Biomedical subjects

Y Azuma

Publications and source records attributed to Y Azuma.

At least 37 records · Page 2Linked to original sources

PubMed2001

Low-intensity pulsed ultrasound accelerates rat femoral fracture healing by acting on the various cellular reactions in the fracture callus.

Low-intensity pulsed ultrasound (LIPUS) has been shown to accelerate fracture healing in both animal models and clinical trials, but the mechanism of action remains unclear. In fracture healing, various consecutive cellular reactions occurred until repair. We investigated whether the advanced effects of LIPUS depended on the duration and timing of LIPUS treatment in a rat closed femoral fracture model to determine the target of LIPUS in the healing process. Sixty-nine Long-Evans male rats that have bilateral closed femoral fractures were used. The right femur was exposed to LIPUS (30 mW/cm2 spatial and temporal average [SATA], for 20 minutes/day), and the left femur was used as a control. Rats were divided into four groups according to timing and duration of treatment (Ph-1, days 1-8; Ph-2, days 9-16; Ph-3, days 17-24; throughout [T], days 1-24 after the fracture). Animals were killed on day 25. After radiographs and microfocus X-ray computed tomography (muCT) tomograms were taken, the hard callus area (HCA), bone mineral content (BMC) at the fracture site, and mechanical torsion properties were measured, and histological analysis was conducted. Interestingly, the maximum torque of the LIPUS-treated femur was significantly greater than that of the controls in all groups without any changes in HCA and BMC. The multiviewing of three-dimensional (3D) muCT reconstructions and histology supported our findings that the partial LIPUS treatment time was able to accelerate healing, but longer treatment was more effective. These results suggest that LIPUS acts on some cellular reactions involved in each phase of the healing process such as inflammatory reaction, angiogenesis, chondrogenesis, intramembranous ossification, endochondral ossification, and bone remodeling.

Animals↗

PubMed2001

Targeted disruption of cadherin-11 leads to a reduction in bone density in calvaria and long bone metaphyses.

The migration and adhesion of osteoblasts requires several classical cadherins. Cadherin-11, one of the classical cadherins, was expressed in mouse osteoblasts in skull bone and femur, revealed by immunohistochemistry. To elucidate the function of cadherin-11 in osteoblastogenesis, cadherin-11 null mutant mice were investigated. Although apparently normal at birth, Alizarin red staining of null mutant mice showed a reduced calcified area at the frontal suture that caused a round-shaped calvaria with increasing animal age to 3 months. Consequently, there was a reduction in bone density at the femoral metaphyses and the diploë of calvaria in null mutant mice. In the in vitro culture of newborn calvarial cells, the calcified area of mutant cells was smaller than those derived from wild-type littermates. These results show that absence of cadherin-11 leads to reduced bone density in some parts of skeletons including calvaria and long bone metaphyses, and thus suggest that cadherin-11 plays roles in the regulation of osteoblast differentiation and in the mineralization of the osteoid matrix.

Animals↗

PubMed2001

Tumor necrosis factor alpha-induced osteoclastogenesis requires tumor necrosis factor receptor-associated factor 6.

Although tumor necrosis factor receptor-associated factor 6 (TRAF6) is required in receptor activator of NF-kappaB-receptor activator of NF-kappaB ligand (RANK-RANKL) signaling for osteoclastogenesis, it has remained unclear whether TRAF6 is crucial in tumor necrosis factor alpha (TNF-alpha)-induced osteoclastogenesis. We examined TRAF6 function in the TNF-alpha-induced osteoclastogenesis by using osteoclast progenitor cells from TRAF6-deficient mice. The results indicated that TNF-alpha did not effectively induce osteoclast differentiation from osteoclast progenitor cells derived from these mice into mature multinucleated osteoclasts, although c-jun N-terminal kinase (JNK) and TNF-alpha activation was observed in osteoclast progenitor cells. Thus, we have provided the first evidence showing that TRAF6 is involved in TNF-alpha-induced osteoclastogenesis.

