Biomedical subjects

Masayoshi Yamaguchi

Publications and source records attributed to Masayoshi Yamaguchi.

At least 55 records · Page 3Linked to original sources

PubMed2004

Inhibitory effect of beta-cryptoxanthin on osteoclast-like cell formation in mouse marrow cultures.

The carotenoid beta-cryptoxanthin has been shown to have an inhibitory effect on bone-resorping factor-stimulated bone resorption in rat bone tissues in vitro. The effect of beta-cryptoxanthin on osteoclast-like cell formation in mouse marrow culture in vitro was investigated. The bone marrow cells were cultured for 7 days in alpha-minimal essential medium containing a bone-resorbing agent [parathyroid hormone (1-34) (PTH), prostaglandin E(2), 1,25-dihydroxyvitamin D(3), lipopolysaccharide, or tumor necrosis factor-alpha (TNFalpha)] with an effective concentration. Osteoclast-like cell formation was estimated by staining for tartrate-resistant acid phosphatase, a marker enzyme of osteoclasts. The presence of PTH (10(-7)M), prostaglandin E(2) (10(-5)M), 1,25-dihydroxyvitamin D(3) (10(-7)M), lipopolysaccharide (10 microg/mL), or TNFalpha (10 ng/mL) induced a remarkable increase in osteoclast-like multinucleated cells. These increases were significantly inhibited in the presence of beta-cryptoxanthin (10(-8) to 10(-6)M). beta-Cryptoxanthin (10(-7) and 10(-6)M) significantly inhibited dibutyryl cyclic adenosine monophosphate (DcAMP) (10(-5)M) or phorbol 12-myristate 13-acetate (PMA) (10(-5)M), an activator of protein kinase C, induced osteoclast-like cell formation. Also, beta-cryptoxanthin (10(-7) and 10(-6)M) had a significant inhibitory effect on osteoclast-like formation induced by receptor activator of NF-kappaB ligand (RANKL) (10 and 20 ng/mL) in the presence of macrophage colony-stimulating factor (M-CSF) (10 and 20 ng/mL). The stimulatory effect of RANKL and M-CSF on osteoclast-like cell formation was significantly enhanced in the presence of PMA, while such an effect was not seen by DcAMP. beta-Cryptoxanthin (10(-6)M) significantly inhibited osteoclast-like cell formation induced by RANKL and M-CSF in the presence of PMA or DcAMP. Moreover, the inhibitory effect of beta-cryptoxanthin on RANKL plus M-CSF-, PTH-, or TNFalpha-induced osteoclast-like cell formation was not observed in the presence of cycloheximide (10(-7)M), an inhibitor of protein synthesis at translational process, or 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole (10(-6)M), an inhibitor of transcription. This study demonstrates that beta-cryptoxanthin has a potent inhibitory effect on osteoclast-like cell formation in mouse marrow culture. The inhibitory action of beta-cryptoxanthin may partly involve in a newly synthesized protein component which is related to RANKL stimulation in osteoclastogenesis.

Animals↗

PubMed2004

beta-Cryptoxanthin stimulates bone formation and inhibits bone resorption in tissue culture in vitro.

