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Biomedical subjects

T Takeuchi

Publications and source records attributed to T Takeuchi.

At least 775 records · Page 43Linked to original sources
PubMed1994

Purification and characterization of a ubenimex (Bestatin)-sensitive aminopeptidase B-like enzyme from K562 human chronic myeloid leukemia cells.

A ubenimex-sensitive aminopeptidase B-like enzyme was purified from the non-membrane-bound fraction of K562 cells by a series of chromatographic procedures and slab-gel electrophoresis. The apparent molecular mass of the enzyme was estimated to be 73 kDa by SDS-PAGE. The aminopeptidase activity was activated by chloride ions and inhibited by Zn2+, Cu2+, Cd2+, and p-chloromercuribenzoic acid. Ubenimex was a potent inhibitor of this aminopeptidase in the nanomolar range. The sequence of the N-terminus of the protein was not determined. Partial amino acid sequencing revealed that the N-terminus of this aminopeptidase B-like enzyme was blocked by acylation. The partial sequences of the two fragments produced by CNBr cleavage and an acylamino acid-releasing reaction showed this enzyme to be a new aminopeptidase.

Amino Acid Sequence↗
PubMed1994

Multisample analysis using an array of microreactors for an alternating-current field-enhanced latex immunoassay.

To develop a rapid and multisample analysis system for latex immunoassay with submicroliters of sample, an array of microreactors were fabricated using micromachining techniques including photolithography, anisotropic etching, and thin gold film deposition. The chamber volume for immunoreactions of a single well was 0.4 microL. An alternating-current (ac) field was used to enhance the rate of the latex agglutination reaction. By applying an ac field for 1 min, alpha-fetoprotein in several samples could be determined simultaneously. The detection limit in this system was approximately 10 pg/mL.

Female↗
PubMed1994

Labeling of v-Src and BCR-ABL tyrosine kinases with [14C]herbimycin A and its use in the elucidation of the kinase inactivation mechanism.

The ansamycin antibiotic, herbimycin A, selectively inactivates cytoplasmic tyrosine kinases, most likely by binding irreversibly to the reactive SH group(s) of kinases. To further investigate the mechanism of herbimycin A action, we attempted to label tyrosine kinases with [14C]herbimycin A. p60v-src and p210BCR-ABL in immune complexes were labeled with [14C]herbimycin A, demonstrating that the antibiotic binds directly to tyrosine kinases. Digestion of [14C]herbimycin A-labeled p60v-src with Staphylococcus aureus V8 protease revealed that the herbimycin A binding site is within the C-terminal 26-kDa fragment of p60v-src, which contains the tyrosine kinase domain. Herbimycin A treatment inhibited labeling of p60v-src by [14C]fluorosulfonylbenzoyl adenosine, an affinity labeling reagent of nucleotide binding sites, indicating that herbimycin A-modified p60v-src cannot interact with ATP. The results suggest that herbimycin A inactivates tyrosine kinases by binding directly to the kinase domain, thereby inhibiting access to ATP.

3T3 Cells↗
PubMed1994

The novel mAb QR6.6 detects a surface molecule on immature thymocytes and inhibits their proliferation on thymic epithelial cells.

We previously reported that the nude mouse-derived splenic T cell clone N-9F exhibits a proliferative response when cultured on thymic stromal cells. This N-9F proliferation is mediated by direct cell-to-cell interactions between T and thymic stromal cells. A thymic epithelial cell clone, SL10.3, also supports N-9F growth. To identify the molecule involved in T cell development in the thymus, we established mAb specific to the N-9F clone. One of these mAb, QR6.6, was found to inhibit the N-9F proliferative response on SL10.3. QR6.6-positive cells were detected in thymus but not in other lymphoid organs such as bone marrow, lymph nodes, or spleen. QR6.6-positive cells accounted for 3 to 5% of the cells in adult thymuses whereas higher percentages were found in neonatal (10-20%) and fetal thymuses (70% at E17 and 10-20% at E15). The positive cells were primarily CD4-8- thymocytes in fetuses and CD4-8- to CD4+8+ thymocytes in adults. The QR6.6 mAb precipitates a 100 kDa molecule from the N-9F clone. The addition of the mAb to fetal thymus organ culture reduces the recovery of cells at culture day 4. It was also found that the mAb inhibits fetal thymocyte proliferation on the SL10.3 thymic epithelial cell line. These results suggest that the 100 kDa molecule detected by the QR6.6 mAb may play a crucial role in the early stage of thymocyte development.

