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Biomedical subjects

S Takeda

Publications and source records attributed to S Takeda.

At least 397 records · Page 22Linked to original sources
PubMed1995

Cloning, sequencing and expression of a novel cDNA encoding human vacuolar ATPase (14-kDa subunit).

A cDNA encoding the 14-kDa subunit of vacuolar ATPase was cloned from human fetal brain. The sequence was composed of 680 nucleotides containing an open reading frame of 357 nucleotides. The deduced peptide sequence consisted of 119 amino acid residues with a calculated molecular weight of 13,369 Da and a pI of 5.19. Overall, this amino-acid sequence was respectively 69% and 70% identical to those of Manduca sexta and Drosophila melanogaster 14-kDa subunits, although the two representatives of Class Insecta were remarkably similar to one another (91% identity). Three regions in particular (the N-terminal, amino acids 5-36; the middle, residues 58-68; and the C-terminal, residues 88-118) were highly conserved. Hence, we think that the 14-kDa subunits have evolved from the same ancestral gene, and that the three conserved regions are important for the structure and function of vacuolar ATPase. A single 0.8-kb band was detected in various human tissues by Northern blot analysis. Since the human 14-kDa subunit is expressed ubiquitously, it might be a housekeeping protein. A separate transcript found in the cDNA library lacked a 6-bp segment in the 5' non-coding region (nucleotides -40 to -35) and also carried a 23C to T (8Thr to Ile) point mutation in the coding region; these minor differences likely reflected normal polymorphism.

Amino Acid Sequence↗
PubMed1995

Detection of K-ras mutations in DNAs isolated from feces of patients with colorectal tumors by mutant-allele-specific amplification (MASA).

K-ras mutations are found in approximately half of all colorectal tumors examined. To explore the possibility of detecting mutated K-ras rapidly and efficiently in DNAs isolated from fecal material, we applied the mutant allele specific amplification (MASA)-PCR method to DNA from feces of patients with colorectal tumors. Among 55 colorectal adenocarcinomas or adenomas examined, 19 were found to carry K-ras mutations in codons 12 or 13. We were able to PCR-amplify DNAs isolated from feces of 15 of these 19 patients, but in only three of the fecal samples, we were able to detect the K-ras mutations corresponding to tumor DNA by MASA and ethidiumbromide staining of the gel. The carcinomas in these three cases were more than 40 mm x 40 mm in size and located in the sigmoid colon or rectum. However, we identified the K-ras mutations in fecal DNAs of additional seven patients by MASA when the gels were blotted and probed with a radio-labeled oligonucleotide; the tumors in those patients had arisen in the distal half of the colon and the smallest of these tumors was only 7 mm x 5 mm. No K-ras mutations were detectable in feces of the remaining five cases, whose tumors were relatively small and/or located in the proximal region. The results suggested that the MASA-PCR system has potential for development as a simple, rapid and noninvasive method for diagnosing the presence of colorectal tumors that carry mutant K-ras alleles, particularly tumors located in the distal colon.

Adenocarcinoma↗
PubMed1995

Ligand-dependent G protein coupling function of amyloid transmembrane precursor.

Amyloid precursor protein (APP), a transmembrane precursor of beta-amyloid, possesses a function whereby it associates with G(o) through its cytoplasmic His657-Lys676. Here we demonstrate that APP has a receptor function. In phospholipid vesicles consisting of baculovirally made APP695 and brain trimeric G(o), 22C11, a monoclonal antibody against the extracellular domain of APP, increased GTP gamma S binding and the turnover number of GTPase of G(o) without affecting its intrinsic GTPase activity. This effect of 22C11 was specific among various antibodies and was observed neither in G(o) vesicles nor in APP695/Gi2 vesicles. In APP695/G(o) vesicles, synthetic APP66-81, the epitope of 22C11, competitively antagonized the action of 22C11. Monoclonal antibody against APP657-676, the G(o) binding domain of APP695, specifically blocked 22C11-dependent activation of G(o). Therefore, APP has a potential receptor function whereby it specifically activates G(o) in a ligand-dependent and ligand-specific manner.

Amyloid beta-Protein Precursor↗
PubMed1995

Control of crystal forms of apoferritin by site-directed mutagenesis.