The effect of beta-cryptoxanthin, which is greatly present in fruits, has not been clarified so far on bone metabolism. The effect of beta-cryptoxanthin on bone formation and bone resorption was investigated in tissue culture in vitro. Rat femoral-diaphyseal (cortical bone) and -metaphyseal (trabecular bone) tissues were cultured for 48 h in Dulbecco's modified Eagle's medium (high glucose, 4.5%) supplemented with antibiotics and bovine serum albumin. The experimental cultures contained 10(-8)-10(-5) M beta-cryptoxanthin. The presence of beta-cryptoxanthin (10(-6) or 10(-5) M) caused a significant increase in calcium content, alkaline phosphatase activity and deoxyribonucleic acid (DNA) content in the diaphyseal and metaphyseal tissues. These increases were completely prevented in the presence of cycloheximide (10(-6) M), an inhibitor of protein synthesis. beta-Carotene (10(-6) or 10(-5) M) or xantine (10(-6) or 10(-5) M) had no effect on the diaphyseal and metaphyseal calcium contents. The bone-resorbing factors parathyroid hormone (1-34) (PTH; 10(-7) M) or prostaglandin E2 (PGE2; 10(-5) M) caused a significant decrease in calcium content in the diaphyseal and metaphyseal tissues. The decrease in bone calcium content induced by PTH or PGE2 was completely inhibited by beta-cryptoxanthin (10(-8)-10(-6) M). In addition, beta-cryptoxanthin (10(-8)-10(-6) M) completely inhibited the PTH (10(-7) M)- or PGE, (10(-5) M)-induced increase in medium glucose consumption and lactic acid production by diaphyseal and metaphyseal tissues. The inhibitory effect of beta-cryptoxanthin (10(-7) M) on PTH (10(-7) M)- or PGE2 (10(-5) M)-stimulated decrease in the diaphyseal calcium content was significantly prevented in the presence of 10(-3) M vanadate, an inhibitor of protein tyrosine phosphatase. Vanadate (10(-3) M) did not have a significant effect on calcium content and lactic acid production in control bone tissues. The present study demonstrates that beta-cryptoxanthin has a direct stimulatory effect on bone formation and an inhibitory effect on bone resorption in tissue culture in vitro.

Alkaline Phosphatase↗

PubMed2004

Regucalcin increases superoxide dismutase activity in rat liver cytosol.

The effect of regucalcin, a regulatory protein in the intracellular signaling process, on superoxide dismutase (SOD) activity in the cytosol of rat liver was investigated. The presence of zinc sulfate (10(-6) or 10(-5) m) or cupric sulfate (10(-6) m) in the enzyme reaction mixture caused a significant increase in SOD activity, indicating that Cu/Zn-SOD may be present in the liver cytosol. SOD activity was significantly increased by the addition of regucalcin (0.1, 0.25, or 0.5 microM) to the reaction mixture. The presence of dithiothreitol (DTT; 0.1, 0.5, or 1.0 mM), a protective reagent for the sulfhydryl group, caused a significant decrease in SOD activity. The effect of regucalcin (0.25 microM) in increasing SOD activity was not seen in the presence of DTT (1.0 mM). Meanwhile, SOD activity was significantly raised by the addition of N-ethylmaleimide (NEM; 0.5 or 1.0 mM), a modifying reagent for the sulfhydryl reagent. Regucalcin (0.25 microM) caused a significant increase in SOD activity in the presence of NEM (1.0 mM). The effect of regucalcin in increasing SOD activity may not involve the sulfhydryl group of SOD. This study demonstrates that regucalcin has an activatory effect on SOD in the liver cytosol of rats.

Animals↗

PubMed2004

Oral administration of beta-cryptoxanthin induces anabolic effects on bone components in the femoral tissues of rats in vivo.

The effect of beta-cryptoxanthin on bone components in the femoral tissues of rats was investigated. Beta-cryptoxanthin was isolated from Satsuma mandarin (Citrus unshiu MARC.). Bone tissues were cultured for 48 h in serum-free Dulbecco's modified Eagle's medium containing either vehicle or beta-cryptoxanthin (10(-7) or 10(-6) M). The presence of beta-cryptoxanthin (10(-7) or 10(-6) M) caused a significant increase in calcium content and alkaline phosphatase activity in the femoral-diaphyseal and femoral-metaphyseal tissues. These increases were completely abolished in the presence of cycloheximide (10(-6) M), an inhibitor of protein synthesis. Thus beta-cryptoxanthin had an anabolic effect on bone calcification in vitro. Moreover, beta-cryptoxanthin (10, 25, or 50 microg/100 g body weight) was orally administered once daily for 7 d to young male rats. The administration of beta-cryptoxanthin (10, 25, or 50 microg/100 g body weight) caused a significant increase in calcium content and alkaline phosphatase activity in the femoral-diaphyseal and femoral-metaphyseal tissues. Femoral-diaphyseal and femoral-metaphyseal DNA contents were significantly increased by the dose of 25 or 50 microg/100 g body weight. A significant increase in metaphyseal DNA content was also seen with the dose of 10 microg/100 g body weight of beta-cryptoxanthin. This study demonstrates that beta-cryptoxanthin has an anabolic effect on bone components in rats in vitro and in vivo.

