Biomedical subjects

A Ikegami

Publications and source records attributed to A Ikegami.

At least 37 records · Page 2Linked to original sources

PubMed1995

Demonstration of isoforms of the estrogen receptor in the bone tissues and in osteoblastic cells.

Expression of isoforms of estrogen receptor (ER) was examined in the bone tissues. Reverse transcriptase-polymerase chain reaction ((RT-PCR) using specific primers for rat ER cDNA was performed with total RNA from rat bone tissues. Then, we sequenced the amplified products after cloning and identified two isoforms of the ER and the wild-type ER. One of the ER mRNA isoforms did not have the region corresponding to exon 4 and the other isoform did not have the region corresponding to both exon 3 and exon 4. These isoforms were designated as ER delta 4 isoform and ER delta 3/4 isoform, respectively. The existence of these isoforms was also confirmed by ROS-17/2.8 osteoblastic osteosarcoma cells. Chloramphenicol acetyltransferase assay showed that these isoforms lost estrogen dependent transactivation activities. We suggest that the ER isoforms may play some roles in the bone metabolism in which estrogen is essential to maintain bone density.

Animals↗

PubMed1995

Mechanical measurements of single actomyosin motor force.

To elucidate the mechanism of force generation by actomyosin motor, a measuring system was constructed, in which an in vitro motility assay was combined with an optical trapping technique. An actin filament of several micron long was attached to a gelsolin-coated polystyrene bead, and was allowed to interact with a small number (approximately 1/1 micron actin filament) of rabbit skeletal heavy meromyosin (an active subfragment of myosin) molecules bound to a nitrocellulose-coated coverglass. The bead position was determined at 33-ms intervals. We measured the force generation event at relatively low (100-400 nM) ATP concentration so that the occurrence of individual force generation events could be detected with our time resolution. The actin-bound bead held in the optical trap moved in a stepwise manner in the direction of the actin filament only in the presence of ATP. At the trap strength of 0.3 pN/nm, the maximum size of the step was 11 nm, and the maximum force associated with the movement was 3.3 pN.

Actins↗

PubMed1994

Demonstration of activin-A in arteriosclerotic lesions.

We previously reported that follistatin, an activin-binding protein, is produced in arteriosclerotic lesions. Here, the expression of activin-A which promotes the growth of vascular smooth muscle cells was examined in arteriosclerotic lesions of WHHL (Watanabe heritable hyperlipidemic) rabbits. Activin-A mRNA was detected in normal aorta by reverse transcriptase-polymerase chain reaction using specific primers for activin-A cDNA and was increased remarkably in arteriosclerotic lesions. In addition, using the cloned rabbit activin-A cDNA, RNA probe was prepared and in situ hybridization histochemistry was performed. Activin-A transcripts were detected abundantly in neointima of the diseased artery. Furthermore, immunohistochemistry also detected activin-A at the protein level. These observations suggest that activin-A is a cytokine expressed in arteriosclerotic lesions and might be involved in the pathogenesis of atherosclerosis.

Activins↗

PubMed1994

Effects of 1 alpha,25-dihydroxyvitamin D3 on macrophage colony-stimulating factor production and proliferation of human monocytic cells.

1 alpha-25-Dihydroxyvitamin D3 [1 alpha,25(OH)2D3] stimulates the proliferation of human monocytes in vitro. In the present study, we investigated a possible role of macrophage colony-stimulating factor (M-CSF) in 1 alpha,25(OH)2D3-induced proliferation of human circulating monocytes and the effects of 1 alpha,25(OH)2D3 on M-CSF production by human monocytic cells. Both 1 alpha,25(OH)2D3 and recombinant human M-CSF increased 2.5-fold the nucleus number of human circulating monocytes on day 6 of the culture. These effects were inhibited by antihuman M-CSF antibody as well as by anti-c-fms antibody, although these antibodies themselves did not affect the nucleus number when added to control culture. These results indicated that M-CSF is required for 1 alpha,25(OH)2D3-stimulated monocyte proliferation. In addition, 1 alpha,25(OH)2D3 stimulated M-CSF secretion from human circulating monocytes. Secretion and mRNA expression of M-CSF by 12-0-tetradecanoylphorbol-13-acetate (TPA)-treated THP-1 cells (human monocytic leukemia cell line) and TPA-treated HL-60 cells (human promyelocytic leukemia cell line) were also increased by 1 alpha,25(OH)2D3. M-CSF secretion from TPA-treated THP-1 cells was increased by 1 alpha,25(OH)2D3 in a dose-dependent and metabolite-specific manner. The present study demonstrates that 1 alpha,25(OH)2D3 is a potent stimulator for M-CSF production by human monocytic cells and that the proliferative effect of 1 alpha,25(OH)2D3 on human monocytes may be attributed, at least in part, to the stimulated secretion of M-CSF.

