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M Takimoto

Publications and source records attributed to M Takimoto.

At least 37 records · Page 2Linked to original sources
PubMed1999

Expression, secretion, and inhibition of angiotensin-converting enzyme in cultured human bronchial epithelial cells.

The purpose of this study was to determine whether angiotensin-converting enzyme is present in cultured human bronchial epithelial cells and which types of epithelial cells possess this enzyme. It is well known that serum promotes squamous differentiation of airway epithelial cell culture in vitro. We found that whole-cell homogenates of both basal (serum-untreated) and squamous-differentiated bronchial epithelial cells degraded hippuryl-L-histidyl-L-leucine, a synthetic substrate for angiotensin-converting enzyme. Analysis of RNA expression by reverse transcription-polymerase chain reaction (RT-PCR) showed the presence of mRNA for angiotensin-converting enzyme in both types of cells. In addition, we found that squamous cells secreted the enzyme into the culture medium more than basal cells did. Angiotensin-converting enzyme inhibitors (imidaprilat, enalaprilat) inhibited the enzyme activity in bronchial epithelial cells with an IC50 of 0.9-3.6 nM. Exogenously added bradykinin was degraded to bradykinin-(1-5), an inactive fragment, in the squamous cell cultures. Our data indicate the presence of angiotensin-converting enzyme in cultured human bronchial epithelial cells and also that the enzyme is secreted by squamous differentiated cells.

Angiotensin Receptor Antagonists↗
PubMed1999

Isolation of cDNAs that cover the entire coding region of a novel human protein D40.

Previously, we have reported a novel protein, D40, that specifically binds to a nuclear factor GCF by two-hybrid system. Northern analysis on tumor cell lines revealed that mRNA of D40 is about 5 kb. To get cDNA clones that cover the total coding region of D40 protein, approximately 5 x 10(5) clones of a human cDNA library were screened, and then several positive clones were obtained. RACE was performed to get the 5' end of the cDNA. DNA sequencing revealed an open reading frame that encodes a protein with 887 amino acids. in vitro transcription and translation analysis of D40 revealed that the molecular weight of the protein is 110 to approximately 130 kda.

Blotting, Northern↗
PubMed1999

Chromosomal assignment of a novel human gene D40.

We have previously reported an identification of a novel human cellular factor, D40. Here, we report the chromosomal localization of the gene that encodes D40. Fluorescent in situ hybridization (FISH) was performed to determine the chromosomal region that D40 gene resides. The chromosomes that derived from normal adult male lymphocytes were hybridized with a mixture of cDNA probes that cover the entire coding region of D40. D40 gene mapped to the long arm of chromosome 15q14-15.

Adult↗
PubMed1998

Suppression of Erk activation and in vivo growth in esophageal cancer cells by the dominant negative Ras mutant, N116Y.

Our previous studies demonstrated that introduction of a dominant negative H-ras mutant, N116Y, inhibits the growth of various types of cancer cells in vitro. In this study, we tested the efficacy of N116Y in blocking the growth of esophageal cancer cells using an adenoviral vector. Infection with N116Y adenovirus, (AdCMV-N116Y), in which N116Y expression is driven by the cytomegalovirus promoter, significantly reduced the in vitro growth of all esophageal cancer cell lines studied. Esophageal cancer cells that contained wild-type K-ras and H-ras (TE8, SGF3, SGF7) were more sensitive to AdCMV-N116Y than HEC46 cells that expressed mutant K-ras protein. Most importantly, direct injection of AdCMV-N116Y into TE8- or SGF3-induced tumors in nude mice suppressed their growth significantly. To examine the suppressive mechanism of N116Y, cell cycle profile and the activation of extracellular signal-regulated kinase 2 (Erk2) were examined by flow cytometry and Western blot analysis, respectively. In TE8 cells, progression into S phase was clearly blocked after infection with AdCMV-N116Y. Infection with AdCMV-N116Y did not strongly suppress the activation of Erk2 after EGF stimulation in serum-starved HEC46 cells, whereas it completely suppressed activation in TE8, SGF3 and SGF7 cells. Our observations suggest that N116Y reduces growth of human esophageal cancer cells and suppresses the activation of Erk2; they also indicate that N116Y is a potential candidate gene for human esophageal cancer gene therapy.

Adenoviridae↗
PubMed1998

Novel mutations of the endothelin B receptor gene in patients with Hirschsprung's disease and their characterization.