Animals↗
PubMed1994

Production of bioactive gastrin from the non-endocrine cell lines CHO and COS-7.

We made a mutated progastrin cDNA construct that contains a cleavage site (-Arg(-4)-Arg(-3)-Lys(-2)-Arg-1) specific for the Kex2-like endoprotease furin, located ahead of the bioactive gastrin. For expressing the mutated progastrin cDNA, we used two non-endocrine cell lines, CHO and COS-7. CHO cells exhibit amidating enzyme activity and levels of amidation enzyme mRNA as high as those in the pituitary-derived endocrine cell line GH3, whereas COS-7 cells have far less amidating activity and lower amounts of mRNA. Mutant progastrin-expressing CHO cells produced mostly amidated gastrin. Gel filtration showed the size of this gastrin corresponded to that of the synthetic human gastrin-17. In contrast, COS-7 cells produced glycine-extended gastrin and only a small amount of amidated gastrin. The difference in the amount of amidated gastrin products produced by the two non-endocrine cell lines is due to differing amounts of the amidation enzyme contained in each cell line.

Amino Acid Sequence↗
PubMed1994

The mutant Eisai hyperbilirubinemic rat is resistant to bile acid-induced cholestasis and cytotoxicity.

We investigated bile flow and biliary excretion of bile acids in the Eisai hyperbilirubinemic rat, a Sprague-Dawley mutant rat with conjugated hyperbilirubinemia, using both in vivo and in vitro models. In vivo bile flow was lower in Eisai hyperbilirubinemic rats than in the control rats before and after taurocholate was infused. After taurocholate was infused, bile acid output was similar in the Eisai hyperbilirubinemic rats and control rats. In the isolated perfused rat liver, biliary excretion of bile acids was higher in the Eisai hyperbilirubinemic rats than in the control rats after a high-dose infusion of taurocholate (0.33 mumol/min/gm liver). Infusion of taurochenodeoxycholate (0.22 mumol/min/gm liver) did not produce cholestasis and did not reduce the biliary excretion of bile acids in the Eisai hyperbilirubinemic rats. Taurochenodeoxycholate significantly increased the phospholipid/bile acid molar ratio and slightly reduced bile acid-induced alkaline phosphatase output into bile. The release of lactate dehydrogenase from the perfused liver 30 min after the start of the taurochenodeoxycholate infusion was 10 times lower in the Eisai hyperbilirubinemic rats than in the control rats (2.0 +/- 0.8 vs. 28.7 +/- 6.8 mU/min/gm liver). When the isolated perfused rat liver was infused with a 1-min pulse of horseradish peroxidase (25 mg), we observed an early and late peak of biliary excretion of horseradish peroxidase. The Eisai hyperbilirubinemic rats showed a significant increase in the late peak.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗
PubMed1994

Tissue-specific expression of mouse tyrosinase gene in cultured chicken cells.

A mouse tyrosinase cDNA has been combined with different promoters and inserted into several replication-competent avian leukosis proviruses and the viruses were transferred into cultured albino chick cells by viral infection. Expression of the tyrosinase gene depended on one of four promoter sequences: the resident constitutive promoter (Rous sarcoma virus long-terminal repeat; RSV-LTR), 471 bp from the mouse tyrosinase gene-associated promoter, 519 bp from the Japanese quail tyrosinase gene associated promoter, or 369 bp from the quail tyrosinase promoter. The infected cells expressed tyrosinase and produced pigment which could be seen with the light microscope. Immunofluorescence microscopy, using an anti mouse tyrosinase T1-specific antibody, also showed the presence of mouse tyrosinase. When infected with the same viral titer, gene expression was highest with the constitutive LTR promoter. The quail tyrosinase promoter, while less efficient than the LTR, was more efficient than the other tyrosinase promoter. Fibroblasts and hepatocytes infected with the construct carrying the constitutive promoter or the truncated quail promoter expressed tyrosinase. The mouse and quail promoters appeared to show tissue-specific expression since fibroblasts and hepatocytes infected with viruses carrying these promoters did not express mouse tyrosinase. Toxicity is associated with constitutive expression of tyrosinase in nonmelanocytes. Therefore the viruses that carry the tissue specific promoters should be useful for in vivo studies.