Animals↗

PubMed2001

Low-intensity pulsed ultrasound initiates bone healing in rat nonunion fracture model.

Low-intensity pulsed ultrasound exposure has been shown clinically to shorten the fracture repair process and to induce healing of nonunions in humans, but its mechanism of action remains unclear. In this study we investigated the effect and mechanism of low-intensity pulsed ultrasound on nonunion fracture healing in rat tibias. A consistently reproducible nonunion was produced in rat tibias by muscle interposition without osteotomy. This model was produced by creating a closed tibial fracture with only the distal end of the tibialis anterior muscle interposed into the fracture site. One limb was noninvasively exposed to low-intensity pulsed ultrasound (a 200-millisecond burst of sine waves of 1.5 MHz, repeating at 1.0 kHz) for 20 minutes daily. The incident intensity was approximately 30 mW/cm2. Rats were killed at intervals between 2 and 6 weeks. The events were assessed by radiographs, microfocus X-ray computed tomograms, and histologic examination. After 6 weeks of exposure, 7 of 14 nonunion fractures showed healing on radiologic assessment. The results of three-dimensional microfocus X-ray computed tomographic reconstruction and histologic examination also supported this finding. On the other hand, all control tibias remained in a state of nonunion during the same period. These results indicate that low-intensity pulsed ultrasound promotes healing in the rat nonunion fracture model.

Animals↗

PubMed2001

Effect of Actinobacillus actinomycetemcomitans protease on the proliferation of gingival epithelial cells.

OBJECTIVE: Actinobacillus actinomycetemcomitans (A. actinomycetemcomitans) has been implicated as an important pathogen in periodontal disease. The aim of this report is to examine the effect of in vitro application of A. actinomycetemcomitans protease on the cell growth, DNA synthesis, and fibronectin level of human gingival epithelial cells. MATERIALS AND METHODS: A. actinomycetemcomitans protease was isolated from the culture supernatant of A. actinomycetemcomitans. Human gingival epithelial cells were incubated in the presence or absence of A. actinomycetemcomitans protease (0.01, 0.1, or I U) for 48 h. The proliferation of cells were tested with the MTT assay and the BrdU assay. The fibronectin level was examined by flow cytometric analysis. RESULTS: The cell growth rate, DNA synthesis rate, and fibronectin level of the gingival epithelial cells that had been incubated with protease, were lower than the respective values in the untreated gingival epithelial cells; as the dose of A. actinomycetemcomitans protease increased, the cell growth rate, DNA synthesis rate, and fibronectin level of the gingival epithelial cells decreased. A. actinomycetemcomitans protease may inhibit the proliferation of human gingival epithelial cells. CONCLUSIONS: This protease may play a key role as a major virulence factor in the development of periodontal disease.

Aggregatibacter actinomycetemcomitans↗

PubMed2000

Immunomodulation of the neutrophil respiratory burst by endomorphins 1 and 2.

Opioid peptides were found to be released from cells of the immune system during inflammation and stress, and were associated with altered immune responses. Production of superoxide anions by PMA-stimulated neutrophils was markedly inhibited in a concentration-dependent manner by preincubation for 15 min with 10(-18) - 10(-6) M of the endogenous opioid peptides endomorphin 1 or 2. Inhibition was prevented by prior treatment with the micro-opioid receptor-selective antagonist beta-funaltrexamine at 10(-12) - 10(-8) M, but not the delta-opioid receptor-selective antagonist naltrindole. In contrast, endomorphins 1 and 2 caused significant potentiation of superoxide anion production in unstimulated neutrophils. These results suggest that the endogenous opioid peptides endomorphins 1 and 2 may modulate the production of superoxide anions in neutrophils via mu-opioid receptors.

Animals↗

PubMed2000

Suppression of azoxymethane-induced colon carcinogenesis in male F344 rats by mandarin juices rich in beta-cryptoxanthin and hesperidin.

