteoblasts. Surprisingly, minocycline induced the expression of DC markers, CD11c and CD86, in BMMs in the presence of RANKL. STAT5 is involved in DC differentiation that is induced by granulocyte–macrophage colony-stimulating factor (GM-CSF). Midostaurin, which is a STAT5 signaling inhibitor and an anti-GM-CSF neutralizing antibody, suppressed the differentiation that was induced by GM-CSF but not by tetracyclines. In vivo, the injection of minocycline into RANKL-injected mice and RANKL-transgenic mice suppressed RANKL-induced osteoclastogenesis and promoted the concomitant appearance of CD11c-positive cells. These results suggest that minocycline prevents bone loss that is induced by local inflammation, including rheumatoid arthritis and periodontitis, through osteoclast-DC-like cell conversion.

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 ß-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.

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.

Deficiency of osteoprotegerin (OPG), a soluble decoy receptor for receptor activator of NF-κB ligand (RANKL), in mice induces osteoporosis caused by enhanced bone resorption but also accelerates bone formation. We examined if bone formation is coupled with bone resorption in OPG-deficient (OPG−/−) mice, using risedronate, an inhibitor of bone resorption. Histomorphometric analysis showed that bone formation-related parameters in OPG−/−mice sharply decreased with the suppression of bone resorption by the daily injection of risedronate for 30 days. OPG−/−mice exhibited high serum alkaline phosphatase activity and osteocalcin concentrations, both of which were decreased to the levels of wild-type mice by risedronate injection. The ectopic bone formation induced by bone morphogenetic protein-2 implantation into OPG−/−mice was not accelerated. These results suggest that bone formation is coupled with bone resorption at local sites in OPG−/−mice. Myeloid differentiation factor 88 (MyD88) plays essential roles in the signaling of the Toll/interleukin-1 (IL-1) receptor family. Toll-IL-1 receptor domain-containing adapter inducing interferon-β (TRIF)-mediated signals are involved in lipopolysaccharide (LPS)-induced MyD88-independent pathways. Using MyD88-deficient (MyD88−/−) mice and TRIF-deficient (TRIF−/−) mice, we examined the roles of MyD88 and TRIF in osteoclast differentiation and function. LPS and IL-1α stimulated osteoclastogenesis in co-cultures of osteoblasts and hemopoietic cells obtained from TRIF−/−mice but not MyD88−/−mice. Bone histomorphometry showed that MyD88−/−mice exhibited osteopenia with reduced bone resorption and formation. These results suggest that the MyD88-mediated signal is essential for the osteoclastogenesis and function induced by IL-1 and LPS, and that MyD88 is physiologically involved in bone turnover.

Abstract Dolomite, a mineral composed of calcium magnesium carbonate (CaMg (CO3)2), is used as a food supplement that supplies calcium and magnesium. However, the effect of magnesium supplementation on bone metabolism in patients with osteoporosis is a matter of controversy. We examined the effects of daily supplementation with dolomite on calcium metabolism in ovariectomized (OVX) rats. Dolomite was administered daily to OVX rats for 9 weeks. The same amount of magnesium chloride as that supplied by the dolomite was given to OVX rats as a positive control. Histological examination revealed that ovariectomy decreased trabecular bone and increased adipose tissues in the femoral metaphysis. Dolomite or magnesium supplementation failed to improve these bone histological features. Calcium content in the femora was decreased in OVX rats. Neither calcium nor magnesium content in the femora in OVX rats was significantly increased by dolomite or magnesium administration. Urinary deoxypyridinoline excretion was significantly increased in OVX rats, and was not affected by the magnesium supplementation. Serum concentrations of magnesium were increased, and those of calcium were decreased, in OVX rats supplemented with dolomite or magnesium. However, there was a tendency toward decreased parathyroid hormone secretion and increased calcitonin secretion in OVX rats supplemented with dolomite or magnesium. Serum 1,25-dihydroxyvitamin D3 and osteocalcin levels were significantly increased in the supplemented OVX rats. These results suggest that increased magnesium intake improves calcium metabolism in favor of increasing bone formation, through the modulation of calcium-regulating hormone secretion.

Myeloid differentiation factor 88 (MyD88) plays essential roles in the signaling of the Toll/interleukin (IL)-1 receptor family. Toll–IL-1 receptor domain-containing adaptor inducing interferon-β (TRIF)-mediated signals are involved in lipopolysaccharide (LPS)-induced MyD88-independent pathways. Using MyD88-deficient (MyD88−/−) mice and TRIF-deficient (TRIF−/−) mice, we examined roles of MyD88 and TRIF in osteoclast differentiation and function. LPS, diacyl lipopeptide, and IL-1α stimulated osteoclastogenesis in cocultures of osteoblasts and hemopoietic cells obtained from TRIF−/− mice, but not MyD88−/− mice. These factors stimulated receptor activator of nuclear factor-κB ligand mRNA expression in TRIF−/− osteoblasts, but not MyD88−/− osteoblasts. LPS stimulated IL-6 production in TRIF−/− osteoblasts, but not TRIF−/− macrophages. LPS and IL-1α enhanced the survival of TRIF−/− osteoclasts, but not MyD88−/− osteoclasts. Diacyl lipopeptide did not support the survival of osteoclasts because of the lack of Toll-like receptor (TLR)6 in osteoclasts. Macrophages expressed both TRIF and TRIF-related adaptor molecule (TRAM) mRNA, whereas osteoblasts and osteoclasts expressed only TRIF mRNA. Bone histomorphometry showed that MyD88−/− mice exhibited osteopenia with reduced bone resorption and formation. These results suggest that the MyD88-mediated signal is essential for the osteoclastogenesis and function induced by IL-1 and TLR ligands, and that MyD88 is physiologically involved in bone turnover.