Animals↗
PubMed1994

Toxoplasma gondii: secretion of a potent nucleoside triphosphate hydrolase into the parasitophorous vacuole.

Toxoplasma gondii is an obligate intracellular parasite capable of invading a wide range of host cells where it residues in a specially modified compartment termed the parasitophorous vacuole. This compartment provides a protected environment for the parasite which enters a rapid growth phase shortly after invasion. To identify functional components of this interface, we have used immunolabeling and cell fractionation to localize the potent nucleoside triphosphate hydrolase (NTPase) produced by Toxoplasma. In extracellular tachyzoites, NTPase was not exposed on the cell surface but was localized in small vesicles, called dense granules, scattered throughout the cell cytoplasm. Shortly following invasion. NTPase was secreted from dense granules and occupied the lumen of the parasitophorous vacuole where it was often associated with the intravacuolar network. NTPase was exclusively found in the supernatant in cell fractionation studies of both extracellular tachyzoites lysed by freeze-thaw and following secretion into the parasitophorous vacuole. These studies provide evidence for the rapid secretion of NTPase into the parasitophorous vacuole, where it may play a key role in processing of nucleotides for purine salvage by the parasite.

Acid Anhydride Hydrolases↗
PubMed1994

Intra-arterial chemotherapy using a reservoir for endocrine-refractory prostate cancer.

For local control in patients with endocrine-refractory prostate cancer, an intra-arterial chemotherapy regimen comprising methotrexate (MTX), Adriamycin (ADM), and cisplatin (CDDP) was evaluated. A total of 19 patients having a mean age of 66.4 +/- 8.8 years and a mean performance status (PS) of 1.3 +/- 1.0 were enrolled. Of these patients, 3 had proved to be resistant to initial endocrine therapy and the remaining 16 had relapsed from disease stabilization after endocrine therapy. The catheter tip was placed in the internal iliac artery in 16 cases, in the common iliac artery in 2 cases, and in the aorta in 1 case after occlusion of the contralateral feeding artery. The intraarterial chemotherapy was performed mainly using MTX (30 mg/m2), ADM (30 mg/m2), and CDDP (50 mg/m2) as one course and was repeated for a mean of 2.9 +/- 2.3 courses. Then, in an outpatient clinic, 5-fluorouracil (5-FU), ADM, or MTX was given intra-arterially as maintenance chemotherapy until re-relapse. As based on the criteria for evaluation of nonsurgical therapy in prostate cancer proposed by the Japanese Urological Association, the prostatic lesion showed a partial response (PR) in 9 cases and no change (NC) in 10 cases. As judged from the response of prostate-specific antigen (PSA), a complete response (CR) was obtained in 6 cases, a PR, in 3 cases; and NC and progressive disease (PD), in 2 cases each. Therefore, the overall response rate was 63%. Improvement in the symptoms was observed in 83% of patients. The duration of the response was 15.1 +/- 10.5 months for the PR cases and 7.4 +/- 5.7 months for the NC cases. Furthermore, the mean survival time observed in the PR group was 38.9 months, which was better than that seen in the NC (16.4 months) and PD (10.5 months) groups. These results suggest that intra-arterial chemotherapy may become an option for the treatment of locally advanced and endocrine-refractory prostate cancers. Using a reservoir, this chemotherapy can be easily given in an outpatient clinic.

Abdominal Muscles↗
PubMed1994

Expression of human tyrosine hydroxylase-chloramphenicol acetyltransferase (CAT) fusion gene in the brains of transgenic mice as examined by CAT immunocytochemistry.