Blotting, Northern↗

PubMed1994

Immunocytochemical identification of androgen receptor in mouse osteoclast-like multinucleated cells.

Expression of androgen receptor (AR) in mouse osteoclast-like multi-nucleated cells (OCs) was examined with immunocytochemical techniques. Murine OCs were obtained by co-culturing mouse osteoblastic cells and bone marrow cells. Three preparations of polyclonal anti-AR antibody which were raised in rabbit against different parts of the human AR were employed for the experiments. Specific staining for AR was demonstrated in the nuclei and the perinuclear area of mouse OCs. This is the first report demonstrating the presence of AR in osteoclast-like cells.

Animals↗

PubMed1994

Cell cycle-dependent expression of estrogen receptor and effect of estrogen on proliferation of synchronized human osteoblast-like osteosarcoma cells.

Dual fluoroimmunohistochemical staining of estrogen receptor (ER) and bromodeoxyuridine was performed in a human osteoblastic osteosarcoma cell line, HOS TE85 cells. ER immunoreactivity was observed preferentially in the nuclei of the cells that were bromodeoxyuridine positive. ER expression at various phases of the cell cycle was investigated in HOS TE85 cells, which were synchronized at the G1/S phase boundary by intermittent exposure to thymidine and hydroxyurea. ER immunoreactivity became detectable in the S phase, decreased in the G2/M and G1 phases, and then reappeared in the S phase of the next cell cycle. Western blot analysis also showed that ER protein exists in these cells and increases in the S phase. Moreover, Northern blot analysis demonstrated that the expression of ER messenger RNA increases in the early S phase, gradually decreases during the progress of the cell cycle, and increases again in the S phase of the subsequent cell cycle. Interestingly, 17 beta-estradiol (10(-8) M) increased cell number and [3H]thymidine incorporation into DNA in the synchronized HOS TE85 cells, whereas this effect was not observed in the nonsynchronized HOS TE85 cells. The present studies suggest that the cell cycle-dependent regulation may contribute to the heterogeneity of ER expression in osteoblastic cells.

Animals↗

PubMed1993

Genomic binding-site cloning reveals an estrogen-responsive gene that encodes a RING finger protein.

Estrogen receptor (ER)-binding fragments were isolated from human genomic DNA by using a recombinant ER protein. Using one of these fragments as a probe, we have identified an estrogen-responsive gene that encodes a putative zinc finger protein. It has a RING finger motif present in a family of apparent DNA-binding proteins and is designated estrogen-responsive finger protein (efp). efp cDNA contains a consensus estrogen-responsive element at the 3' untranslated region that can act as a downstream estrogen-dependent enhancer. Moreover, efp is regulated by estrogen as demonstrated at both the mRNA and the protein level in ER-positive cells derived from mammary gland. These data suggest that efp may represent an estrogen-responsive transcription factor that mediates phenotypic expression of the diverse estrogen action. Thus, the genomic binding-site cloning may be applicable for isolation of the target genes of other transcription factors.

Amino Acid Sequence↗

PubMed1993

Vascular smooth muscle cells as target for estrogen.

ER mRNA was detected as 6.0 kb band by Northern blot analysis in vascular smooth muscle cells (VSMC) derived from rat aorta. The presence of ER mRNA in VSMC was confirmed by reverse transcriptase-polymerase chain reaction using specific primers for rat ER cDNA. In addition, the immunocytochemistry of ER was performed in VSMC using a monoclonal anti-ER antibody which recognizes DNA-binding domain of ER. The immunoreactivity was distributed in the cytoplasm as well as in the nuclei. Thus, the expression of ER in VSMC was demonstrated at both the protein and the mRNA level. Furthermore, the expression of c-fos mRNA in VSMC was found up-regulated by 17 beta-estradiol treatment within 30 min. The observation that VSMC possess ER and respond to estrogen supports the idea that estrogen may directly influence vascular cell system through the ER.