Surface charges of protein molecules are not only important to biological functions but also crucial to the molecular assembly responsible for crystallization. Appropriate alteration in the surface charge distribution of a protein molecule induces new molecular alignment in the proper direction in the crystal and, hence, controls the crystal form. Apoferritin molecules are known to crystallize in two- and three-dimensional forms in the presence of cadmium ions, which bridge neighboring protein molecules. Here we report a controlled transformation of the apoferritin 2-D crystal by site-directed mutagenesis. In mutant apoferritin, two amino acid residues binding a cadmium-ion through their negative charge, were replaced by one type of nonionic amino acid residues. The amino acid residues, Asp-84 and Gln-86 in the sequence of recombinant (i.e., wild-type) horse L-apoferritin, were replaced by Ser. The wild-type apoferritin yielded a hexagonal lattice 2-D crystal in the presence of cadmium ions. In contrast, the mutant apoferritin yielded two types of oblique crystals independent of the presence of cadmium ions. Image reconstruction of electron micrographs of the mutant crystals made clear that the mutant apoferritin molecules oriented themselves with the 2-fold symmetry axis perpendicular to the crystal plane in both crystals, while the wild-type apoferritin molecules oriented themselves with the 3-fold symmetry axis perpendicular to the crystal plane. The changes of crystal forms and molecular orientation in the 2-D crystals were well explained by a change of the electrostatic interactions induced by the mutagenesis.

Animals↗
PubMed1995

Effects of methanol on styrene metabolism among workers occupationally exposed at low concentrations.

A survey was conducted in the second half of a work week on 39 male workers who were occupationally exposed to styrene in combination with methanol and methyl acetate during the production of plastic buttons. Time-weighted average exposure during an 8-h shift to styrene (Sty-A) and methyl acetate was monitored by carbon cloth-equipped personal samplers and to methanol by water-equipped ones. Urine samples were collected near the end of the shift and analyzed for mandelic (MA-U) and phenylglyoxylic acids (PhGA-U) by HPLC. Geometric mean styrene concentration was 12.4 ppm (micrograms/g) with the maximum of 46 ppm, whereas the values for methanol and methyl acetate in combination were 23.5 ppm and 229 ppm, respectively. The relationship of MA-U and PhGA-U with Sty-A was examined by linear regression analysis. The equations for the regression lines were compared with the results from a previous survey (Ikeda et al. 1983) in which workers were exposed only to styrene, and the methods employed were identical with that in the present study. The comparison showed no evidence to suggest that styrene metabolism is suppressed by coexposure to methanol and methyl acetate at low concentrations below the current occupational exposure limit of 200 ppm.

Acetates↗
PubMed1995

Increases in pulmonary artery pressure and cardiac output due to the inhibition of nitric oxide synthesis during operative stress.

Pulmonary artery pressure (PAP), cardiac output (CO), and urinary nitrate, a stable endproduct of nitric oxide (NO), were measured pre- and postoperatively in eight patients who underwent esophagectomy for squamous cell carcinoma of the thoracic esophagus. A significant elevation of PAP and CO on the day of operation (POD 0) was accompanied by a low concentration of urinary nitrate. A reduction in PAP and CO, and an increase in nitrate to the preoperative levels, were found on PODs 2 and 3, respectively, but urinary nitrate decreased again after POD 3. Consequently, the changes in PAP and CO were closely correlated with the nitrate concentration. These results suggest that operative stress inhibited NO synthesis with a transitory induction of endogenous NO synthesis postoperatively.

Aged↗
PubMed1995

A 10-year survivor with unresectable hepatic metastases from sigmoid colon carcinoma treated with regional chemotherapy.

We treated a man with unresectable hepatic metastases from sigmoid colon carcinoma who has since survived for more than 10 years. A sigmoidectomy with lymph node dissection was performed and a continuous hepatic arterial infusion of 5-fluorouracil (5-FU) with intermittent infusion of mitomycin C (MMC) was administered for about 3 months after this operation. The total doses of 5-FU and MMC were 16 g and 84 mg, respectively. Tegafur also was administered orally at a dose of 600 mg/day for about 8 months. The carcinoembryonic antigen (CEA) level (which had reached 4,409 ng/ml preoperatively) normalized 4 months after surgery, and still remains normal. Very few patients with unresectable hepatic metastases survive for 5 or more years. However, regional chemotherapy can be effective in some patients.