Abstract: The ZrO2 films were deposited onto a Si(100) substrate using an alternate reaction of ZrCl4 and O2 under atmospheric pressure. It is found that the growth rate of ZrO2 film depends on the growth conditions, such as growth temperature, partial pressure of the sources being supplied, and exposure time of the substrate to the gaseous sources. Self-limiting growth of the ZrO2 was achieved in the range of the growth temperature of 673–923 K. The x-ray diffractogram of the ZrO2 films showed a typical diffraction pattern assigned to the tetragonal polycrystalline phase. The obtained ZrO2 films were of smooth and uniform surface. It was found that the [O]/[Zr] ratio of the ZrO2 films are similar to that of the ZrO2 bulk.

Bone-resorbing osteoclasts are formed from hemopoietic cells of the monocyte–macrophage lineage under the control of bone-forming osteoblasts. We have cloned an osteoblast-derived factor essential for osteoclastogenesis, the receptor activator of NF-κB ligand (RANKL). Synovial fibroblasts and activated T lymphocytes from patients with rheumatoid arthritis also express RANKL, which appears to trigger bone destruction in rheumatoid arthritis as well. Recent studies have shown that T lymphocytes produce cytokines other than RANKL such as IL-17, granulocyte–macrophage colony-stimulating factor and IFN-γ, which have powerful regulatory effects on osteoclastogenesis. The possible roles of RANKL and other cytokines produced by T lymphocytes in bone destruction are described.

Preparation of InN thin films has been examined using an atmospheric pressure halide chemical vapor deposition technique. It was found that the quality of the InN pillar crystal film grown on a Si(100) substrate is significantly dependent upon the ratio of NH₃∶InCl₃ used as source materials. Hall mobility decreases as the NH₃∶InCl₃ ratio is decreased, while the carrier concentration increases. This is explained in terms of the formation of nitrogen vacancies. A decrease of the NH₃∶InCl₃ ratio causes the increase of nitrogen defects in the InN film. This also increases the number of electrons being trapped by the defects, while their mobility is reduced because of the electrons being scattered at the vacancies.

Preparation of quartz (rock crystal) films has been studied by an atmospheric pressure chemical vapor deposition method using Si(OC₂H₅)₄ and O₂ as starting materials in the presence of gaseous HCl. The films were deposited onto a sapphire (0001) substrate at 600–850 °C with a growth rate of 0.3–3.0 µm h−1. Their X-ray diffraction profiles showed an intense diffraction peak at 50.6° assigned to the (0003) diffraction of quartz with a hexagonal structure suggesting epitaxial growth. The full-width at half-maximum was 10.0 min for the quartz film prepared at 850 °C. Reflection high-energy electron diffraction measurements showed a diffraction pattern similar to that of a single crystal of quartz. It was found by energy-dispersive analysis of the X-rays that the [O]/[S] ratios of the deposited films were in the range of 1.94–1.97, independent of the growth temperature. Fourier transform infrared spectra exhibited a weak absorption band at 3585 cm−1, suggesting OH radicals to be present as an impurity. The refractive index of the quartz epitaxial films prepared at 850 °C was 1.528.

Antigen‐specific T‐cells infiltrate the heart and play an important role in the myocardial damage involved in acute myocarditis and dilated cardiomyopathy. To investigate the roles of the co‐stimulatory molecules CD30/CD30L, CD27/CD27L, OX40/OX40L, and 4‐1BB/4‐1BBL, which belong to the tumor necrosis factor (TNF) receptor/ligand superfamily, in the development of acute viral myocarditis, the expression of these molecules was first analysed in the hearts of mice with acute myocarditis induced by Coxsackievirus B3 (CVB3) in vivo. Secondly, the induction of these molecules was evaluated on cultured cardiac myocytes treated with interferon (IFN)‐γ and the interleukin (IL)‐6 production by cultured cardiac myocytes was analysed by stimulation with monoclonal antibodies (MAbs) against these molecules in vitro. Thirdly, the effects of in vivoadministration of anti‐CD30L, anti‐CD27L, anti‐OX40L, or anti‐4‐1BBL MAb on the development of acute viral myocarditis were examined. CVB3‐induced myocarditis resulted in the induction of CD30L and 4‐1BBL on the surface of cardiac myocytes, confirmed by treatment with IFN‐γ in vitro. CD27L and OX40L were constitutively expressed on cardiac myocytes in vivoand in vitro. Anti‐CD30L and anti‐4‐1BBL MAbs stimulated IL‐6 production by cardiac myocytes in vitro. Furthermore, in vivoanti‐4‐1BBL MAb treatment significantly decreased the myocardial inflammation, whereas the other MAbs did not. These findings suggest that TNF ligand superfamily co‐stimulatory molecules, especially 4‐1BBL, play an important role in the development of acute viral myocarditis and raise the possibility that immunotherapy with anti‐4‐1BBL MAb may be of benefit in acute viral myocarditis. Copyright © 2001 John Wiley & Sons, Ltd.

Cubic Fe₄N epitaxial layers have been grown on a MgO (100) substrate by atmospheric pressure halide vapor-phase epitaxy using FeCl₃ and NH₃ as the starting materials. The full width at half maximum of the X-ray (200) diffraction peak for the Fe₄N epitaxial layer was about 0.05°. Transmission electron diffraction measurements revealed a diffraction pattern similar to that of a single crystal with cubic structure. The saturation magnetization and coercive force of an Fe₄N epitaxial layer deposited at 600 °C were 182.0 emu g−1 and 30 Oe, respectively. It was found, for the first time, that the epitaxial layer of Fe₄N displayed anomalous light reflectivity modulated by an external magnetic field.

