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Biomedical subjects

M Takimoto

Publications and source records attributed to M Takimoto.

At least 55 records · Page 3Linked to original sources

Expression of GCF protein is dependent on the cell cycle.

GCF is reported to be a transcriptional regulator that binds to specific GC-rich sequences in the epidermal growth factor (EGF) receptor gene promotor and to represses its transcription. In this study, to examine the expression of GCF in cell cycle, we have developed antibodies against GCF protein. The anti-GCF antibody recognized a major band with a molecular weight of approximately 100 kda present in human Hela cell line by immunoprecipitation followed by Western blotting. Although GCF proteins were detected throughout the cell cycle, it reached the highest level in G1 phase, and fell down to the lowest level during G2/M phase. Thus, the expression of GCF is regulated in a cell cycle-dependent manner, suggesting a potential role of GCF in the control of cell growth.

Binding Sites↗

Re-examination of 5' end of cDNA of transcription factor GCF.

N-terminal region of transcriptional factor GCF was re-examined as the published amino acid sequence is extremely basic. 5'Rapid Amplification of cDNA End (5' RACE) analysis was performed and the result revealed that sequence of the first 308 bases of published GCF cDNA does not conform to the one obtained by 5' RACE. Sequence of genomic GCF DNA is consistent with the new cDNA sequence but not with the published one. Results of ordinal RT-PCR analyses are consistent with that of 5' RACE.

Base Sequence↗

Lack of endothelin ETB receptor binding and function in the rat with a mutant ETB receptor gene.

Congenital aganglionosis rat is a mutant with an autosomal recessive gene (sl). Recent studies have revealed that the endothelin ETB receptor gene of sl/sl rat has a deletion of 301-bp region spanning exon 1 and intron 1 corresponding to the first and the second transmembrane domains of the receptor. In the present experiments, we examined the functions of ETB receptors in the sl/sl rats. In the membranes of cerebellum, heart, and lung of control (+/+ and sl/+) rats, ET-1 induced a monophasic, competitive displacement of [125I]ET-1 binding, whereas ET-3, IRL 1620, and BQ-123 showed biphasic displacement. In the membranes of sl/sl rats, in contrast, ET-1, BQ-123, ET-3, and IRL 1620 showed only monophasic displacement. Scatchard analysis revealed a single [125I]ET-3 binding site in the membrane of control heart but not in the sl/sl rat heart, and the specific binding sites for [125I]ET-1 in both control and sl/sl rat hearts. In the control rat aorta but not in the sl/sl rat aorta, ET-3 induced endothelium-dependent relaxation. These results suggest that sl/sl rats do not have functional ETB receptors.

Animals↗

White blood cell count, C-reactive protein and erythrocyte sedimentation rate in respiratory syncytial virus infection of the lower respiratory tract.

Laboratory findings such as white blood cell (WBC) count, C-reactive protein (CRP) concentration and erythrocyte sedimentation rate (ESR) level in patients with bronchiolitis, bronchopneumonia and lobar pneumonia caused by respiratory syncytial virus (RSV) were studied. The diagnosis of having RSV infection of the lower respiratory tract was made on the presence of RSV antigen in nasopharyngeal specimens by means of enzyme immunoassay, on chest X-ray appearances and clinical manifestations. The WBC counts in the lobar pneumonia cases (n = 25, 12,288 +/- 6296/mm3) were significantly greater than those for the bronchiolitis (n = 52, 9562 +/- 2720/mm3) and bronchopneumonia (n = 43, 8369 +/- 3714/mm3) cases. The concentrations of CRP in lobar pneumonia cases (n = 25, 6.5 +/- 7.3 mg/dL) were significantly greater than those in the bronchiolitis (n = 52, 1.9 +/- 2.0 mg/dL) and bronchopneumonia (n = 43, 2.1 +/- 2.4 mg/dL) cases. The ESR levels in the lobar pneumonia cases (n = 24, 43.8 +/- 29. mm/h) were also significantly higher than those in the bronchiolitis (n = 34, 20.1 +/- 12.3 mm/h) and bronchopneumonia (n = 40, 24.7 +/- 15.9 mm/h) cases. There were no significant differences in the WBC counts, the CRP concentrations and ESR levels between the bronchiolitis and bronchopneumonia cases. These results suggest that the RSV lobar pneumonia cases are coinfected with some bacterial organisms more heavily than in the RSV bronchiolitis and bronchopneumonia cases.