Administration, Oral↗

PubMed2004

Suppressive effect of regucalcin on protein phosphatase activity in the heart cytosol of normal and regucalcin transgenic rats.

The role of regucalcin, a regulatory protein in intracellular signaling pathway, in the regulation of protein phosphatase activity in the heart muscle cytosol was investigated by using normal (wild-type) and regucalcin transgenic (TG) rats. Protein phosphatase activity was assayed in a reaction mixture containing the cytosolic protein in the presence of phosphotyrosine, phosphoserine, and phosphothreonine. The addition of calcium chloride (10 and 20 microM) in the enzyme reaction mixture caused a significant increase in protein phosphatase activity toward three phosphoaminoacids. Trifluoperazine (10 and 20 microM), an antagonist of calmodulin, completely inhibited calcium (10 microM) addition-increased protein phosphatase activity toward three phosphoaminoacids. Moreover, the calcium (10 microM)-increased enzyme activity toward phosphoserine and phosphothreonine was significantly enhanced by the addition of calmodulin (2.5 or 5 microg/ml). Such an enhancement was not seen in the presence of phosphotyrosine. Regucalcin (10(-9) and 10(-8) M) significantly inhibited protein phosphatase activity toward three phosphoaminoacids in the presence of ethylene glycol bis (2-aminoethlether) N,N,N',N'-tetraacetic acid (EGTA; 1 mM), without Ca2+ addition. The inhibitory effect of regucalcin (10(-10)-10(-8) M) was also seen in the presence of calcium chloride (10 microM). Western blot analysis showed a remarkable expression of regucalcin protein in the cytosol of heart of regucalcin TG female rats as compared with that of wild-type female rats. Protein phosphatase activity toward three phosphoaminoacids was significantly decreased in the heart cytosol of TG rats. The enhancing effect of calcium (10 microM) addition on protein phosphatase activity toward three phosphoaminoacids was not seen in the heart cytosol of TG rats. This study demonstrates that endogenous regucalcin plays a suppressive role in the regulation of protein phosphatase activity in rat heart cytoplasm.

Animals↗

PubMed2004

Expression of regucalcin in rat bone marrow cells: involvement of osteoclastic bone resorption in regucalcin transgenic rats.

The expression of regucalcin in rat bone marrow cells was investigated. The expression of regucalcin mRNA in the bone marrow cells of normal (wild-type) rat was shown by using reverse transcription-polymerase chain reaction (RT-PCR) with a specific primer of regucalcin cDNA. Regucalcin protein was detected in the marrow cells of normal (wild-type) rats using Western blot analysis. Regucalcin levels were significantly increased in the marrow cells of regucalcin transgenic (TG) male and female rats with increasing age (5-36 weeks old). When the marrow cells obtained from normal or regucalcin TG rats (36-week-old) were cultured for 7 days, the number of tartrate-resistant acid phosphatase (TRACP), a marker enzyme of osteoclasts, positive multinucleated cells (MNCs) were significantly increased in the marrow culture of regucalcin TG rats. This increase was remarkable in female TG rats as compared with male TG rats. The effect of parathyroid hormone [human PTH (1-34); 10(-7) M] or 1,25-dihydroxyvitamin D3 [1,25(OH)2D3; 10(-7) M] in stimulating TRACP-positive MNCs formation was significantly enhanced in female regucalcin TG rats. Calcium content in the femoral-diaphyseal and -meta-physeal tissues was significantly decreased in regucalcin TG rats (10- or 36-week-old). This decrease was greater in female than in male. Femoral-metaphyseal deoxyribonucleic acid (DNA) content was significantly reduced in regucalcin TG male and female rats (36-week-old). Moreover, serum inorganic phosphorus, triglyceride, HDL-cholesterol, and albumin concentrations were significantly increased in regucalcin TG female rats (36-week-old), while serum calcium, zinc, and glucose concentrations were not significantly altered in TG male and female rats. In TG male rats, serum triglyceride and HDL-cholesterol concentrations were significantly raised. This study demonstrates that regucalcin is expressed in rat bone marrow cells, and that osteoclastic bone resorption is stimulated in regucalcin TG rats with increasing age. Also, regucalcin TG aged rats was found to induce serum metabolic disorder.