Transforming growth factor β (TGF‐β) is abundant in bone and has complex effects on osteolysis, with both positive and negative effects on osteoclast differentiation, suggesting that it acts via more than one mechanism. Osteoclastogenesis is determined primarily by osteoblast (OB) expression of the tumor necrosis factor (TNF)‐related molecule receptor activator of NF‐κB ligand (RANKL) and its decoy receptor osteoprotegerin (OPG), which are increased and decreased, respectively, by osteolytic factors. A RANKL‐independent osteoclastogenic mechanism mediated by TNF‐α has also been shown. Therefore, we investigated TGF‐β effects on osteoclast formation in culture systems in which osteoclastogenic stimulus is dependent on OBs and culture systems where it was provided by exogenously added RANKL or TNF‐α. Both OPG and TGF‐β inhibited osteoclast formation in hemopoietic cell/OB cocultures, but the kinetics of their action differed. TGF‐β also inhibited osteoclastogenesis in cocultures of cells derived from OPG null (opg−/−) mice. TGF‐β strongly decreased RANKL messenger RNA (mRNA) expression in cultured osteoblasts, and addition of exogenous RANKL to TGFβ‐inhibited cocultures of opg−/−cells partially restored osteoclastogenesis. Combined, these data indicate that the inhibitory actions of TGF‐β were mediated mainly by decreased OB production of RANKL. In contrast, in the absence of OBs, TGF‐β greatly increased osteoclast formation in recombinant RANKL‐ or TNF‐α‐stimulated cultures of hemopoietic cells or RAW 264.7 macrophage‐like cells to levels several‐fold greater than attainable by maximal stimulation by RANKL or TNF‐α. These data suggest that TGF‐β may increase osteoclast formation via action on osteoclast precursors. Therefore, although RANKL (or TNF‐α) is essential for osteoclast formation, factors such as TGF‐β may powerfully modify these osteoclastogenic stimuli. Such actions may be critical to the control of physiological and pathophysiological osteolysis.

Transforming growth factor β (TGF‐β) is abundant in bone and has complex effects on osteolysis, with both positive and negative effects on osteoclast differentiation, suggesting that it acts via more than one mechanism. Osteoclastogenesis is determined primarily by osteoblast (OB) expression of the tumor necrosis factor (TNF)‐related molecule receptor activator of NF‐κB ligand (RANKL) and its decoy receptor osteoprotegerin (OPG), which are increased and decreased, respectively, by osteolytic factors. A RANKL‐independent osteoclastogenic mechanism mediated by TNF‐α has also been shown. Therefore, we investigated TGF‐β effects on osteoclast formation in culture systems in which osteoclastogenic stimulus is dependent on OBs and culture systems where it was provided by exogenously added RANKL or TNF‐α. Both OPG and TGF‐β inhibited osteoclast formation in hemopoietic cell/OB cocultures, but the kinetics of their action differed. TGF‐β also inhibited osteoclastogenesis in cocultures of cells derived from OPG null (opg−/−) mice. TGF‐β strongly decreased RANKL messenger RNA (mRNA) expression in cultured osteoblasts, and addition of exogenous RANKL to TGFβ‐inhibited cocultures of opg−/−cells partially restored osteoclastogenesis. Combined, these data indicate that the inhibitory actions of TGF‐β were mediated mainly by decreased OB production of RANKL. In contrast, in the absence of OBs, TGF‐β greatly increased osteoclast formation in recombinant RANKL‐ or TNF‐α‐stimulated cultures of hemopoietic cells or RAW 264.7 macrophage‐like cells to levels several‐fold greater than attainable by maximal stimulation by RANKL or TNF‐α. These data suggest that TGF‐β may increase osteoclast formation via action on osteoclast precursors. Therefore, although RANKL (or TNF‐α) is essential for osteoclast formation, factors such as TGF‐β may powerfully modify these osteoclastogenic stimuli. Such actions may be critical to the control of physiological and pathophysiological osteolysis.

SaOS‐4/3, a subclone of the human osteosarcoma cell line SaOS‐2, established by transfecting the human parathyroid hormone/parathyroid hormone‐related protein (PTH/PTHrP) receptor complementary DNA (cDNA), supported osteoclast formation in response to PTH in coculture with mouse bone marrow cells. Osteoclast formation supported by SaOS‐4/3 cells was completely inhibited by adding either osteoprotegerin (OPG) or antibodies against human macrophage colony‐stimulating factor (M‐CSF). Expression of messenger RNAs (mRNAs) for receptor activator of NF‐κB ligand/osteoclast differentiation factor (RANKL/ODF) and both membrane‐associated and secreted forms of M‐CSF by SaOS‐4/3 cells was up‐regulated in response to PTH. SaOS‐4/3 cells constitutively expressed OPG mRNA, expression of which was down‐regulated by PTH. To elucidate the mechanism of PTH‐induced osteoclastogenesis, SaOS‐4/3 cells were spot‐cultured for 2 h in the center of a culture well and then mouse bone marrow cells were uniformly plated over the well. When the spot coculture was treated for 6 days with both PTH and M‐CSF, osteoclasts were induced exclusively inside the colony of SaOS‐4/3 cells. Osteoclasts were formed both inside and outside the colony of SaOS‐4/3 cells in coculture treated with a soluble form of RANKL/ODF (sRANKL/sODF) in the presence of M‐CSF. When the spot coculture was treated with sRANKL/sODF, osteoclasts were formed only inside the colony of SaOS‐4/3 cells. Adding M‐CSF alone failed to support osteoclast formation in the spot coculture. PTH‐induced osteoclast formation occurring inside the colony of SaOS‐4/3 cells was not affected by the concentration of M‐CSF in the culture medium. Mouse primary osteoblasts supported osteoclast formation in a similar fashion to SaOS‐4/3 cells. These findings suggest that the up‐regulation of RANKL/ODF expression is an essential step for PTH‐induced osteoclastogenesis, and membrane‐ or matrix‐associated forms of both M‐CSF and RANKL/ODF are essentially involved in osteoclast formation supported by osteoblasts/stromal cells.