Blood Sedimentation↗

ETA and ETB receptors cooperate in DNA synthesis via opposing regulations of cAMP in human lung cell line.

We investigated the contribution of endothelin type A (ETA) and ETB receptors on ET-induced DNA synthesis in CCD-18Lu cells, a human lung cell line possessing both ETA and ETB (ETA/ETB ratio: 9:1). ET-1 (0.05-2 nM) potently induced [3H]thymidine incorporation by 2- to 14-fold over the basal level. An ETA-selective antagonist, FR139317, inhibited 0.2 nM ET-1-induced DNA synthesis dose dependently, showing complete inhibition at 1 microM. ET-3 was inactive up to 2 nM. In contrast, ETB-selective antagonists, 100 nM of BQ-788 or IRL 2500, partially (30-60%) inhibited 0.2 nM ET-1-induced DNA synthesis. Stimulation of either ETA or ETB evoked the increases in intracellular Ca2+ concentration ([Ca2+]i). ETB-mediated but not ET-1-induced [Ca2+]i increase was pertussis toxin (PTX) sensitive. Adenosine 3',5'-cyclic monophosphate (cAMP) formation via ETA was observed in PTX-treated cells, whereas the inhibition of isoproterenol-stimulated cAMP formation via ETB was observed in PTX-untreated cells. Like the ETB-selective antagonists, PTX treatment or dibutyryl cAMP partially (50-70%) inhibited ET-1-induced DNA synthesis. These data suggest that 1) ET-1 induces DNA synthesis predominantly through ETA, via PTX-insensitive G protein; 2) ETA-mediated cAMP formation inhibits DNA synthesis; and 3) stimulation of ETB coupling to Gi protein modulates ETA-mediated DNA synthesis by inhibiting cAMP formation.

Cell Division↗

Endothelin-A receptor-mediated prostanoid secretion via autocrine and deoxyribonucleic acid synthesis via paracrine signaling in human bronchial epithelial cells.

It is known that serum promotes squamous differentiation of airway epithelial cell culture in vitro. We investigated which types of epithelial cells produce endothelins (ETs) and ET-evoked cellular responses in cultured human bronchial epithelial cells (HBECs). Squamous cells, not basal cells (nondifferentiated), secrete ET-1 actively. Specific binding of [125I]ET-1, not [125I]ET-3, was observed in squamous as well as basal cells, demonstrating the existence of only the ETA receptor. ET-1 together with epidermal growth factor stimulated synergistic DNA synthesis in basal cells, whereas ET-1 alone did not. Serum failed to induce DNA synthesis in HBECs, indicating terminal differentiation into squamous cells. ET-1 (1-100 nM) dose-dependently stimulated PGE2 (0.6-2.8 ng/10(5) cells) and thromboxane B2 release (4-30 pg/10(5) cells) from squamous HBECS, but not from basal cells. Western blot analysis showed that both squamous and basal HBECs expressed inducible and endothelial nitric oxide synthase, whereas ET-1 failed to stimulate nitric oxide synthase expression. Our findings suggest that secreted ET-1 from squamous cells evokes the release of prostanoids in an autocrine manner, and stimulates DNA synthesis in basal cells as a comitogen in a paracrine manner. Thus, it is likely that ET-1 secreted from HBECs plays an important role as a local, autocrine, and paracrine modulator in airway responses.

Azepines↗

[Modifications of fractional uptake method for 123I-IMP].

We intended to improve the fractional uptake (FU) method which was developed to quantify cerebral blood flow using 123I-IMP without blood sampling. The quantification of cardiac output (CO) using first-pass data was adapted to FU method while the original FU method used CO which was estimated from the body surface area of patients. Time-radioactivity curves of the lungs and brain were separately obtained by a small-field-of-view gamma camera. In 24 cases, mean cerebral blood flow (mCBF) obtained by the modified FU method showed the better correlation (r = 0.833, P< 0.001) to mCBF measured by the Patlak plot with 99mTc-HMPAO than the original FU method (r=0.667, p<0.01). With these modifications, the reliability of FU method could be improved and the modified FU method might be performed in the other institutions.

Adult↗

A maxi Cl- channel coupled to endothelin B receptors in the basolateral membrane of guinea-pig parietal cells.