Animals↗

PubMed2004

Receptor activator of NF-kappaB ligand-stimulated osteoclastogenesis in mouse marrow culture is suppressed by zinc in vitro.

Zinc has been shown to have an inhibitory effect on osteoclastic bone resorption in vitro. This study was undertaken to determine whether the inhibitory action of zinc on osteoclastogenesis is related to receptor activator of NF-kappaB ligand (RANKL), which plays a pivotal role in differentiation from pre-osteoclasts to osteoclasts. Mouse marrow cells were cultured for 3 days in alpha-minimal essential medium containing lipopolysaccharide (LPS), tumor necrosis factor-alpha (TNFalpha), or RANKL, which stimulates osteoclastogenesis; then zinc sulfate was added to the culture medium containing each osteoclastogenesis-stimulating factor, and the cells were further incubated for 4 days. Osteoclast-like cell formation was estimated by staining for tartrate-resistant acid phosphatase (TRACP), a marker enzyme of osteoclasts. The presence of LPS (10 micro g/ml), TNFalpha (10 ng/ml), or RANKL (10 or 20 ng/ml) with M-CSF (10 or 20 ng/ml) induced a remarkable increase in osteoclast-like multinucleated cells (MNCs). The stimulatory effect of LPS was not significantly altered by the addition of zinc sulfate (10(-6)-10(-4) M). Meanwhile, TNFalpha- or RANKL-induced osteoclast-like cell formation was significantly inhibited in the presence of zinc sulfate (10(-6)-10(-4) M). The effect of zinc sulfate (10(-4) M) in inhibiting RANKL-induced osteoclast-like cell formation was completely abolished in the presence of cycloheximide (10(-7) M), an inhibitor of translation in protein synthesis, or 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole (DRB; 10(-6) M), an inhibitor of transcription. These results suggest that the inhibitory action of zinc on osteoclastogenesis is partly due to suppressing signaling pathway which is related to RANKL stimulation in osteoclast development.

Animals↗

PubMed2004

Role of regucalcin in liver nuclear function: binding of regucalcin to nuclear protein or DNA and modulation of tumor-related gene expression.

Regucalcin is a regulatory protein in the intracellular signaling pathway which is related to regulation of nuclear function. In this study the binding of regucalcin to nuclear proteins or DNA in vitro was examined. The results of the Far-Western analysis showed the existence of protein components which bind to regucalcin in the nucleus isolated from rat liver. Whether regucalcin binds deoxyribonucleic acid (DNA) was analyzed using DNA cellulose in vitro. Regucalcin was incubated in reaction mixture containing DNA cellulose, and DNA-binding regucalcin was detected using Western blot analysis for regucalcin. The results showed that regucalcin binds DNA in vitro. Moreover, the expression of c-src, p53, and Rb mRNAs was examined in the cloned rat hepatoma H4-II-E cells cultured for 24 or 48 h in the presence of fetal bovine serum (10%), using reverse transcription-polymerase chain reaction (RT-PCR). The expression of oncogene c-src mRNA was significantly suppressed in the hepatoma cells (transfectants), overexpressing regucalcin. Meanwhile, the expression of the tumor suppressor gene p53 or Rb mRNA was significantly enhanced in transfectants. This study may support the view that regucalcin modulates the transcriptional process by binding to protein and DNA in the nucleus.

Animals↗

PubMed2004

Characterization of protein tyrosine phosphatase activity in rat liver microsomes: suppressive effect of endogenous regucalcin in transgenic rats.