SaOS‐4/3, a subclone of the human osteosarcoma cell line SaOS‐2, established by transfecting the human parathyroid hormone/parathyroid hormone‐related protein (PTH/PTHrP) receptor complementary DNA (cDNA), supported osteoclast formation in response to PTH in coculture with mouse bone marrow cells. Osteoclast formation supported by SaOS‐4/3 cells was completely inhibited by adding either osteoprotegerin (OPG) or antibodies against human macrophage colony‐stimulating factor (M‐CSF). Expression of messenger RNAs (mRNAs) for receptor activator of NF‐κB ligand/osteoclast differentiation factor (RANKL/ODF) and both membrane‐associated and secreted forms of M‐CSF by SaOS‐4/3 cells was up‐regulated in response to PTH. SaOS‐4/3 cells constitutively expressed OPG mRNA, expression of which was down‐regulated by PTH. To elucidate the mechanism of PTH‐induced osteoclastogenesis, SaOS‐4/3 cells were spot‐cultured for 2 h in the center of a culture well and then mouse bone marrow cells were uniformly plated over the well. When the spot coculture was treated for 6 days with both PTH and M‐CSF, osteoclasts were induced exclusively inside the colony of SaOS‐4/3 cells. Osteoclasts were formed both inside and outside the colony of SaOS‐4/3 cells in coculture treated with a soluble form of RANKL/ODF (sRANKL/sODF) in the presence of M‐CSF. When the spot coculture was treated with sRANKL/sODF, osteoclasts were formed only inside the colony of SaOS‐4/3 cells. Adding M‐CSF alone failed to support osteoclast formation in the spot coculture. PTH‐induced osteoclast formation occurring inside the colony of SaOS‐4/3 cells was not affected by the concentration of M‐CSF in the culture medium. Mouse primary osteoblasts supported osteoclast formation in a similar fashion to SaOS‐4/3 cells. These findings suggest that the up‐regulation of RANKL/ODF expression is an essential step for PTH‐induced osteoclastogenesis, and membrane‐ or matrix‐associated forms of both M‐CSF and RANKL/ODF are essentially involved in osteoclast formation supported by osteoblasts/stromal cells.

Abstract:: We have isolated osteoclast precursors (OCPs) from cocultures of mouse calvarial cells and bone marrow cells without adding any osteotropic factors. OCPs expressed Mac-1, Mac-2, and Gr-1 antigens but not osteoclast markers such as tartrate-resistant acid phosphatase (TRAP) and calcitonin receptors, and they differentiated into TRAP-positive cells within 48 h on a fixed calvarial cell layer pretreated with osteotropic factors such as 1α,25-dihydroxyvitamin D3. In the present study, we investigated the regulatory mechanisms of OCP formation from hemopoietic cells and TRAP-positive cell formation from OCPs. Calvarial osteoblasts obtained from macrophage-colony stimulating factor (M-CSF) -deficient op/op mice failed to support OCP formation or the differentiation of OCPs into TRAP-positive cells. Both OCP formation and TRAP-positive cell formation supported by osteoblasts were completely inhibited by osteoclastogenesis inhibitory factor (OCIF, also called OPG), which is a decoy receptor of osteoclast differentiation factor (ODF; also called TRANCE, RANKL, and OPGL). When bone marrow cells were cultured for 4 days with soluble ODF (sODF/sRANKL) together with M-CSF, OCPs were formed even in the absence of osteoblasts. When OCPs were treated with sODF/sRANKL and M-CSF in the absence of osteoblasts, they differentiated into TRAP-positive cells within 48 h even in the presence of hydroxyurea. Northern blotting analysis revealed that osteoblasts constitutively expressed a certain level of ODF/RANKL mRNA. These results indicated that M-CSF and sODF/sRANKL produced by osteoblasts are two essential factors for both OCP formation and TRAP-positive osteoclast formation.

Hexagonal ZnO films have been grown on a sapphire(0001) substrate by atmospheric pressure halide vapor phase epitaxy using ZnO buffer layers. The full width at half maximum of the X-ray (0002) diffraction peak for the ZnO films with the buffer layer was found to be smaller than that of the ZnO films without the buffer layer. Reflection high-energy electron diffraction measurements of the former revealed a diffraction pattern similar to that of a single crystal. The photoluminescence spectra showed a strong peak at 370 nm up to 180 K.

Osteoclast differentiation factor (ODF, also called RANKL/TRANCE/OPGL) stimulates the differentiation of osteoclast progenitors of the monocyte/macrophage lineage into osteoclasts in the presence of macrophage colony-stimulating factor (M-CSF, also called CSF-1). When mouse bone marrow cells were cultured with M-CSF, M-CSF–dependent bone marrow macrophages (M-BMMφ) appeared within 3 d. Tartrate-resistant acid phosphatase–positive osteoclasts were also formed when M-BMMφ were further cultured for 3 d with mouse tumor necrosis factor α (TNF-α) in the presence of M-CSF. Osteoclast formation induced by TNF-α was inhibited by the addition of respective antibodies against TNF receptor 1 (TNFR1) or TNFR2, but not by osteoclastogenesis inhibitory factor (OCIF, also called OPG, a decoy receptor of ODF/RANKL), nor the Fab fragment of anti–RANK (ODF/RANKL receptor) antibody. Experiments using M-BMMφ prepared from TNFR1- or TNFR2-deficient mice showed that both TNFR1- and TNFR2-induced signals were important for osteoclast formation induced by TNF-α. Osteoclasts induced by TNF-α formed resorption pits on dentine slices only in the presence of IL-1α. These results demonstrate that TNF-α stimulates osteoclast differentiation in the presence of M-CSF through a mechanism independent of the ODF/RANKL–RANK system. TNF-α together with IL-1α may play an important role in bone resorption of inflammatory bone diseases.