We have reported protective effects of dietary administration of a powder "CHRP" containing high amounts of beta-cryptoxanthin and hesperidin prepared from a Satsuma mandarin (Citrus unshiu Marc.) juice on azoxymethane (AOM)-induced rat aberrant crypt foci through suppression of crypt cell proliferation and/or induction of detoxifying enzymes. In the present study, we investigated the modifying effects of a commercial Satsuma mandarin (Citrus unshiu Marc.) juice (MJ) and those of MJ2 and MJ5, which were prepared from MJ and are richer in beta-cryptoxanthin and hesperidin than MJ, on the occurrence of colonic tumors induced by AOM in male F344 rats. Rats were given 2 weekly s.c. injections of AOM (20 mg/kg body weight) to induce colonic neoplasms. They also received MJ, MJ2, or MJ5 as a drinking water at night for 36 weeks, starting 1 week after the last dosing of AOM. AOM exposure produced colonic adenocarcinoma with an incidence of 69% and a multiplicity of 0.76 +/- 0.57/rat at week 38. MJ, MJ2, and MJ5 administration significantly reduced the frequency of colonic carcinoma [MJ: 35% (49% reduction), p < 0.02; MJ2: 20% (64% reduction), p = 0.0028; and MJ5: 15% (78% reduction), p < 0.00021] and multiplicity [MJ: 0.40 +/- 0.58 (47% reduction), p < 0.05; MJ2: 0.25 +/- 0.43 (67% reduction), p < 0.005; and MJ5: 0.15 +/- 0.36 (80% reduction), p < 0.001]. Also, the numbers of cancer cells positive for proliferative cell nuclear antigen (PCNA) and cyclin D1 in colonic tumors were lowered by these treatments. In addition, treatment with MJ, MJ2, or MJ5 significantly increased apoptotic index in colonic adenocarcinoma. These findings might suggest effective chemopreventive ability of MJs, especially MJ5, in colon tumorigenesis.

Adenocarcinoma↗

PubMed2000

Toll-like receptor 4-mediated signal pathway induced by Porphyromonas gingivalis lipopolysaccharide in human gingival fibroblasts.

The lipopolysaccharide (LPS) secreted by Porphyromonas gingivalis is implicated in the initiation and progression of periodontitis. Human gingival fibroblasts (HGFs) are the major constituent of gingival connective tissue. In this study, we examined the expression of Toll-like receptor 4 (TLR4) on HGFs by flow cytometric analysis, and studied the signal transduction induced by LPS stimulation of HGFs by enzyme-linked immunosorbent assay, Western blotting, and immunoprecipitation. We show that LPS binds to HGFs, and that HGFs express TLR4 and myeloid differentiation primary response gene 88 (MyD88). P. gingivalis LPS-induced interleukin (IL)-1 production in HGFs was inhibited by anti-TLR4 antibody. P. gingivalis LPS treatment of HGFs activated several intracellular proteins including protein tyrosine kinases, and upregulated the expression of IL-1 receptor-associated kinase (IRAK), nuclear factor-kappaB (NF-kappaB), and activating protein-1 (AP-1), and these events were suppressed by anti-TLR4 monoclonal antibody. Our findings suggest that the binding of P. gingivalis LPS to TLR4 on HGFs activates various second messenger systems.

Antibodies, Monoclonal↗

PubMed2000

Tumor necrosis factor-alpha induces differentiation of and bone resorption by osteoclasts.

Osteoclast progenitors differentiate into mature osteoclasts in the presence of receptor activator of NF-kappaB (RANK) ligand on stromal or osteoblastic cells and monocyte macrophage colony-stimulating factor (M-CSF). The soluble RANK ligand induces the same differentiation in vitro without stromal cells. Tumor necrosis factor-alpha (TNF-alpha), a potent cytokine involved in the regulation of osteoclast activity, promotes bone resorption via a primary effect on osteoblasts; however, it remains unclear whether TNF-alpha can also directly induce the differentiation of osteoclast progenitors into mature osteoclasts. This study revealed that TNF-alpha directly induced the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs), which produced resorption pits on bone in vitro in the presence of M-CSF. The bone resorption activity of TNF-alpha-induced MNCs was lower than that of soluble RANK ligand-induced MNCs; however, interleukin-1beta stimulated this activity of TNF-alpha-induced MNCs without an increase in the number of MNCs. In this case, interleukin-1beta did not induce TRAP-positive MNC formation. The osteoclast progenitors expressed TNF receptors, p55 and p75; and the induction of TRAP-positive MNCs by TNF-alpha was inhibited completely by an anti-p55 antibody and partially by an anti-p75 antibody. Our findings presented here are the first to indicate that TNF-alpha is a crucial differentiation factor for osteoclasts. Our results suggest that TNF-alpha and M-CSF play an important role in local osteolysis in chronic inflammatory diseases.