1. To study endothelin (ET) receptors in guinea-pig stomach, ET-binding assays and in vitro autoradiography were performed on fundic cell suspensions and on sections of the fundus, respectively. ETA and ETB receptor subtypes were found to coexist in the parietal cells. 2. Endothelin 1 (ET-1) added to the (basolateral) bathing solution was found to activate noisy whole-cell Cl- currents within about 1 min in both single, isolated parietal cells and those within gastric glands obtained from the fundus. 3. ET-1-induced Cl- currents were rapidly blocked by a Cl- channel blocker (NPPB) added to the (basolateral) bathing solution in a concentration-dependent manner with a half-maximum inhibition concentration of 33 microM. 4. The anion selectivity sequence of the ET-1-induced conductance was I- > Br- > Cl- > F-, corresponding to Eisenman's sequence I. 5. Changes in extracellular pH between 5 and 8 did not affect the ET-1-induced activation of Cl- currents. 6. Similar activating effects were also observed with ET-3 and a specific ETB receptor agonist (IRL1620). An ETB receptor antagonist (IRL1720) prevented the ET-1 effect, whereas an ETA-selective antagonist (FR139317 or BQ123) failed to antagonize the ET-1 effect. 7. In the whole-cell mode, unitary Cl- channel events could be observed in association with ET-1-activated macroscopic currents. The single-channel conductances were around 200 and 350 pS at negative and positive membrane potentials, respectively. 8. It is concluded that gastric parietal cells of guinea-pig possess pH-insensitive 'maxi' Cl- channels coupled to ETB receptors in the basolateral membrane.

Animals↗

[Thrombocytopenic purpura associated with primary human herpesvirus-6 infection].

We report here a case of a 5-month-old infant with thrombocytopenic purpura associated with serologically confirmed primary human herpesvirus-6 (HHV-6) infection. His platelet count decreased to 17,000/mm3 on the 3rd day from the onset: Bone marrow aspiration showed that the megakaryocyte count was 129/mm3 and almost all of the them were of the non-platelet forming type. The IgG antibody to HHV-6 by indirect immunofluorescent assay in the sera in acute and convalescent phases were < 10x and 160x, respectively. IgM antibody to HHV-6 in the acute phase was positive. Platelet-associated IgG was also positive. We diagnosed this case as having thrombocytopenic purpura associated with primary HHV-6 infection. Steroid therapy was started for his thrombocytopenic purpura from the 4th day after the alleviation of his fever. His platelet count increased without side effects and became within the normal range without relapse despite discontinuation of steroid therapy. Thrombocytopenic purpura might be one of the complications of primary HHV-6 infection.

Antibodies, Viral↗

[Respiratory tract infection and respiratory syncytial virus in young infants].

We investigated the role of respiratory syncytial virus (RSV) in respiratory tract infections of up-to-3-months old infants. This prospective study was carried out from April 1993 to March 1994. Detection of RSV antigen in nasopharyngeal specimens was done by enzyme immunoassay using TESTPACK RSV at our out patient clinic. During this study period, 65 young infants with respiratory tract infection visited our clinic. Seventeen patients (26%) were diagnosed as having RSV infection. Fifteen out of the 17 infants with RSV infection were observed in spring and winter. Fourteen out of the 17 infants with RSV infection had acute bronchiolitis or pneumonia. In contrast to RSV infection, only three patients out of the 48 infants with non-RSV infection had lower respiratory tract infection. In conclusion, the majority of young infants infected with RSV suffered from bronchiolitis or pneumonia.

Antigens, Viral↗

[Comprehensive evaluation of pharmacokinetic and clinical studies on tazobactam/piperacillin in pediatric field].