In acute myocarditis and dilated cardiomyopathy, it has previously been reported that antigen‐specific T‐cells infiltrate the heart and play an important role in the myocardial damage involved. For antigen‐specific T‐cell activation to occur, it is necessary for the T‐cell to receive co‐stimulatory signals provided by co‐stimulatory molecules expressed on the antigen‐presenting cell (APC), as well as the main signal provided by binding of the T‐cell receptor (TCR) to the antigen. To investigate the roles for the co‐stimulatory molecules B7‐1 and B7‐2 in the development of chronic ongoing viral myocarditis, firstly the expression of B7‐1/B7‐2 was analysed in the hearts of A/J mice with myocarditis induced by Coxsackievirus B3 (CVB3). Secondly the induction of B7‐1/B7‐2 on cultured cardiac myocytes treated with interferon (IFN)‐γ was evaluated. Thirdly the effects of the in vivoadministration of anti‐B7‐1/B7‐2 monoclonal antibodies (MAbs) on the survival of mice with viral myocarditis were examined. CVB3‐induced myocarditis resulted in enhanced expression of B7‐1/B7‐2 on cardiac myocytes. The expression of B7‐1/B7‐2 on cardiac myocytes could be induced by IFN‐γ in vitro. In vivoanti‐B7‐1 MAb treatment significantly prolonged the survival of mice with myocarditis, whereas anti‐B7‐2 MAb treatment abrogated the protective effect of anti‐B7‐1. These findings indicate that distinct roles for B7‐1 and B7‐2 antigens are involved in the development of viral myocarditis and raise the possibility of immunotherapy with anti‐B7‐1 MAb to prevent T‐cell‐mediated cardiac myocyte injury and to improve the prognosis of viral myocarditis. Copyright © 1999 John Wiley & Sons, Ltd.

T-cell-mediated myocardial damage has been shown to be involved in acute myocarditis and dilated cardiomyopathy. It is necessary for T-cells to receive a co-stimulatory signal as well as the main signal through the T-cell receptor for antigen-specific T-cell activation to occur. To investigate the roles of the co-stimulatory molecules CD40/CD40L, CD30/CD30L, CD27/CD27L, and OX40/OX40L, which belong to the tumour necrosis factor (TNF) receptor/ligand superfamily, in the development of chronic ongoing myocarditis, the expression of CD40, CD30L, CD27L, and OX40L was analysed in the hearts of A/J mice with myocarditis induced by Coxsackie virus B3 (CVB3). The expression of CD40L, CD30, CD27, and OX40 was also examined on the infiltrating cells. Furthermore, the induction of CD40, CD30L, CD27L, and OX40L was evaluated on cultured cardiac myocytes treated with interferon (IFN)-γ. CVB3-induced myocarditis resulted in the induction of CD40 and CD30L on the surface of cardiac myocytes. Induction of CD40 and CD30L on cardiac myocytes was confirmed by treatment with IFN-γ in vitro. CD27L and OX40L were expressed on cardiac myocytes in vivo and in vitro. The expression of CD27L and OX40L on cardiac myocytes was increased, at least partly, by CVB3-induced myocarditis in vivo. Many infiltrating cells expressed CD27 and OX40, whereas much smaller numbers expressed CD40L and CD30. The induction of these molecules, especially CD40 and CD30L, on cardiac myocytes strongly suggests that cardiac myocytes may co-stimulate T-cells and induce cytokine production by T-cells and humoral immune responses. This may play an important role in the pathogenesis of the resulting myocardial damage. Copyright © 1999 John Wiley & Sons, Ltd.

T‐cell‐mediated myocardial damage has been shown to be involved in acute myocarditis and dilated cardiomyopathy. It is necessary for T‐cells to receive a co‐stimulatory signal as well as the main signal through the T‐cell receptor for antigen‐specific T‐cell activation to occur. To investigate the roles of the co‐stimulatory molecules CD40/CD40L, CD30/CD30L, CD27/CD27L, and OX40/OX40L, which belong to the tumour necrosis factor (TNF) receptor/ligand superfamily, in the development of chronic ongoing myocarditis, the expression of CD40, CD30L, CD27L, and OX40L was analysed in the hearts of A/J mice with myocarditis induced by Coxsackie virus B3 (CVB3). The expression of CD40L, CD30, CD27, and OX40 was also examined on the infiltrating cells. Furthermore, the induction of CD40, CD30L, CD27L, and OX40L was evaluated on cultured cardiac myocytes treated with interferon (IFN)‐γ. CVB3‐induced myocarditis resulted in the induction of CD40 and CD30L on the surface of cardiac myocytes. Induction of CD40 and CD30L on cardiac myocytes was confirmed by treatment with IFN‐γ in vitro. CD27L and OX40L were expressed on cardiac myocytes in vivoand in vitro. The expression of CD27L and OX40L on cardiac myocytes was increased, at least partly, by CVB3‐induced myocarditis in vivo. Many infiltrating cells expressed CD27 and OX40, whereas much smaller numbers expressed CD40L and CD30. The induction of these molecules, especially CD40 and CD30L, on cardiac myocytes strongly suggests that cardiac myocytes may co‐stimulate T‐cells and induce cytokine production by T‐cells and humoral immune responses. This may play an important role in the pathogenesis of the resulting myocardial damage. Copyright © 1999 John Wiley & Sons, Ltd.