The role of regucalcin, a regulatory protein in intracellular signaling system, in the regulation of protein phosphatase activity in rat liver microsomes was investigated. Protein phosphatase activity torward phosphotyrosine, phosphoserine, and phosphothreonine was assayed in a reaction mixture containing the microsomal protein. Protein phosphatase activity toward phosphotyrosine was strong as compared with that of the enzyme activity toward phosphoserine and phosphothreonine, indicating the existence of protein tyrosine phosphatase. Protein phosphatase activity toward three phosphoaminoacids was significantly enhanced by the addition of both calcium chloride (10 micro M) and calmodulin (2.5 or 5 micro g/ml) in the reaction mixture. The presence of ethylene glycol bis (2-amino-ethylether) N, N, N', N'-tetracetic acid (EGTA; 0.1, 1 or 2 mM) or trifluoperazine (TFP; 10, 20 or 50 micro M), an antagonist of calmodulin, did not have a significant effect on protein phosphatase activity toward phosphotyrosine without calcium addition. Microsomal protein tyrosine phosphatase activity was not changed by okadaic acid (10(-6)-10(-4) M). The enzyme activity was significantly decreased by vanadate (10, 50 or 100 micro M). The addition of regucalcin (0.25 or 0.5 micro M) in the reaction mixture caused a significant inhibition of protein tyrosine phosphatase activity in liver microsomes. Western blot analysis showed a remarkable increase in regucalcin protein level in the liver microsomes of regucalcin transgenic (TG) rats. Protein tyrosine phosphatase activity was significantly suppressed in the liver microsomes of TG rats. This study demonstrates that protein tyrosine phosphatase activity is found in the liver microsomes, and that the enzyme activity is suppressed by regucalcin.

Animals↗

PubMed2004

Bone loss in regucalcin transgenic rats: enhancement of osteoclastic cell formation from bone marrow of rats with increasing age.

Bone loss was previously shown to be induced in the femoral tissue of regucalcin transgenic (TG) rats. Regucalcin is expressed in rat bone marrow cells and its expression is enhanced in regucalcin TG rats. This study was undertaken to determine the change in osteoclastic bone resorption in regucalcin TG rats with increasing age. Femoral-diaphyseal and -metaphyseal tissues were obtained from normal (wild-type) and regucalcin TG rats aged 5, 14, 25 or 50 weeks. Calcium content in the femoral-diaphyseal and -metaphyseal tissues was significantly decreased in regucalcin TG male and female rats aged 5, 14, 25 or 50 weeks as compared with the value obtained from normal rats with each age. When the marrow cells obtained from normal or regucalcin TG rats were cultured for 7 days, the number of tartrate-resistant acid phosphatase (TRACP), a marker of osteoclasts, positive multinucleated cells (MNCs) were significantly increased in the marrow culture of regucalcin TG male and female rats aged 5, 14, 25 or 50 weeks. The effect of parathyroid hormone [human PTH (1-34); 10(-7) M] or 1,25-dihydroxyvitamin D3 [1,25(OH)2D3; 10(-7) M] in stimulating TRACP-positive MNC formation was significantly enhanced in regucalcin TG male and female rats aged 14 or 25 weeks. This study demonstrates that osteoclastic bone resorption is stimulated in regucalcin TG male and female rats with increasing age.

Acid Phosphatase↗

PubMed2004

Hyperlipidemia is induced in regucalcin transgenic rats with increasing age.

Regucalcin plays an important role as a regulatory protein in intracellular signaling pathway in many cells. Regucalcin transgenic (TG) rats have been shown to induce a remarkable increase in serum triglyceride and HDL-cholesterol concentrations at the age of 36 weeks (35). Furthermore, this was investigated in regucalcin TG rats with increasing age (14, 25, 36 or 50 weeks). Serum triglyceride or HDL-cholesterol concentration was markedly increased in regucalcin TG male and female rats at 14, 25, 36 or 50 weeks of age. Serum-free fatty acid concentration was significantly elevated in regucalcin TG male and female rats at 25, 36 or 50 weeks. In the TG female rats, a significant increase in serum free fatty acid concentration was also observed at 14 weeks of age, while it was not seen in the TG male rats. Serum-free cholesterol concentration was significantly increased in regucalcin TG female rats at 14, 25, 36 or 50 weeks. Such an increase was not induced in the TG male rats. Moreover, serum calcium concentration was significantly raised in regucalcin TG male and female rats at 50 weeks of age. Also, serum albumin concentration was significantly elevated in regucalcin TG female rats at 25, 36, or 50 weeks of age. Such an increase was not observed in the TG male rats. Serum zinc, glucose or urea nitrogen concentration was not significantly altered in TG male and female rats. This study demonstrates that hyperlipidemia is uniquely induced in regucalcin TG rats with increasing age.