Animals↗

PubMed1993

Immunohistochemical detection and northern blot analysis of estrogen receptor in osteoblastic cells.

Expression of estrogen receptor (ER) was studied in MC3T3-E1 cells (mouse osteoblastic cell line), HOS TE85 cells (human osteosarcoma cell line), and primary osteoblastic cells derived from mouse calvaria with immunohistochemical techniques. The staining of ER was readily detectable in MC3T3-E1 cells, HOS TE85 cells, and primary osteoblastic cells by using a monoclonal anti-ER antibody that recognizes the DNA binding domain of ER. The immunoreactivity was distributed in the cytoplasm as well as in the nuclei. 17 beta-Estradiol (10(-8) M) did not alter this staining pattern. The expression of ER was confirmed by Northern blot analysis using rat ER cDNA probe, which revealed a 6.5 kb band in MC3T3-E1 cells and a 6.2 kb band in HOS TE85 cells. The mRNA level of ER was not altered by 17 beta-estradiol (10(-8) M). The immunohistochemical studies showed that ER was not detectable in all cells but in a small population of each cell type. This study is the first report to demonstrate the presence of ER immunohistochemically, and our results suggest the heterogeneity of ER expression among osteoblastic cells.

3T3 Cells↗

PubMed1993

Effects of a new diphenylpiperazine calcium antagonist, KB-2796, on cerebral ischemic neuronal damage in rats.

1. The effects of KB-2796, a new diphenylpiperazine calcium antagonist, on the mitochondrial dysfunction and energy metabolism deficits were examined in the ischemic rat brain. 2. KB-2796 (30 mg/kg, p.o.), administered 60 min prior to decapitation, improved the reduced respiratory activity of mitochondria obtained from rat brain 5 min after decapitative ischemia. 3. KB-2796 (30 mg/kg, p.o.), administered 60 min prior to ischemic insult, improved both the reductions in pyruvate and ATP and prevented increases in the lactate/pyruvate ratio induced by 30-min forebrain ischemia in rats with 4-vessel occlusion (4-VO). 4. The effect of KB-2796 on local cerebral glucose utilization (LCGU) was examined by a quantitative autoradiographic 2-[14C]deoxyglucose method in normal and 4-VO rats. 5. Postischemic LCGU measured 24 hr after reperfusion in the forebrain, in particular in the cortex, thalamus, geniculate body, hippocampus, caudate-putamen, nucleus accumbens, colliculus, and corpus callosum, was below the normal control value. 6. KB-2796 (1 mg/kg, i.v.), administered 1 min prior to the injection of 2-[14C]deoxyglucose, improved the reductions in LCGU that were produced by cerebral ischemia in the cortex, thalamus, geniculate body, caudate-putamen, nucleus accumbens and substantia nigra, but did not affect LCGU in normal rats. 7. These findings suggest that KB-2796 minimized the deficits in brain energy metabolism produced by ischemia; this agent may therefore be a valuable therapeutic drug in cerebrovascular-related disorders.

Animals↗

PubMed1992

Vitamin K2 modulates proliferation and function of osteoblastic cells in vitro.

A human osteosarcoma cell line, HOS TE85 cells, and a mouse osteoblastic cell line, MC3T3-E1 cells, were cultured for 3 days in a medium containing various concentrations of menaquinone-4 (vitamin K2). As a result, the proliferation of HOS cells was suppressed by vitamin K2 in a dose dependent manner up to 56% of control by 10(-7)M of vitamin K2 and that of MC3T3-E1 cells was suppressed to 84% of control by 10(-6)M of vitamin K2. Vitamin K2 increased alkaline phosphatase activity in both kinds of cells. Warfarin counteracted the effect of vitamin K2 on osteoblastic cell proliferation. Our results show that vitamin K2 modulates proliferation and function of osteoblastic cells by some mechanisms including gamma-carboxylation system.

Animals↗

PubMed1992

[Hemorheological effect of KB-2796, a new Ca2+ antagonist].