We previously reported that hypoxia caused rapid activation of RAS/mitogen-activated protein kinase (MAPK) pathway, two other stress-activated MAPK family members, stress-activated protein kinase (SAPK) and p38MAPK, and Src family tyrosine kinases, p60c-srcand p59c-fynin cultured rat cardiac myocytes. In this study, to elucidate how hypoxia affects adhesive interaction between cardiac myocytes and extracellular matrix (ECM), we investigated the molecular mechanism of the activation of focal adhesion-associated tyrosine kinases p125FAKand paxillin. Here, we show that hypoxia induced tyrosine phosphorylation of p125FAKand paxillin and that hypoxia-induced activation of p125FAKwas accompanied by its increased association with adapter proteins Shc and GRB2, and non-receptor type tyrosine kinase p60c-src.Furthermore, hypoxia caused subcellular translocation of p125FAKfrom perinuclear sites to the focal adhesions. These results strongly suggest that p125FAKis one of the most important components in hypoxia-induced intracellular signaling in cardiac myocytes and may play a pivotal role in adhesive interaction between cardiac myocytes and ECM.

Recently, we demonstrated that pulsatile mechanical stretch induced rapid secretion of vascular endothelial growth factor (VEGF) by cultured rat cardiac myocytesin vitro.To investigate whether pulsatile stretch activates intracellular signaling in cardiac myocytes, we examined the activation of mitogen-activated protein kinase (MAPK) family members and focal adhesion kinase (p125FAK) in cultured rat cardiac myocytes. We found that pulsatile stretch rapidly phosphorylated p44/p42 MAPKs (extracellular signal-regulated protein kinase [ERK] 1/2), stress-activated protein kinase (SAPK), p38MAPK, and p125FAK. The stretch-induced activation of ERKs was at least partly mediated by VEGF, which was shown to be induced by transforming growth factor (TGF)-β, and was also partly dependent on tyrosine kinases as well as protein kinase C (PKC). These data provide the direct evidence that pulsatile stretch can activate intracellular signaling in cardiac myocytes and that this was at least partly mediated by VEGF, which may play a role in cardiac adaptation to mechanical overload.

Osteoclasts, the multinucleated giant cells that resorb bone, develop from monocyte-macrophage lineage cells. Osteoblasts or bone marrow stromal cells have been suggested to be involved in osteoclastic bone resorption. The recent discovery of new members of the tumor necrosis factor (TNF) receptor-ligand family has elucidated the precise mechanism by which osteoblasts/stromal cells regulate osteoclast differentiation and function. Osteoblasts/stromal cells express a new member of the TNF-ligand family “osteoclast differentiation factor(ODF)/osteoprotegerin ligand (OPGL)/TNF-related activation-induced cytokine (TRANCE)/receptor activator of NF-kB ligand (RANKL)” as a membrane associated factor. Osteoclast precursors which possess RANK, a TNF receptor family member, recognize ODF/OPGL/TRANCE/RANKL through cell-to-cell interaction with osteoblasts/stromal cells, and differentiate into osteoclasts in the presence of macrophage colony-stimulating factor. Mature osteoclasts also express RANK, and their bone-resorbingactivity is also induced by ODF/OPGL/TRANCE/RANKL which osteoblasts/stromal cells possess. Osteoprotegerin (OPG)/osteoclastogenesis inhibitory factor (OCIF)/TNF receptor-like molecule 1 (TR1) is a soluble decoy receptor for ODF/OPGL/ TRANCE/RANKL. Activation of NF-kB and c-Jun N-terminal kinase through the RANK-mediated signaling system appears to be involved in differentiation and activation of osteoclasts.

In acute myocarditis and dilated cardiomyopathy, it has previously been reported that antigen-specific T-cells infiltrate the heart and play an important role in the myocardial damage involved. For antigen-specific T-cell activation to occur, it is necessary for the T-cell to receive co-stimulatory signals provided by co-stimulatory molecules expressed on the antigen-presenting cell (APC), as well as the main signal provided by binding of the T-cell receptor (TCR) to the antigen. To investigate the roles for the co-stimulatory molecules B7-1 and B7-2 in the development of chronic ongoing viral myocarditis, firstly the expression of B7-1/B7-2 was analysed in the hearts of A/J mice with myocarditis induced by Coxsackievirus B3 (CVB3). Secondly the induction of B7-1/B7-2 on cultured cardiac myocytes treated with interferon (IFN)-γ was evaluated. Thirdly the effects of the in vivo administration of anti-B7-1/B7-2 monoclonal antibodies (MAbs) on the survival of mice with viral myocarditis were examined. CVB3-induced myocarditis resulted in enhanced expression of B7-1/B7-2 on cardiac myocytes. The expression of B7-1/B7-2 on cardiac myocytes could be induced by IFN-γ in vitro. In vivo anti-B7-1 MAb treatment significantly prolonged the survival of mice with myocarditis, whereas anti-B7-2 MAb treatment abrogated the protective effect of anti-B7-1. These findings indicate that distinct roles for B7-1 and B7-2 antigens are involved in the development of viral myocarditis and raise the possibility of immunotherapy with anti-B7-1 MAb to prevent T-cell-mediated cardiac myocyte injury and to improve the prognosis of viral myocarditis. Copyright © 1999 John Wiley & Sons, Ltd.