To evaluate the pharmacokinetic and clinical effects of the newly developed combination antibiotic tazobactam/piperacillin (TAZ/PIPC, YP-14) on various infections in pediatric field, a study group was organized, and a joint research by 17 institutions and their related hospitals was conducted. Informed consents of subjects were obtained prior to the study. The results obtained in this study are as follows: 1. Blood concentration and urinary excretion Pharmacokinetics of TAZ/PIPC was studied in children at doses of 25 and 50 mg/kg through intravenous injection or intravenous drip infusion. With intravenous injection, maximum blood concentrations (Cmax's) of TAZ and PIPC were achieved 5 minutes after the administration. Cmax's of TAZ were 26.9 micrograms/ml with 25 mg/kg and 45.1 micrograms/ml with 50 mg/kg, and those of PIPC were 131.0 and 199.6 micrograms/ml, respectively. Values of the total area under the blood concentration curve (AUC's) of TAZ were 14.2 micrograms.hr/ml with 25 mg/kg and 26.1 micrograms.hr/ml with 50 mg/kg, and those of PIPC were 64.0 and 112.8 micrograms.hr/ml, respectively; thus dose dependency was observed with both TAZ and PIPC. The Cmax's of desethyl piperacillin (DEt-PIPC), the active metabolite of PIPC, achieved at 60 minutes after administration, were 1.2 and 2.0 micrograms/ml, respectively. The AUC's of DEt-PIPC were 2.6 and 4.2 micrograms.hr/ml, respectively. The half-lives (T 1/2's) of TAZ were 0.60 and 0.54 hour, respectively, and those of PIPC were 0.62 and 0.65 hour, respectively. In the first 6 hours after the initiation of administration, the cumulative recovery rates of TAZ in the urine were 46.7 and 56.0% respectively, those of PIPC were 46.1 and 57.2%, respectively, and those of DEt-PIPC were 5.9 and 3.0%, respectively. With intravenous drip infusion, the Cmax's of both TAZ and PIPC were achieved at the completion of drip; the Cmax's of TAZ were 12.1 micrograms/ml with 25 mg/kg and 28.9 micrograms/ml with 50 mg/kg, and those of PIPC were 54.6 and 137.9 micrograms/ml, respectively. The AUC's of TAZ were 11.6 micrograms.hr/ml with 25 mg/kg and 25.6 micrograms.hr/ml with 50 mg/kg, and those of PIPC were 49.0 and 117.2 micrograms.hr/ml, respectively; thus dose dependency was observed with both TAZ and PIPC. Cmax's of DEt-PIPC, achieved at 60 minutes after completion of drip, were 0.9 and 1.7 micrograms/ml, respectively. The AUC's of DEt-PIPC were 2.0 and 3.8 micrograms.hr/ml, respectively. The half-lives (T 1/2's) of TAZ were 0.59 and 0.62 hour, respectively, and those of PIPC were 0.58 and 0.57 hour, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Adolescent↗

Regulation of ion transport by endothelins in rat colonic mucosa: effects of an ETA antagonist (FR139317) and an ETB agonist (IRL1620).

Rat colonic mucosa contains ETA and ETB receptors with Kd values for endothelin (ET)-1 of 32 and 11 pM and maximal binding capacities of 277 and 181 fmol/mg protein, respectively. In muscle-stripped rat colon without tonic nerve activity in Ussing chambers, the serosal addition of ET-1, ET-3 and IRL1620 inhibited amiloride-sensitive noncoupled Na+ entry and enhanced diphenylamine-2-carboxylate-sensitive Cl- secretion, producing a sustained decrease and a transient increase in the short-circuit current (Isc) and the transepithelial conductance, respectively. EC50 values of ET-1, ET-3 and IRL1620 and the maximal changes in Isc were 2.0, 10.2 and 10.9 nM and -12.7, -7.0 and -7.1 muA/cm2, respectively for the Na+ entry; these values were 50, 220 and 225 nM and +57.3, +47.3 and +21.3 muA/cm2, respectively, for the Cl- secretion. FR139317 (100 nM) inhibited ET-1-induced Na+ and Cl- movements, shifting the concentration-response curves to the right (EC50 = 25 nM and 1 microM, respectively), and inhibited ET-3 (> 100 nM)-induced Cl- movement, decreasing the maximal response to 35%, but it did not inhibit either ET-3-induced Na+ movement nor IRL1620-induced Na+ and Cl- movements. The removal of serosal Ca++ reduced 100 nM ET-1- and IRL1620-evoked changes in Isc by 50% and 70% for the Na+ entry and by 80% and 100% for the Cl- secretion, respectively. Indomethacin (1 microM) also reduced changes in Isc by 30% and 70% for the Cl- secretion but did not affect the Na+ entry. Our results show that ETA and ETB receptors regulate Na+ and Cl- transport by different mechanisms.

Animals↗

[Strategies for the detection and the identification of tumor suppressor genes].

The methods for the detection and the identification of tumor suppressor genes are characterized and classified as follows: Firstly; by the comparison of expressed genes or proteins between tumor cells and normal cells. Secondly; by the gene transfer. Thirdly; by the identification of proteins which are involved in the biochemical process for cell growth suppression and DNA repair. Human genome contains dozens of tumor suppressor genes and tumor suppression-related genes which have not yet been cloned. Positional cloning is the most reliable method for the cloning of these genes. However, it cost huge amount of labor and time. New methods developed in recent years, such as differential mRNA display or two hybrids system, would facilitate the identification and the molecular cloning of novel tumor suppressor genes.