The hemorheological effect of KB-2796, 1-[bis(4-fluorophenyl)methyl]-4-(2,3,4,-trimethoxybenzyl)piperazine dihydrochloride, was studied in guinea pigs and rabbits in comparison with those of flunarizine (FNZ) and pentoxifylline (PXF). KB-2796 and FNZ at 10-100 microM dose-dependently prevented crenation of rabbit erythrocytes induced by the Ca2+ ionophore A23187. After incubation of guinea pig whole blood at 37 degrees C, blood micropore-filterability decreased and blood viscosity increased with the progress of erythrocyte crenation. After a 4-hr incubation, KB-2796 inhibited erythrocyte crenation and decreased blood filterability at a concentration of 30 microM, and it increased blood viscosity at 10 microM. Treatment with FNZ (30 microM) and PXF (100 microM) also inhibited erythrocyte crenation and decreased blood filterability. Intravenous administration of KB-2796 at 3 mg/kg significantly inhibited the decrease of blood micropore-filterability after occlusion of the bilateral carotid arteries in rabbits. Although FNZ (3 mg/kg, i.v.) had no effect, PXF (3 mg/kg, i.v.) produced significant inhibition. These results suggest that KB-2796 prevents increase of blood viscosity and decrease of blood filterability by inhibiting the crenation of erythrocytes and suggest that this effect may be useful for the improvement of hemorheology in ischemic disease.

Animals↗

PubMed1992

Neurochemical investigation on the effects of a new diphenylpiperazine calcium antagonist, KB-2796, on the central dopaminergic system of rats.

The effects of KB-2796, a new diphenylpiperazine calcium antagonist, on the striatal dopaminergic system of rats were investigated in comparison with various calcium antagonists and the dopamine antagonist chlorpromazine. The inhibiting effect of KB-2796 on [3H]spiperone binding to striatal membranes in vitro was weaker than those of chlorpromazine and the other diphenylpiperazine analogues, flunarizine and cinnarizine, and more potent than those of verapamil and nicardipine. Diltiazem and nifedipine were inactive. KB-2796 (30, 100 mg/kg, p.o.) had no effect on Kd and Bmax values of in vitro [3H]spiperone specific binding to striatal membranes obtained from the rat at 36 hr and 7 days after repeated administration for 18 days, whereas flunarizine (30 mg/kg, p.o.) and chlorpromazine (3 mg/kg, p.o.) increased Bmax values by 47% and 31%, respectively, at 36 hr, but not at 7 days after the final administration. At 1 hr after the single administration, KB-2796 (30, 100 mg/kg, p.o.) had no effect on the content of dopamine and its metabolites in the striatum, whereas flunarizine (30 mg/kg, p.o.) and chlorpromazine (3 mg/kg, p.o.) increased the level of homovanillic acid. These results indicate that flunarizine may affect dopaminergic neurotransmission by partially blocking dopamine D2 receptors, while KB-2796 has negligible in vivo effect on the dopaminergic system.

Animals↗

PubMed1992

[Immunohistochemical study of collagen type III in adenomyosis].

To clarify the mechanism of the origin of adenomyosis, we investigated the immunohistochemical distribution of collagen type III in the human endometrium and the adenomyosis throughout the menstrual cycle. 1) Collagen type III was localized in the stroma of normal endometrium and ectopic endometrium, and in the myometrium. No staining of collagen type III was observed in the endometrial gland. 2) The intensity of staining for collagen type III in the basal layer of normal endometrium was stronger than those in the functional layer. The staining in the proliferative phase was stronger than that in the secretory phase. 3) The intensity of staining of collagen type III in ectopic endometrium paralleled that in the basal layer of normal endometrium. 4) The intensity of staining of collagen type III in the myometrium did not change throughout the menstrual cycle. These results suggest that close similarities exist between stromal cells in normal endometrium and in ectopic endometrium from the viewpoint of the extracellular matrix.

Collagen↗

PubMed1991

Behavioral and biochemical effects of a novel calcium antagonist, KB-2796, on the central dopaminergic system.