Animals↗

PubMed2000

Anabolic response of mouse bone-marrow-derived stromal cell clone ST2 cells to low-intensity pulsed ultrasound.

The effects of 20-min exposure to low-intensity, pulsed ultrasound were investigated in ST2 cells of bone marrow stromal origin. They responded to ultrasound with elevated levels of IGF mRNAs, osteocalcin, and bone sialoprotein mRNAs. The upregulated expression of these messages appeared in a biphasic manner, with the first peak resistant to the protein synthesis inhibitor cycloheximide, and a second peak that was eliminated by NS398, an inhibitor of the inducive prostaglandin G/H synthase (cyclooxygenase-2). A cumulative effect of mechanical loading called the memory effect, which has been observed in vivo, can be explained from such a biphasic anabolic reaction mediated by prostaglandins. The upregulation of IGF or osteocalcin mRNAs can be observed even at 24 h after the initiation of the 20-min exposure to ultrasound. Our results suggest that this low-intensity, pulsed ultrasound, which has been clinically used to accelerate the healing processes of fractured bone, induces a direct anabolic reaction of osteogenic cells, leading to bone matrix formation.

Animals↗

PubMed2000

Alteration of sugar chains on alpha(1)-acid glycoprotein secreted following cytokine stimulation of HuH-7 cells in vitro.

In inflammation, hepatocytes secrete several proteins into serum, the so-called acute phase proteins. In addition to increased serum levels of alpha(1)-acid glycoprotein (AGP), there are also changes in the composition of its sugar chains. To investigate the cytokine-stimulated alteration of sugar chains on AGP, we cultured the human hepatoma cell line HuH-7 cells in serum-free medium (IS-RPMI) with and without IL-1beta and IL-6, and analyzed AGP secretion into the medium. AGP was increased during stimulation with both IL-1beta and IL-6, although the effect of IL-1beta was more pronounced. Lectin-binding assay for secreted AGP also indicated significant increases in the binding activities to Aleuria aurantia lectin (AAL), Sambucus sieboldiana agglutinin (SSA), and concanavalin-A (ConA). In particular, AAL binding activity increased with higher expression of the sialyl Lewis X (sLe(X)) antigen, NeuAc alpha2-3 Gal beta1-4 (Fuc alpha1-3) GlcNAc-R. Thus, the increase in AGP fucosylation may be correlated with the increase of sLe(X). The present results indicate that the serum-free culture of HuH-7 cells provides a useful model for investigating the secretion of proteins from hepatocytes, and the effects of cytokines on the changes in sugar chains of glycoproteins in vitro.

Carbohydrate Conformation↗

PubMed2000

Comparison of inhibitory effects of local anesthetics on immune functions of neutrophils.

Immunological effects of a variety of local anesthetics on adhesion, phagocytosis, and the production of superoxide anion and hydrogen peroxide by neutrophils were compared. Neutrophils were isolated by peritoneal lavage from rats, 4 h after injection of 1% glycogen. Lidocaine, mepivacaine, procaine, prilocaine and tetracaine at 1 mg/ml inhibited adhesion, phagocytosis, and the production of superoxide anion and hydrogen peroxide in neutrophils. Moreover, lidocaine, mepivacaine, procaine and prilocaine at 0.1 mg/ml inhibited the production of superoxide anion and hydrogen peroxide but not adhesion or phagocytosis. In contrast, tetracaine at 0.1 mg/ml inhibited phagocytosis, and the production of superoxide anion and hydrogen peroxide but not adhesion. At 0.01 mg/ml, however, tetracaine inhibited the production of superoxide anion and hydrogen peroxide; in contrast, other drugs failed to affect neutrophil function. These results suggest that the local anesthetics may affect adhesion, phagocytosis, and the production of superoxide anion and hydrogen peroxide by neutrophils.