We have produced transgenic (Tg) mice carrying 5.0-kb fragment from the 5'-flanking region of the human tyrosine hydroxylase (hTH) gene fused to a reporter gene, chloramphenicol acetyltransferase (CAT) [Sasaoka et al. (1992) Mol Brain Res 16: 274-286]. In the brain of the Tg mice, CAT expression has been observed in catecholaminergic (CAnergic) neurons and also in non-CAnergic neurons. The aim of the present study is to examine in detail the cell-type specific expression of the hTH-CAT fusion gene in the brain of the Tg mice, by use of immunohistochemistry for CAT, TH, and aromatic L-amino acid decarboxylase (AADC). CAT-immunoreactive cells were found in CAnergic brain regions which contained TH-positive cells, and also in non-CAnergic brain regions which contained no TH-labeled cells. The non-CAnergic brain regions that represented CAT-stained cells were further divided into two groups: (i) regions containing AADC-labeled cells, for example, bed nucleus of the stria terminalis, nucleus suprachiasmaticus, mammillary body, nucleus raphe dorsalis, inferior colliculus, and nucleus parabrachialis, and (ii) regions containing no AADC-positive cells, for example, main olfactory bulb (except A16), accessory olfactory bulb, nucleus olfactorius anterior, caudoputamen, septum, nucleus accumbens, hippocampus, medial nucleus of the amygdala, entorhinal cortex, nucleus supraopticus, and parasubiculum. The results indicate that the 5.0-kb DNA fragment flanking the 5' end of the hTH gene may contain the element(s) specific for neuron-specific TH expression but which may be insufficient to attenuate ectopic expression.

Animals↗
PubMed1994

Mechanism of transient mental nerve paraesthesia in sagittal split mandibular ramus osteotomy.

We investigated the mechanism involved in paraesthesia associated with sagittal split mandibular ramus osteotomy by three-dimensional computed tomography (3-D CT). Ten female patients underwent this procedure between 1988 and 1991. The inferior alveolar neuro-vascular bundles remained intact during the sagittal osteotomy in all cases. We examined the changes in the shape of the foramen mandibulae over a period of 6 months during which the transient mental nerve paraesthesia was recovered, and studied the distance from the foramen mandibulae to the spina mentalis (F-S distance) as measured on 3-D film. The postoperative 3-D CT scan showed bone resorption in front of the foramen mandibulae, and the F-S distance was shortened by an average of 2.94 mm. These findings suggest that possible causes of the paraesthesia is due to compression of the nerve trunk resulting from posterior movement of the mandibular ramus.

Adolescent↗
PubMed1994

Postnatal reference growth curves for very low birth weight infants.

To construct standard growth curves for Japanese infants of very low birth weight (VLBW) with birth weights of 500-1499 g, we reviewed longitudinal data provided by 54 neonatal intensive care units in Japan. A total of 382 surviving singleton infants, appropriate for gestational age infants, and who were free of neurological sequelae at more than 2.5 years of age, were enrolled. Growth curves, including body weight, head circumference and body length were generated for four ranges of birth weight: 500-749 g, 750-999 g, 1000-1249 g, and 1250-1499 g. When compared with previously published growth data from western countries, Japanese infants of VLBW showed greater weight loss, regained birth weight more slowly, and exhibited smaller average gains in weight, head circumference, and body length. The growth curves reported in western countries may not be useful as reference standards of early postnatal growth in Japan. The new growth curves are a more accurate reflection of current in-hospital growth trends in Japan.

Asian People↗
PubMed1994

Dysgenesis of melanocytes and cochlear dysfunction in mutant microphthalmia (mi) mice.

In order to evaluate the cytological homology of intermediate cells and melanocytes, and to investigate the function of melanocytes in the inner ear, hearing acuity and cochlear pathology were studied in three strains of mice, namely, wild type mice (+/+), albino mice without melanin (c2J/c2J), and microphthalmia mice with no melanocytes (mibw/mibw). Our histochemical data indicated that intermediate cells showed cytological characteristics almost identical to those of melanocytes and that disorders of melanin and/or melanocytes were reflected in the stria vascularis of each mouse. While c2J/c2J presented the same normal hearing acuity and normal structure of the stria vascularis as +/+, the hearing acuity of mibw/mibw mutants was severely impaired. Their stria vascularis was abnormally thin, lacking intermediate cells. According to these results, lack of melanin has little influence on hearing acuity; however, the absence of intermediate cells or melanocytes causes severe hearing loss, presumably due to a strial dysfunction.

Animals↗
PubMed1994

Crocidolite asbestos increased 8-hydroxydeoxyguanosine levels in cellular DNA of a human promyelocytic leukemia cell line, HL60.