Cloning, Molecular↗

Identification of cellular proteins that bind the central conserved region of p53.

The bacterial fusion protein between glutathione S-transferase and the central conserved region of human p53(GST-p53) was purified and fixed on the beads and then used in the binding assay with radiolabeled cell extract from human hepatocarcinoma cell line, Hep3B. The binding assay disclosed the presence of cellular proteins that interact with GST-p53 but not with GST. SV40 large T antigen abrogated the bindings of two cellular proteins with molecular weights of 50 kda and 40 kda. The binding of the proteins to p53 was observed in a cell cycle-dependent manner. These two proteins are candidate cellular proteins which regulate the function of p53.

Amino Acid Sequence↗

The role of respiratory syncytial virus in acute bronchiolitis in small children in northern Japan.

Respiratory syncytial virus (RSV) plays an important role in acute bronchiolitis, which is life threatening in some infants. We investigated the epidemiology of RSV acute bronchiolitis in children less than 3 years old in northern Japan. From April 1991 to March 1993, 162 infants with acute bronchiolitis were hospitalized in our pediatric wards. The diagnosis of RSV acute bronchiolitis was based on the typical clinical manifestations and the presence of RSV antigen in their nasopharyngeal specimens or the rise of the RSV antibody titer. 124 out of 162 patients (76.5%) were diagnosed as having RSV acute bronchiolitis. 43.5% of patients with RSV acute bronchiolitis were 6 months old or less. The epidemic of RSV acute bronchiolitis commenced in October, peaked in December and ended in summer. RSV is quite prevalent in infants with acute bronchiolitis in northern Japan.

Acute Disease↗

[Epidemiological study on respiratory syncytial virus lower respiratory tract infections in northern Hokkaido, Japan].

We carried out an epidemiological study on lower respiratory tract infections, concerning the respiratory syncytial virus (RSV) infection, in northern Hokkaido, Japan, during the 2-year-study period from April 1991 to March 1993. There were 467 hospitalized patients with lower respiratory tract infections. We examined the presence of RSV antigen in their nasopharyngeal specimens of 456 out of 467 patients (97.6%) by enzyme immunoassay. The diagnosis of Mycoplasma pneumoniae infection was done by the rise of antibody titer, performed with complement fixation method, in the sera of the acute and convalescent phases. There were 133 patients with RSV infection (28.9%), 45 patients with Mycoplasma pneumoniae infection (9.8%) and 7 patients with measles virus infection (1.5%). RSV played an important role in the lower respiratory tract infections in northern Hokkaido, Japan. The epidemic of RSV infection was observed in spring and early winter in 1991 and only in early winter in 1992. The number of patients with RSV infection from April 1991 to March 1992 was greater than that from April 1992 to March 1993. In contrast with RSV infection, the epidemic of Mycoplasma pneumoniae infection was observed in 1992. We speculated that the RSV and Mycoplasma pneumoniae infections might interfere with each other.

Adolescent↗

ETA and ETB receptors on single smooth muscle cells cooperate in mediating guinea pig tracheal contraction.

We investigated the distribution of endothelin A (ETA) and ETB receptors in single smooth muscle cells and their contribution to ET-induced contractions of guinea pig trachea. ETA and ETB receptors were detected in smooth muscle membranes (maximum binding capacities of 810 and 360 fmol/mg protein and dissociation constants of 38 and 5.1 pM for 125I-labeled ET-1 and 125I-ET-3, respectively) and visualized autoradiographically in primary cultured cells. ET-1 and ET-3 evoked concentration-dependent increases in intracellular Ca2+ concentration and smooth muscle tension. The half-maximally effective concentrations of ET-1 and ET-3 at inducing contractions were 1.9 and 2.7 nM, respectively. The Ca2+ responses showed tachyphylaxis to both ETs after stimulation with ET-1, but only to ET-3 after stimulation with ET-3. Consecutive applications of ET-3 and ET-1 (10 nM each) classified the cells into ETA dominant (approximately 30%) responding to only ET-1, ETB dominant (approximately 20%) responding to only ET-3, and ETA- and ETB-possessing (approximately 50%) cells responding to both. The ETA antagonist, 10 microM BQ-123, attenuated ET-1-induced contractions but did not affect the ET-3-induced contractions. The results indicate that both receptors coexist in a major population of smooth muscle cells and cooperate in mediating ET-1-induced contractions.

Animals↗