Interleukin-1 (IL-1) is one of the most potent bone-resorbing factors involved in bone loss associated with inflammation. We previously reported that IL-1 prolonged the survival of multinucleated osteoclast-like cells (OCLs) formed in cocultures of murine osteoblasts/stromal cells and bone marrow cells via the prevention of spontaneously occurring apoptosis. It was reported that macrophage colony-stimulating factor (M-CSF/CSF-1) prolongs the survival of OCLs without the help of osteoblasts/stromal cells. The present study was conducted to determine whether IL-1 also directly induces the multinucleation and activation of OCLs. Mononuclear osteoclast-like cells (prefusion osteoclasts; pOCs) were purified using the “disintegrin” echistatin from cocultures of murine osteoblastic cells (MB 1.8 cells) and bone marrow cells. Both IL-1 and M-CSF prolonged the survival and induced the multinucleation of pOCs through their respective receptors. However, actin ring formation (a functional marker of osteoclasts) by multinucleated cells was observed in the pOC cultures treated with IL-1, but not those treated with M-CSF. We previously reported that enriched multinucleated OCLs as well as pOCs placed on bone/dentine slices formed few resorption pits, but their pit-forming activity was greatly increased by the addition of osteoblasts/stromal cells. Here, pit-forming activity of both pOCs and enriched OCLs placed on dentine slices was induced by adding IL-1, even in the absence of osteoblasts/stromal cells. M-CSF failed to induce pit-forming activity in pOC and enriched OCL cultures. These results indicate that IL-1 induces the multinucleation and bone-resorbing activity of osteoclasts even in the absence of osteoblasts/stromal cells.

In murine acute viral myocarditis, natural killer (NK) cells infiltrate the heart first, followed by activated T‐cells, which play an important role in the pathogenesis of the myocardial damage. Because of their multipotential effects, cytokines are thought to play a role in the induction and development of these immune processes. To clarify in more detail the precise mechanism of the cytokine networks involved, the expression of various cytokine mRNAs has been investigated in myocardial cells infected with Coxsackievirus B3 (CVB3) in vivoand in vitroby a semiquantitative polymerase chain reaction (PCR) method. Interleukin (IL)‐1α, IL‐1β, IL‐6, tumour necrosis factor (TNF)‐α, and TNF‐β were expressed almost throughout the early phase of virus infection with some variations. IL‐2, IL‐3, IL‐4, IL‐10, interferon (IFN)‐γ, granulocyte/macrophage colony stimulating factor (GM‐CSF), and IL‐2 receptor (IL‐2R) were mainly expressed by the infiltrating cells. TNF‐α, TNF‐β, and IL‐1β were also expressed partly by the infiltrating cells. T‐helper (Th)1‐related cytokines (IL‐2, IFN‐γ, and TNF‐β) were more strongly expressed than Th2‐related cytokines (IL‐4 and IL‐10) in vivo, indicating that the Th cells which infiltrated the heart and mediated the immune responses in the early phase of acute myocarditis were mainly of Th1‐type. © 1997 by John Wiley & Sons, Ltd.

A chemical equilibrium model is applied to the growth of the InxGa1−xN alloy grown by metalorganic vapor-phase epitaxy (MOVPE). The equilibrium partial pressures and the phase diagram of deposition are calculated for the InxGa1−xN alloy. The vapor-solid distribution relationship is discussed in comparison with the experimental data reported in the literature. It is shown that the solid composition of the InxGa1−xN alloy grown by MOVPE is thermodynamically controlled and that the incorporation of group III elements into the solid phase deviates from a linear function of the input mole ratio of the group III metalorganic sources under the conditions of high mole fraction of decomposed NH3(high value of α), high temperature and low input VIIIratio. The origin of the deviation of the solid composition from the linear relation is also discussed.

Evidence has accumulated that vascular endothelial growth factor (VEGF) is expressed in the heart, and its expression is markedly increased in response to hypoxia. Recently, it was shown that pulsatile myocardial stretchin vivomarkedly enhanced VEGF mRNA level in the heart. To investigate whether pulsatile mechanical stretch really stimulates VEGF expression by cardiac myocytes, using anin vitropreparation, we examined the secretion of VEGF into the culture media from cardiac myocytes subjected to pulsatile stretch. We found that pulsatile mechanical stretch induced rapid secretion of VEGF by cultured rat cardiac myocytes and mRNA expression of VEGF and VEGF receptors in the cardiac myocytes. We also found that the stretch-induced secretion of VEGF was at least in part mediated by TGF-β. These data provide the direct evidence that mechanical overload itself can induce VEGF secretion by cardiac myocytes, which may play a role in ameliorating the relative myocardial hypoxia.

We previously reported a procedure to obtain a preparation containing a large number of mouse osteoclast (OCL)‐like multinucleated cells (MNCs) formed in cocultures of mouse osteoblastic and bone marrow cells in the presence of 1α,25‐dihydroxyvitamin D3[1α,25‐(OH)2D3]. The MNCs satisfied major criteria of OCLs, such as tartrate‐resistant acid phosphatase (TRAP) activity, acid production, calcitonin (CT) receptors, and the ability to form resorption pits on bone slices. In this report, we describe a simple resorption assay system using MNC preparations. After culturing MNC preparations or disaggregated rat OCL preparations on dentin slices, they were stained with Mayer's hematoxylin. The stained area corresponded exactly with the resorption pits visualized by scanning electron microscopy and were measured using an image analysis system attached to a light microscope. Pit formation by MNCs was gradually enhanced by reducing the medium pH (pH 7.5 < 7.2 < 6.9). The plan area resorbed by MNCs increased linearly for up to 72 h. These results are very similar to those obtained with OCL preparations. In multiple standard assays with MNC preparations, more than 250 MNCs could be placed on a dentin slice, and the total area resorbed to a level of up to 9% of the whole surface within 48 h. In contrast, in multiple assays with OCL preparations, it was not easy to place more than 50 OCLs on a slice and the resorbed area was only 0.7% of the surface. The resorbing activity of the MNC preparation expressed by the resorbed area per a TRAP‐positive MNC (4.4 ± 0.4 × 10−3μm2) was greater than that of the OCL preparation (1.9 ± 0.4 × 10−3μm2). Pit formation by MNCs was dose dependently inhibited by salmon and human CT (ED50: salmon CT, 10−14M; human CT, 3 × 10−13M). Bafilomycin A1, a specific inhibitor of vacuolar type H+‐ATPase, also inhibited pit formation by MNCs at concentrations of 10−9M and above. In contrast, estrogen at 10−12–10−6M had no significant inhibitory effect on pit formation in this assay system. The assay system presented here is sensitive and reproducible and will be useful for examining the effects of various natural factors and synthetic compounds on osteoclastic bone resorption. We have previously established a culture system for examining MNC formation. Therefore, it is now possible to examine the effects of various test compounds on the recruitment of OCLs and pit formation by the same OCLs in a series of experiments.