The effect of a novel calcium antagonist, KB-2796, on the central dopaminergic system was behaviorally and biochemically studied in mice and compared with that of flunarizine, nicardipine and chlorpromazine. Neither KB-2796 nor nicardipine had an inhibitory effect on apomorphine-induced cage climbing and turning behavior in mice with unilateral lesions in the striatum, even at a high dose of 100 mg/kg p.o. Both drugs slightly inhibited methamphetamine-induced increases in locomotor activity and turning behavior but only at a high dose (100 mg/kg p.o.). KB-2796 and nicardipine did not affect the content of dopamine (DA), norepinephrine, and serotonin, or the content of their metabolites homovanillic acid (HVA), dihydroxyphenylacetic acid (DOPAC), and 5-hydroxyindoleacetic acid, or the DA turnover rate in the forebrain of mice. However, flunarizine and chlorpromazine inhibited behavioral changes induced by apomorphine or methamphetamine in a dose-dependent manner, increased the content of DOPAC and HVA and accelerated the DA turnover rate in mouse brain. These results suggest that KB-2796 has a negligible effect on the central dopaminergic system.

Animals↗

PubMed1991

Induced dipole moment of purple membranes. Theoretical analysis.

The induced dipole moment of a purple membrane parallel to the membrane surface was analysed based on the two-dimensional fluctuation theory of counterions. The observed polarizability previously measured by electric dichroism and its dependence on the membrane size were derived from theory for the case where the interaction between counterions was screened. The screening effect of salts on the induced dipole moment was estimated from the theory on diffuse double layers.

Journal Article↗

PubMed1990

Effect of partial delipidation of purple membrane on the photodynamics of bacteriorhodopsin.

The effect of lipid-protein interaction on the photodynamics of bacteriorhodopsin (bR) was investigated by using partially delipidated purple membrane (pm). When pm was incubated with a mild detergent, Tween 20, the two major lipid components of pm, phospholipids and glycolipids, were released in different ways: the amount of phospholipids released was proportional to the logarithm of the incubation time; the release of glycolipids became noticeable after the release of approximately 2 phospholipids/bR, but soon leveled off at approximately 50% of the initial content. It was found that the thermal decay of the photocycle intermediate N560 was inhibited by the removal of less than 2 phospholipids per bR. This inhibition was partly explained by an increase in the local pH near the membrane surface. More significant changes in the bR photoreactions were observed when greater than 2 phospholipids/bR were removed: (1) the extent of light adaptation became much smaller, and this reduction correlated with the release of glycolipids; (2) N560 became difficult to detect; (3) the M412 intermediate, which is characterized by a pH-insensitive lifetime, was replaced by a long-lived M-like photoproduct with a pH-sensitive lifetime. The heavy delipidation apparently altered the mechanism by which the deprotonated Schiff base receives a proton. An important conformational change in the protein moiety is suggested to take place during the M412 state, this conformational change being inhibited in the rigid lipid environment.

Bacteriorhodopsins↗

PubMed1989

Independent flexible motion of submolecular domains of the Ca2+,Mg2+-ATPase of sarcoplasmic reticulum measured by time-resolved fluorescence depolarization of site-specifically attached probes.

The Ca2+-transporting ATPase of rabbit skeletal muscle sarcoplasmic reticulum was site-specifically labeled with either N-(1-anilinonaphth-4-yl)maleimide (ANM) or 5-[[(iodoacetamido)-ethyl]amino]naphthalene-1-sulfonate (IAEDANS), and the segmental motion of submolecular domains of the ATPase molecule was examined by means of time-resolved and steady-state fluorescence anisotropy measurements. The ANM-binding domain showed wobbling with a rotational relaxation time phi = 69 ns in the absence of free Ca2+ without any independent wobbling of the ANM moiety. The IAEDANS-binding domain showed a significantly slower wobbling with phi = 190 ns in the absence of Ca2+. The present results demonstrated for the first time that the ATPase molecule is composed of distinct domains whose mobilities are considerably different from each other. The binding of Ca2+ to the transport site increased the segmental motion of ANM-labeled domain, leading to a phi value of 65 ns. Solubilization of the ANM-labeled SR membranes by deoxycholate led to a further increase in the segmental flexibility (phi = 48 ns in the absence of free Ca2+), indicating that the mobility of the ANM-binding domain was considerably restricted through interaction with the membrane. The mobility of the ANM-binding domain of solubilized ATPase was also increased to some extent upon binding of Ca2+.

Adenosine Triphosphatases↗