Hirschsprung's disease (HSCR) is a congenital intestinal disease, characterized by the absence of ganglion cells in the distal portion of the intestinal tract. Recently, three susceptibility genes have been identified in HSCR, namely the RET protooncogene, the endothelin B (ETB) receptor gene (EDNRB), and the endothelin-3 (ET-3) gene (EDN3). To investigate whether mutations in EDNRB could be related with HSCR in non-inbred populations in Japan, we examined alterations of the gene in 31 isolated patients. Three novel mutations were detected as follows: two transversions, A to T and C to A at nucleotides 311 (N104I) and 1170 (S390R), respectively, and a transition, T to C at nucleotide 325 (C109R). To analyze functions of these mutant receptors, they were expressed in Chinese hamster ovary cells. S390R mutation did not change the binding affinities but caused the decreases in the ligand-induced increment of intracellular calcium and in the inhibition of adenylyl cyclase activity, showing the impairment of the intracellular signaling. C109R receptors were proved to be localized near the nuclei as an unusual 44-kDa protein with the extremely low affinity to endothelin-1 (ET-1) and not to be translocated into the plasma membrane. On the other hand, N104I receptors showed almost the same binding affinities and functional properties as those of the wild type. Therefore, we conclude that S390R and C109R mutations could cause HSCR but that N104I mutation might be polymorphous.

Adenylyl Cyclase Inhibitors↗
PubMed1998

Motor discoordination and increased susceptibility to cerebellar injury in GLAST mutant mice.

To study the function of GLAST, a glutamate transporter highly expressed in the cerebellar Bergmann astrocytes, the mouse GLAST gene was inactivated. GLAST-deficient mice developed normally and could manage simple coordinated tasks, such as staying on a stationary or a slowly rotating rod, but failed more challenging task such as staying on a quickly rotating rod. Electrophysiological examination revealed that Purkinje cells in the mutant mice remained to be multiply innervated by climbing fibres even at the adult stage. We also found that oedema volumes in the mutant mice increased significantly after cerebellar injury. These results indicate that GLAST plays active roles both in the cerebellar climbing fibre synapse formation and in preventing excitotoxic cerebellar damage after acute brain injury.

ATP-Binding Cassette Transporters↗
PubMed1998

Endothelin-1 enhances nitric oxide-induced cell death in cultured vascular smooth-muscle cells.

Increased expression of endothelin-1 (ET-1) immunoreactivity is demonstrated in the active atherosclerotic plaque. Here we show that both ETA and ETB receptors are expressed in rat vascular smooth-muscle cells (VSMCs). ET-1 binding to ETB receptors enhances nitric oxide-induced cell death in VSMCs. These findings suggest that ET-1 may participate in the mechanism of cell death (apoptosis) in the plaque through activation of ETB-mediated pathways and that a selective ETB receptor antagonist could be useful in preventing acute plaque alterations, such as plaque rupture.

Amino Acid Sequence↗
PubMed1998

Both ET(A) and ET(B) receptors are involved in mitogen-activated protein kinase activation and DNA synthesis of astrocytes: study using ET(B) receptor-deficient rats (aganglionosis rats).

Endothelin (ET) is known to be a potent mitogen in astrocytes. However, the contribution and signalling pathway of ET(A) and/or ET(B) receptor to the proliferation of astrocytes remain unclear. We investigated ET-induced DNA synthesis in astrocytes using ET(B) receptor-deficient mutant rats (aganglionosis rats: sl/sl). Western blotting with anti-ET receptor subtype-specific antibodies and Scatchard analysis of binding revealed that ET(B) receptor expression in astrocytes depended on gene dosage (+/+: sl/+: sl/sl=2: 1:0), whereas ET(A) receptor expression was unchanged among the three genotypes. ET-1 (10 nM) stimulated [3H]thymidine incorporation and mitogen-activated protein kinase (MAP kinase) activity not only in +/+ via both ET(A) and ET(B) receptors, but also in sl/sl astrocytes via ET(A) receptor with about half the extent of those observed in +/+ astrocytes. Treatment with pertussis toxin (PTX) suppressed the ET-1-induced increases in the incorporation and MAP kinase activity in +/+, but not sl/sl astrocytes, indicating that the ET(B) receptor-, but not the ET(A) receptor-, mediated pathway to DNA synthesis involves PTX-sensitive G proteins, e.g. Gi and/or Go (Gi/o). In +/+ astrocytes, ET-1 (1 nM) stimulated cAMP accumulation, and the ET(B) receptor-selective agonist IRL 1620 (1 nM) suppressed 10 microM forskolin-induced cAMP accumulation, suggesting Gs coupling to the ET(A) receptor and Gi/o coupling to the ET(B) receptor. On the other hand, ET-1 did not increase cAMP accumulation in sl/sl astrocytes, although ET-1 (1 nM) suppressed the forskolin-induced response, suggesting Gi/o coupling to the ET(A) receptor. Our results suggest the possibility that the selectivity of G protein for ET(A) receptor is changed from Gs to Gi/o in ET(B) receptor-deficient astrocytes.