Crocidolite, one of the most carcinogenic asbestos fibers, induces the release of reactive oxygen species (ROS) from neutrophils and macrophages. Using HPLC combined with electrochemical detection, we determined that 8-hydroxydeoxyguanosine (8OHdG), a molecule typical of mutagenic oxidative DNA damage, was induced in the cellular DNA of a human promyelocytic leukemia cell line, HL60, incubated with crocidolite. Crocidolite increased 8OHdG in the cellular DNA of phorbol myristate acetate (PMA)-differentiated HL60, which phagocytosed crocidolite. PMA-differentiated HL60 released ROS spontaneously, as determined by ESR with 5,5-dimethylpyrrolone-N-oxide as a spin trap. However, the release of ROS from the cell line did not increase after the addition of crocidolite. The addition of superoxide dismutase at a sufficient concentration to scavenge ROS released from the cell did not inhibit the 8OHdG increase induced by crocidolite. Cytochalasin B, which inhibited phagocytosis, did not inhibit the release of ROS. However, it inhibited the crocidolite-induced 8OHdG increase by 48.3%. Contrary to PMA-differentiated HL60, undifferentiated HL60 neither phagocytosed crocidolite nor showed a crocidolite-induced increase in 8OHdG formation. The 8OHdG increase induced by crocidolite was not correlated with ROS release, but with the internalization of crocidolite, suggesting that the increase was not due to an increase in ROS release from the cell but was due to the conversion of relatively inert ROS to highly reactive ROS, such as hydroxyl radicals, by crocidolite that was internalized and close to DNA.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1994

Evaluation of 8-hydroxydeoxyguanosine, a typical oxidative DNA damage, in human leukocytes.

8-Hydroxydeoxyguanosine (8-OHdG) is a typical form of oxidative DNA damage which causes mutation in vitro and in vivo. We investigated potential factors confounding 8-OHdG determination and, based on the results, then determined the 8-OHdG levels in human peripheral blood leukocytes. 8-OHdG was detected electrochemically after extraction of DNA from the cells without the use of phenol by a DNA extractor under helium. In the preliminary experiments, the mononuclear leukocytes (MN) in blood samples obtained from 19 laboratory workers and students were separated from the polymorphonuclear leukocytes (PMN) with Mono-Poly resolving medium. The 8-OHdG in the MN (1.157 +/- 0.414 molecules per 10(5) deoxyguanosine) did not differ significantly from that in PMN (1.131 +/- 0.418). The effect of red blood cells (RBC) on 8-OHdG formation during DNA extraction was then examined by adding RBC to the human lymphoblastoid cell line FA72. Addition of RBC at ratios of up to 4 RBC per FA72 cell did not increase 8-OHdG levels, while addition at a RBC/FA72 cell ratio of 20 increased the 8-OHdG level 1.43-fold over that without RBC. The potential effect of histidine, a scavenger of both hydroxyl radicals and singlet oxygen, on reduction of artificial 8-OHdG formation during DNA extraction was examined during DNA extraction in the human promyelocytic leukemia cell line HL60. Addition of His decreased the 8-OHdG level dose-dependently (30% reduction at 30 mM His concentration). Based on these results, we determined the 8-OHdG levels in human leukocyte samples obtained from 79 healthy male factory workers aged 24-59 years. The leukocyte fraction containing both MN and PMN was separated from RBC with Mono-Poly resolving medium and DNA was extracted from the leukocytes in the presence of 30 mM His. The mean 8-OHdG level in these samples was 1.072 +/- 0.230. To evaluate the reliability of the assay, FA72 was used as a standard sample in all assay determinations and the 8-OHdG levels of both the leukocyte samples and the FA72 sample(s) were measured in each determination. The inter- and intra-assay coefficients of variation (CV) were calculated to be 14.4% (n = 14) and 3.9-13.5% (n = 3-5 per assay) respectively. The 8-OHdG level was measured twice in 19 leukocyte samples; the value at the first determination was not correlated with that at the second determination. The range of 8-OHdG levels in the samples was relatively small compared with the CV of the assay.(ABSTRACT TRUNCATED AT 400 WORDS)

8-Hydroxy-2'-Deoxyguanosine↗