We have reported that numerous tartrate‐resistant acid phosphatase‐positive osteoclast‐like multinucleated cells (TRAP+MNCs) are formed when mouse osteoblastic cells and spleen cells are cocultured in the presence of 1α25‐dihydroxyvitamin D3[1α,25‐(OH)2D3] (Endocrinology 123:2600, 1988). In this study, we prepared a TRAP+MNC population using a modified coculture system and examined its osteoclastic properties. TRAP+MNCs were formed in cocultures of mouse osteoblastic cells and marrow cells on 10 cm collagen gelcoated dishes. The TRAP+MNC population was prepared by treating the dishes with 0.2% bacterial collagenase followed by density gradient centrifugation. The yield of TRAP+MNCs was 20,000–40,000 cells per dish, much higher than that of osteoclasts (OCLs) isolated from neonatal rat bones (∼ 1000 cells per head). The purity of TRAP+MNCs was 5.6 ± 0.6% in cell number and about 30% in the number of nuclei. The recovery of TRAP+MNCs after density gradient centrifugation was 30–40%. Acid production by MNCs was demonstrated by vital staining with acridine orange. Numerous resorption pits were formed when the MNC population was cultured for 48 h on bone slices. Autoradiography using [125I]salmon calcitonin (CT) showed abundant CT binding in most TRAP+MNCs. Saturation analysis of [125I]salmon CT indicated a dissociation constant Kdfor TRAP+MNCs of 8.9 ± 0.7 × 1010M and 16.5 ± 1.5 ± 106binding sites per cell. These results were similar to the value (3.5 × 10−10M) and the number of binding sites (3.3 × 106per cell) in isolated rat OCLs. Displacement curves for [125I]salmon CT with unlabeled salmon and human CT were similar in MNC and OCL preparations. Salmon and human CT increased cAMP production (maximal response: salmon CT at 10−10M, human CT at 10−8M; ED50: salmon CT, 2.2 × 10−11M, human CT, 1.3 × 10−9M) in the MNC preparation. These results indicate that a large number of mouse TRAP+MNCs possessing OCL characteristics can be easily prepared from in vitro cultures. This procedure will facilitate examination of mammalian OCL functions.

In situ monitoring of the growth process in atomic layer epitaxy (ALE) is essential to understand the growth mechanism. In this paper, our recent study of the ALE growth mechanism investigated by means of two in situ monitoring methods, a gravimetric method using a microbalance and an optical method using surface photo-absorption (SPA), is reviewed. In situ and real-time monitoring of the growth rate on a monolayer scale, in situ observation of the growth process during ALE optically, the determination of surface chemical species during ALE growth, and the reaction mechanism that proceeds on the substrate surface during ALE are discussed for GaAs ALE growth using the GaCl source.

The As desorption from an (001) GaAs surface in atomic layer epitaxy (ALE) is investigated by a real-time in situ gravimetric monitoring system. The direct gravimetric information from the growing surface is monitored during an ALE cycle. It is shown that the growth rate decreases to 0.75 ML/cycle with increasing H2purge time according to the As desorption from the surface, and two kinds of stable reconstructions exist in the ALE process. Based on the results, a growth model is proposed for the growth of the smooth ALE surface on a (2 × 4) reconstructed surface.

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 previously reported that both hypoxia and hypoxia followed by reoxygenation (hypoxia/reoxygenation) rapidly and sequentially activate mitogen-activated protein kinase kinase kinase (MAPKKK) activity of Raf-1. This was followed by the sequential activation of MAP kinase kinase (MAPKK), MAP kinases (p42mapkand p44mapk), and S6 kinase (p90rsk). In this study, we demonstrated that both hypoxia and hypoxia/reoxygenation caused rapid activation of Src family tyrosine kinases, p60c-srcand p59c-fyn, which are upstream mediators of MAP kinase activation. This was followed by the activation of p21ras. Because Src family tyrosine kinases are known to be cell-surface-associated kinases and upstream regulators of p21ras, these results strongly suggested that activation of Src family tyrosine kinases plays a key role in triggering intracellular signaling cascades in cardiac myocytes in response to hypoxia and hypoxia/reoxygenation.

Osteoclasts which derive from hemopoietic cells are multinucleated cells responsible for bone resorption. We found that cyclopiazonic acid (CPA), thapsigargin (TG), and 2,5-di-(t-butyl)-1,4-hydroquinone (BHQ) induced osteoclast-like cell (OCL) formation in cocultures of mouse calvaria-derived stromal cells and hemopoietic cells such as bone marrow cells and spleen cells. OCLs induced by these compounds showed typical characteristics of osteoclasts such as tartrate-resistant acid phosphatase activity and pit forming activity. These compounds are known as endoplasmic reticulum (ER)/sarcoplasmic reticulum (SR) Ca2+-ATPase inhibitors that increase intracellular Ca2+levels by inhibiting Ca2+-ATPase activity located in the membrane of ER/SR. Ca2+-ionophores such as ionomycin which increase intracellular Ca2+levels also stimulated OCL formation in the cocultures. Differentiation of hemopoietic cells into OCLs induced by these compounds required the presence of calvarial cells. These results indicate that an increase of intracellular Ca2+levels may be a part of signaling pathways to induce osteoclast differentiation in the presence of calvarial cells.