Aging↗

PubMed2004

Regucalcin increases superoxide dismutase activity in the heart cytosol of normal and regucalcin transgenic rats.

The effect of regucalcin, a regulatory protein in the intracellular signaling system, on superoxide dismutase (SOD) activity in the heart cytosol of normal rats and regucalcin transgenic (TG) rats was investigated. The addition of regucalcin (10(-10) to 10(-8) M) with a physiologic concentration in the enzyme reaction mixture containing the heart cytosol obtained from normal rats caused a significant increase in SOD activity, indicating that regucalcin directly activates the enzyme. The effect of regucalcin (10(-8) M) in increasing SOD activity was not seen in the presence of dithiothreitol (DTT; 0.1 or 1.0 mM), a protecting reagent for sulfhydryl group, or N-ethylmaleimide (NEM; 0.1 or 1.0 mM), a modifying reagent for sulfhydryl group, in the reaction mixture, indicating that regucalcin does not affect the sulfhydryl group. The addition of zinc sulfate (10(-6) to 10(-4) M) in the reaction mixture did not cause a significant change in SOD activity, while the enzyme activity was markedly decreased in the presence of cupric sulfate (10(-6) to 10(-4) M). The activatory effect of regucalcin (10(-8) M) on SOD was seen in the presence of zinc (10(-4) M), while not observed in the presence of copper (10(-4) M). Moreover, SOD activity was significantly enhanced in the heart cytosol of regucalcin TG rats as compared with that of normal rats. This study demonstrates that regucalcin increases SOD activity in the heart cytosol of rats, and that its effect is not related to the sulfhydryl group of enzymes.

Animals↗

PubMed2004

Expression of albumin in bone tissues and osteoblastic cells: involvement of hormonal regulation.

The expression of albumin in bone-related cells was investigated. Bone marrow cells and femoral-diaphyseal and -metaphyseal tissues were obtained from normal rat femur. Western blot analysis for protein in the lysate of bone marrow cells showed expression of albumin. The production of albumin was found in the bone marrow cells and femoral-diaphyseal and -metaphyseal tissues obtained from rat femur. When the femoral-diaphyseal and -metaphyseal tissues were cultured for 48 h in a serum-free medium in vitro, albumin was greatly released into culture medium from the bone tissues. The expression of albumin in osteoblastic MC3T3-E1 cells was investigated by Western blot analysis. Osteoblastic cells were cultured for 48 h in a serum-free medium containing either vehicle, parathyroid hormone (1-34) (human PTH; 10(-7) M), 17beta-estradiol (10(-9) M), insulin-like growth factor-I (IGF-I; 10(-8) M) or zinc sulfate (10(-4) M). The production of albumin in osteoblastic cells was significantly enhanced in the presence of PTH, IGF-I, 17beta-estradiol or zinc sulfate. These factors stimulated significantly the release of albumin from osteoblastic cells into culture medium. The effect of IGF-I (10(-8) M) in increasing the production of albumin in osteoblastic cells was completely prevented in the presence of cycloheximide (10(-7) M), an inhibitor of protein synthesis, or 5, 6-dichloro-1-beta-D-ribofuranosyl-benzimidazole (DRB; 10(-6) M), an inhibitor of transcriptional activity. This study demonstrates that albumin is expressed in bone marrow cells and osteoblastic cells, and that the production of albumin in osteoblastic cells is enhanced by various bone anabolic factors in vitro.

3T3 Cells↗

PubMed2004

Role of albumin in osteoblastic cells: enhancement of cell proliferation and suppression of alkaline phosphatase activity.