Amino Acid Sequence↗
PubMed1998

28-day repeated oral toxicity study of a hypolipidemic agent, NK-104 in rats.

NK-104, an inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase, was administered orally to Wistar rats at a dose of 2, 10, 50 or 100 mg/kg for 28 days consecutively, and the toxicity of NK-104 and its recovery with 2 weeks cessation of drug treatment were examined. As major clinical signs, loose stool, diarrhea, crouching and emaciation were observed in both sexes at 50 or 100 mg/kg, and all females at 100 mg/kg died or became moribund due to severe emaciation before the completion of treatment. The suppression of body weight gain or decrease in body weight was observed in the female dose group at 50 mg/kg and both sexes at 100 mg/kg. Decreased food intake was observed in both sexes at 100 mg/kg. Moreover, an increase in cholinesterase (Ch.E) in the male dose groups at 50 and 100 mg/kg and an increase in glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) in the female dose group at 50 mg/kg were observed in blood chemistry testing. Macroscopic examination showed thickening of the forestomach mucosa in the groups of 10 mg/kg or more. Microscopic examination revealed hyperkeratosis and hypertrophy of the spinous layer associated with both cell infiltration of the mucosal propria and submucosal edema. In addition, skeletal muscle lesions including atrophy, vacuolation and focal necrosis were observed in the female dose groups at 50 and 100 mg/kg. The above-mentioned microscopic changes were not observed on cessation of drug treatment. The non-toxic dose level of NK-104 in the 28-day repeated oral toxicity study using rats was determined to be 2 mg/kg.

Administration, Oral↗
PubMed1998

Six-month repeated oral toxicity study of NK-104 in rats.

NK-104 is a novel potent inhibitor of the rate-limiting enzyme in cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, and has been shown to be a highly effective agent in lowering blood cholesterol. In the present study, NK-104 was orally administered to Wistar rats at a dose of 0.3, 1, 3 or 010 mg/kg for 6 months for examination of toxicity. Additional recovery groups of 8 rats each of both sexes receiving 0 and 10 mg/kg were maintained without treatment for 1 month in order to assess recovery. As a result, no toxicological changes were observed in general signs, body weight, food intake, ophthalmological examination, urinalysis, hematological and blood chemical examinations for organ weights. An autopsy revealed thickening of the forestomach mucosa in both sexes at a dose of 1 mg/kg or more. This change was microscopically recognized as hyperkeratosis and hypertrophy of the spinous layer associated with both cell infiltration of the mucosal propria and edema of sub-mucosa in the forestomach in both sexes at doses of 3 and 10 mg/kg. Forestomach changes were not observed in any cases after 1 month cessation of drug treatment. The non-toxic dose of NK-104 in the 6-month repeated oral toxicity study in rats is estimated to be 1 mg/kg/day.

Administration, Oral↗
PubMed1997

Identification and characterization of a novel trans-membrane protein gene, pdh1, from Schizosaccharomyces pombe.

We have cloned a new gene, pdh1, from genomic DNA of fission yeast Schizosaccharomyces pombe. pdh1 is actively transcribed as 1400-nucleotide mRNA in vegetatively growing cells and can code for a 226 amino acid polypeptide (pdh1p). Computational structural prediction has revealed that the pdh1p is a highly hydrophobic protein with seven transmembrane domains. The prediction has also detected a possible C-kinase phosphorylation site within the longest hydrophilic loop.

Amino Acid Sequence↗
PubMed1997

Cocaine-induced liver injury in mice is mediated by nitric oxide and reactive oxygen species.

The modulating effects of nitric oxide (NO) and reactive oxygen species on cocaine-induced hepatotoxicity were examined by measuring plasma alanine aminotransferase activity and by carrying out histological studies. Liver injury was induced by a single injection of cocaine in adult male ICR mice. Pretreatment with aminoguanidine (an inhibitor of NO synthase), N-methyl-D-glucamine dithiocarbamate complex with iron ion (II) (Fe2+(MGD)2, a trapping reagent of NO) or deferoxamine complex with iron ion (III) (Fe3+-deferoxamine, a scavenger of NO) produced a marked inhibition of the hepatotoxicity induced by cocaine. In addition, pretreatment with allopurinol (an inhibitor of xanthine oxidase) and 1,3-dimethylthiourea (a scavenger of hydroxyl radical) also produced a potent inhibition. These findings suggest that a hydroxyl radical produced by the reaction of NO and superoxide anion (O2-) via peroxynitrite may be involved in the pathogenesis of cocaine hepatotoxicity.