Anesthetics, Local↗

PubMed2000

Effects of ultrasound and 1,25-dihydroxyvitamin D3 on growth factor secretion in co-cultures of osteoblasts and endothelial cells.

It has been shown that low-intensity pulsed ultrasound (US) accelerates fracture healing in animal models and in clinical studies. However, the mechanism by which US accelerates fracture healing remains unclear. Systemic factors and several growth factors, such as platelet-derived growth factor (PDGF), are thought to be involved in the process of fracture healing. In the present study, we examined the effects of US and 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] on growth factor secretion in a co-culture system of human osteoblastic cells (SaOS-2) and endothelial cells (HUVEC). US was applied to cultured cells for 20 min daily for four consecutive days. US treatment increased the PDGF-AB level in the conditioned media. 1,25-(OH)2D3 (1 x 10(-8) M) also enhanced PDGF-AB secretion. The secretion of PDGF-AB was synergistically increased by the combination of US and 1,25-(OH)2D3. These results suggest that the stimulation of growth factor secretion from cells by US and 1,25-(OH)2D3 treatment may be involved in the acceleration of fracture healing.

Analysis of Variance↗

PubMed2000

The role of Ran in nuclear function.

There have been three major advances in the understanding of the Ran pathway during the past two years: first, a general model for Ran's function in nuclear transport has been proposed and extensively tested. Second, crystal structures for many proteins that regulate or interact with Ran have been reported, which provide molecular details of how Ran works. Third, it has been documented that Ran regulates mitotic spindle assembly in a transport-independent fashion.

Animals↗

PubMed2000

Effects of the mycelial extract of cultured Cordyceps sinensis on in vivo hepatic energy metabolism and blood flow in dietary hypoferric anaemic mice.

The beneficial effects of a traditional Chinese medicine, Cordyceps sinensis (Cs), on mice with hypoferric anaemia were evaluated by NMR spectroscopy. Experimental hypoferric anaemia was induced in mice by feeding with an Fe-free diet for 6 weeks. They were then given extract from cultured Cs (200 mg/kg body weight daily, orally) and were placed on an Fe-containing recovery diet (35 mg Fe/kg diet) for 4 weeks. In vivo 31P and 2H NMR spectra acquired noninvasively and quantitatively at weekly intervals were used to evaluate hepatic energy metabolism and blood flow in the mice. During the 4-week Cs-extract treatment, consistent increases were observed in liver beta-ATP: inorganic phosphate value by liver 31P NMR spectroscopy, representing the high energy state, and in blood-flow rate as determined by 2H NMR spectroscopy of deuterated water (D2O) uptake after intravenous injection of D2O. The haematological variables (the packed cell volume and the haemoglobin level) and the hepatic intracellular pH, which was determined from the NMR chemical shift difference between the inorganic phosphate peak and the alpha-phosphate peak of ATP, were not significantly different between Cs-extract-treated and control mice. As blood flow and energy metabolism are thought to be linked, the Cs-extract-increased hepatic energy metabolism in the dietary hypoferric anaemic mice was concluded to be due to increased hepatic blood flow.

Adenosine Triphosphate↗

PubMed2000

Decrease in cell surface sialic acid in etoposide-treated Jurkat cells and the role of cell surface sialidase.