The role of albumin in osteoblastic cells was investigated. Osteoblastic MC3T3-E1 cells with subconfluent monolayers were cultured for 24 to 72 h in a medium without fetal bovine serum (FBS) in the presence or absence of albumin (0.25, 0.5, or 1.0 mg/ml of medium). The number of osteoblastic cells was significantly increased by culture for 24 to 72 h in the presence of albumin (1.0 mg/ml). The effect of albumin in increasing cell number was completely abolished in the presence of PD98049 (10(-7) M), staurosporine (10(-7) M), or dibucaine (10(-7) M), which is an inhibitor of various protein kinases. Also, the stimulating effect of albumin on cell proliferation was completely prevented in the presence of cycloheximide (10(-7) M), an inhibitor of protein synthesis, or 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), an inhibitor of transcriptional activity. DNA content was significantly increased in the cells cultured with albumin (1.0 mg/ml) addition for 24 to 72 h. Alkaline phosphatase activity in the cells, which participates in osteoblastic calcification, was significantly decreased by the culture with albumin (0.25, 0.5, or 1.0 mg/ml) for 24 to 72 h. The expression of insulin-like growth factor-I (IGF-I) and transforming growth factor-beta1 mRNAs using transcription-polymerase chain reaction (RT-PCR) analysis in osteoblastic cells was not significantly altered by culture for 48 h with albumin (1.0 mg/ml). This study demonstrated that albumin had a role in the regulation of osteoblastic cell function.

Albumins↗

PubMed2003

Overexpression of regucalcin modulates tumor-related gene expression in cloned rat hepatoma H4-II-E cells.

Regucalcin is a regulatory protein in intracellular signaling pathway which is related to various protein kinases and protein phosphatases in many cells. The effect of regucalcin on the expression of tumor-related genes was investigated in the cloned rat hepatoma H4-II-E cells and the hepatoma cells (transfectants) overexpressing regucalcin. Hepatoma cells were cultured for 24-72 h in the presence of fetal bovine serum (FBS; 10%). The proliferation of hepatoma cells was significantly suppressed at 24-72 h of culture in regucalcin transfectants as compared with that of wild-type or mock-type cells. Western blot analysis showed that regucalcin was markedly expressed in transfectants. The expression of c-myc, c-fos, c-jun, Ha-ras, and p53 mRNAs was determined using reverse transcription-polymerase chain reaction (RT-PCR). Of these genes, the expression of c-myc or Ha-ras mRNAs was significantly suppressed in regucalcin transfectants. The suppression of c-myc mRNA expression in transfectants was confirmed by using Northern blot analysis; significant suppression was seen at 24, 48, or 72 h of culture in the presence of 10% FBS. Culture with 10% FBS significantly enhanced c-myc mRNA expression in the hepatoma cells (wild-type) as compared with that of 1% FBS. The enhancement was significantly abolished in the transfectants. Meanwhile, the expression of p53 mRNA in the hepatoma cells was significantly enhanced in regucalcin-overexpressing hepatoma cells. This study demonstrates that the expression of oncogene c-myc and Ha-ras mRNA in hepatoma cells overexpressing regucalcin is suppressed, and that the tumor suppression gene p53 is enhanced in the transfectants.

Animals↗

PubMed2003

Suppressive effect of endogenous regucalcin on nitric oxide synthase activity in cloned rat hepatoma H4-II-E cells overexpressing regucalcin.

The role of endogenous regucalcin, which is a regulatory protein in calcium signaling, in the regulation of nitric oxide (NO) synthase activity in the cloned rat hepatoma H4-II-E cells was investigated. Hepatoma cells were cultured for 24-72 h in the presence of fetal bovine serum (FBS; 10%). NO synthase activity in the 5,500 g supernatant of cell homogenate was significantly increased by the addition of calcium chloride (10 microM) and calmodulin (2.5 microg/ml) in the enzyme reaction mixture. The presence of trifluoperazine (TFP; 50 microM), an antagonist of calmodulin, inhibited the effect of calcium (10 microM) addition in increasing NO synthase activity, indicating the existence of Ca(2+)/calmodulin-dependent NO synthase in hepatoma cells. NO synthase activity was significantly decreased by the addition of regucalcin (10(-8) or 10(-7) M) in the reaction mixture without or with Ca(2+)/calmodulin addition. The effect of regucalcin (10(-7) M) in decreasing NO synthase activity was also seen in the presence of TFP (50 microM) or EGTA (1 mM). The presence of anti-regucalcin monoclonal antibody (10-50 ng/ml) in the reaction mixture caused a significant elevation of NO synthase activity. NO synthase activity was significantly suppressed in the hepatoma cells (transfectants) overexpressing regucalcin. This decrease was completely abolished in the presence of anti-regucalcin monoclonal antibody (50 ng/ml) in the reaction mixture. Moreover, the effect of Ca(2+)/calmodulin addition in increasing NO synthase activity in the hepatoma cells (wild-type) was completely prevented in transfectants. The present study demonstrates that endogenous regucalcin has a suppressive effect on NO synthase activity in the cloned rat hepatoma H4-II-E cells.