Allopurinol↗
PubMed1997

Epilepsy and exacerbation of brain injury in mice lacking the glutamate transporter GLT-1.

Extracellular levels of the excitatory neurotransmitter glutamate in the nervous system are maintained by transporters that actively remove glutamate from the extracellular space. Homozygous mice deficient in GLT-1, a widely distributed astrocytic glutamate transporter, show lethal spontaneous seizures and increased susceptibility to acute cortical injury. These effects can be attributed to elevated levels of residual glutamate in the brains of these mice.

ATP-Binding Cassette Transporters↗
PubMed1997

Endothelin evokes efflux of glutamate in cultures of rat astrocytes.

Excessive release of glutamate, from glial cells as well as neurons, is thought to be a major cause of neuronal death in ischemia. To investigate glutamate release from glial cells, we measured glutamate efflux from cultures of rat astrocytes preloaded with L-[3H]glutamate. Glutamate efflux was induced by either 60 mM KCI or Na+-free medium, suggesting that the efflux is due to the reversed operation of a Na+- and K+-coupled glutamate uptake machinery. While investigating various neuropeptides and neurotransmitters, we found that endothelin (ET) specifically induced efflux of glutamate. Northern blot analysis and binding study showed that the ET type B receptor (ET(B)-R) subtype was expressed two to three times more densely than the ET type A receptor (ET(A)-R) in astrocytes. The ET(B)-R antagonist IRL 2500 partially inhibited efflux of glutamate induced by 1 nM ET-1 in a concentration-dependent manner, causing a maximal inhibition of 60% at 1 microM. However, the ET(A)-R antagonist BQ-123 did not cause significant inhibition even at 10 microM. Combination of both antagonists completely inhibited the ET-1-induced efflux. These results indicate that both receptor subtypes are involved in efflux of glutamate with a major contribution from the ET(B)-R. Our findings suggest that ET, which is known to be released in ischemia, may exacerbate neurodegeneration by stimulating efflux of glutamate.

Animals↗
PubMed1997

[Brain uptake ratio as an index of cerebral blood flow obtained with 99mTc-ECD].

A new index of cerebral blood flow (brain uptake ratio, BUR) using 99mTc-ECD was developed and evaluated in 66 patients (132 cerebral hemispheres). BUR was calculated from brain count in anterior planar image (60-80 sec after injection of 99mTc-ECD) divided by the summation of the count of aortic arch during first transit of radionuclide. BUR correlated well with brain perfusion index (BPI) obtained with Patlak plot method (r = 0.960, p < 0.001). In conclusion, BUR is useful as a simple and non-invasive index reflecting cerebral blood flow.

Adolescent↗
PubMed1997

[Extramedullary plasmacytoma with multiple metastasis following a maxillary plasmacytoma].

A 67-year old man noticed swelling of left maxillary in October 1992. Surgical biopsy of the left maxillary tumor revealed the plasmacytoma at the department of oral surgery. The Tumor disappeared after radiation therapy. He was admitted with dizziness and tarry stool in June 1994. We found left cervical and abdominal paraaortic lymph node swellings by the computer tomography. He died inspite of chemotherapy in December. Autopsy showed that plasma cells diffusely and nodularly invased in all alimentary tract with multiple ulcerations. Invasion was also observed in the liver, lungs, thyroid, heart, kidneys, and adrenals. The plasma cells in the liver showed IgG kappa type by tissue immunostain. Diffuse invasion of extramedullary plasmacytoma is very rare in alimentary tract and many other organs. This case may be value to delineate the nature of this disease.

Aged↗
PubMed1997

[A case of the old injured vena cava superior found during the operation of lung cancer].

We operated a 78-year-old male with lung cancer and unstable angina pectoris. During the operation of right upper lobe, a mass, with size of 2 cm in diameter was detected on the right postero-lateral side of the superior vena cava, and was resected. Postoperative pathological examination suggested that the mass resulted from an injury made by a Swan-Ganz catheter used before operation. Thus, as insertion of a Swan-Ganz catheter through the right subclavian vein may involve risk, so we concluded that the right internal jugular vein or femoral vein may be preferable as a route of first choice for insertion.

Aged↗