The present study investigated the mechanism underlying alterations of cell surface sugar chains of Jurkat cells by inducing apoptosis with etoposide, an inhibitor of topoisomerase II. Within 3 h of etoposide treatment, flowcytometric analysis revealed a decrease in Maackia amurensis agglutinin recognized alpha2,3-linked sialic acid moieties and an increase in Ricinus communis agglutinin recognized galactose. The results suggested that asialo-sugar chains on glycoconjugates were rapidly induced on the etoposide-treated cell surface. To clarify the desialylation mechanism, we studied alpha2,3-sialyltransferase mRNA expression and the activity of sialidase on the cell surface during etoposide-induced apoptosis. The expression of hST3Gal III and hST3Gal IV mRNAs were down-regulated and sialidase activity on the cell surface increased threefold within 2 h of etoposide treatment. Moreover, the decrease in alpha2,3-linked sialic acid levels was significantly suppressed in the presence of 2,3-dehydro-2-deoxy-N-acetylneuraminic acid, an inhibitor of sialidase. These results suggested that activation or exposure of sialidase on the cell surface was induced by etoposide treatment and was the main cause of the decrease in sialic acids.

Apoptosis↗

PubMed2000

Insulin receptor substrate-1 in osteoblast is indispensable for maintaining bone turnover.

Insulin receptor substrates (IRS-1 and -2) are essential for intracellular signaling by insulin and IGF-I, anabolic regulators of bone metabolism. Mice lacking the IRS-1 gene IRS-1(-/-) showed severe osteopenia with low bone turnover. IRS-1 was expressed in osteoblasts, but not in osteoclasts, of wild-type (WT) mice. IRS-1(-/-) osteoblasts treated with insulin or IGF-I failed to induce tyrosine phosphorylation of cellular proteins, and they showed reduced proliferation and differentiation. Osteoclastogenesis in the coculture of hemopoietic cells and osteoblasts depended on IRS-1 expression in osteoblasts and could not be rescued by IRS-1 expression in hemopoietic cells in the presence of not only IGF-I but also 1,25(OH)(2)D(3). In addition, osteoclast differentiation factor (RANKL/ODF) was not induced by these factors in IRS-1(-/-) osteoblasts. We conclude that IRS-1 deficiency in osteoblasts impairs osteoblast proliferation, differentiation, and support of osteoclastogenesis, resulting in low-turnover osteopenia. Osteoblastic IRS-1 is essential for maintaining bone turnover, because it mediates signaling by IGF-I and insulin and, we propose, also by other factors, such as 1,25(OH)(2)D(3).

Animals↗

PubMed2000

Effect of the novel prostaglandin A1 derivative TEI-6363 on ROS17/2.8 cell differentiation in vitro.

The effect of TEI-6363 (5-[E-4-N,N-dimethylaminophenylmethylene]-4-hydroxy-2-[1-methyl imidazole-2-ilthio]-4-[4-phenylbutyl]-2-cyclopentenone), a chemically synthesized prostaglandin A1 derivative, on cell proliferation and osteoblastic differentiation was investigated concurrently. ROS17/2.8 cells (a rat osteosarcoma-derived cell line) were treated with TEI-6363 at two concentrations, 10(-7) and 10(-6) M, and viable cells were counted to assess cytotoxic effects and determine the growth curve. After 96 h of treatment, there was no evidence of any effect of TEI-6363 on cell viability at either concentration. However, a clear inhibitory effect on cell proliferation was observed after treatment with 10(-6) M TEI-6363 for 24 h or longer. A pulse-treatment experiment showed that TEI-6363 induced the inhibition of proliferating ROS17/2.8 cells 24 h after addition. The inhibition of proliferation was associated with G1-arrest demonstrated by flow cytometric analysis, and incorporation of [3H]thymidine by ROS17/2.8 cells was decreased. Osteoblastic differentiation (assessed on the basis of increased alkaline phosphatase activity and collagen synthesis) was induced by TEI-6363 treatment at 10(-6) M following G1-arrest and inhibition of cell proliferation. These results suggest that TEI-6363 arrested the cell cycle of ROS17/2.8 cells at the G1 phase and induced osteoblastic differentiation. These results did not appear to be dependent on a marked cytotoxic effect.

Alkaline Phosphatase↗