Animals↗

PubMed2003

Enhancement of albumin expression in bone tissues with healing rat fractures.

The characterization of 66 kDa protein molecule, a major protein component which is produced from femoral-diaphyseal tissues with fracture healing (Igarashi and Yamaguchi [2002] Int. J. Mol. Med. 9:503-508), was investigated. Weaning rats were killed at 7 and 14 days after femoral fracture. When the femoral-diaphyseal tissues with fracture healing were cultured for 48 h in a serum-free medium, many proteins in the bone tissues were released into the medium. Analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that a protein molecule of approximately 66 kDa was markedly increased in culture medium from bone tissues with fracture healing. N-terminal sequencing of 66 kDa protein indicated that its N-terminus was identical to that of rat albumin. Western blot analysis of medium 66 kDa protein showed expression of albumin. This expression was significantly enhanced by fracture healing. The expression of albumin was seen in the diaphyseal (cortical bone) and metaphyseal (trabecular bone) tissues of rat femur. When the femoral-diaphyseal tissues obtained at 7 days after femoral fracture were cultured in a serum-free medium containing either vehicle, parathyroid hormone (1-34) (10(-7) M), insulin-like growth factor-I (10(-8) M) or zinc acexamate (10(-4) M), medium albumin was significantly increased in the presence of those bone-stimulating factors. The addition of albumin (0.5 or 1.0 mg/ml of medium) caused a significant increase in calcium and deoxyribonucleic acid contents in the femoral-diaphyseal and -metaphyseal tissues obtained from normal rats in vitro. The present study demonstrates that fracture healing induces a remarkable production of albumin which is a major protein component produced from femoral-diaphyseal tissues of rats, and that albumin has an anabolic effect on bone components.

Albumins↗

PubMed2003

Inhibitory role of regucalcin in the regulation of nitric oxide synthase activity in rat brain cytosol: involvement of aging.

Regucalcin is shown to play a regulatory role in intracellular signaling system in many cells. The protein has been reported to be present in rat brain neurons. The role of regucalcin in the regulation of nitric oxide (NO) synthase activity in the brain cytosol of female young and aged rats was investigated. The presence of regucalcin (10(-9) and 10(-8) M) in the enzyme reaction mixture caused a significant decrease in NO synthase activity in the absence or presence of both calcium chloride (10 micro M) and calmodulin (2.5 micro g/ml). The effect of regucalcin (10(-8) M) in decreasing brain cytosolic NO synthase activity was not seen in the presence of Nw-nitro-L-argine methyl ester (NAME) (10(-4) M), trifluoperazine (20 micro M) or EGTA (1 mM). Regucalcin protein levels were significantly lowered in the brain cytosol of aged (50 weeks old) rats as compared with that of young (5 weeks old) rats. Brain cytosolic NO synthase activity was significantly reduced with increasing age. In aged rat brain cytosol, regucalcin caused a significant decrease in NO synthase activity. The presence of anti-regucalcin monoclonal antibody (10-50 ng/ml) caused a significant increase in NO synthase activity in the brain cytosol of young and aged rats. This elevation was completely blocked by the addition of regucalcin (10(-8) M). The present study demonstrates that endogenous regucalcin has an inhibitory effect on NO synthase activity in the brain cytosol of young and aged rats.

Age Factors↗