Adenocarcinoma↗
PubMed1995

Evaluation of noise-induced hearing loss by reference to the upper limit of hearing.

We have defined the upper limit of hearing as the maximum audible frequency measured with fixed intensity and changing frequency. We have previously established the standard upper limit ageing curves from the normal age variation in the upper limit of hearing. In the present study, we sought to clarify the effects of occupational noise on the upper limit of hearing. We measured the upper limit of hearing in 239 healthy male workers (478 ears) exposed to intensive occupational noise. Their age variation in the upper limit of hearing was compared with the standard upper limit ageing curves in males. There were statistically significant deteriorations. Even if the ears that had normal hearing levels (35 dB or less) were selected, deterioration in the upper limit of hearing was noticeable. The upper limit of hearing may serve as clinically useful information on the hearing impairment that precedes noticeable hearing impairment in conventional audiometry for workers exposed to intensive occupational noise.

Adolescent↗
PubMed1995

Expression of nm23-H1 gene product in thyroid, ovary, and breast cancers.

The nm23 gene product is one of several possible mediators of cancer invasion and metastasis. As the amounts of nm23-H1 mRNA and gene product are reduced in metastatic lymph nodes from patients with papillary carcinoma of the thyroid, we examined the expression of nm23 gene product in 115 thyroid cancers, 78 ovarian cancers, 63 breast cancers, and metastatic lymph node tissues by immunohistochemistry. It was found that nm23-H1, but not nm23-H2 gene product, was expressed in primary sites of thyroid, ovarian, and breast cancers, except for medullary and anaplastic carcinomas of the thyroid, but expressed only weakly or poorly in metastatic lymph nodes. Although nm23-H1 gene product expression was lower in anaplastic and medullary carcinomas of the thyroid, there was no significant difference in nm23-H1 gene product expression among histological types of ovarian and breast cancers. Our data indicate that the nm23-H1 gene product may play a role in metastasis in these hormone-producing organs and that other factors may be involved in metastasis of anaplastic and medullary carcinomas of the thyroid.

Breast Neoplasms↗
PubMed1995

Regulation of hepatic macrophage function by oral administration of xiao-chai-hu-tang (sho-saiko-to, TJ-9) in rats.

The effect of Xiao-Chai-Hu-Tang (Sho-saiko-to, TJ-9), the extract of a mixture of 7 herbs, on hepatic macrophage function was studied using rats. Hepatic macrophages were activated by injection of Corynebacterium parvum or 70% partial hepatectomy. Oral administration of TJ-9 for 3 weeks did not affect the ability of these macrophages to produce superoxide anions evaluated in situ by liver perfusion with nitro blue tetrazolium (NBT) and phorbol myristate acetate (PMA). However, the similar administration of TJ-9 attenuated the blocking of the activation after partial hepatectomy produced by pretreatment with gum arabic, a polysaccharide of high molecular weight. When gum arabic was added to the medium of rat hepatic macrophages cultured with normal rat sera, their ability to produce superoxide anions was reduced in a dose-related manner. This reduction was attenuated by changing the sera to the sera obtained from rats given oral doses of TJ-9 for 3 weeks. These results suggest that TJ-9 may improve the blocked function of hepatic macrophages in activation.

Administration, Oral↗
PubMed1995

Tubulin dynamics in neuronal axons of living zebrafish embryos.

The mechanism of cytoskeletal protein transport, especially the question of what kind of form the cytoskeletal proteins assume during transport in neurons in situ, has been an important, as yet unsettled issue. To clear up this matter, we adopted the embryonic zebrafish as a living animal model and applied the fluorescence recovery after photobleaching (FRAP) method. The zebrafish embryo is appropriate for this kind of study because of its transparency during the early developmental stage, allowing the observation of neurons that incorporate the microinjected fluorescent tubulin directly under fluorescence microscopy. FRAP revealed no movement of the bleached zone proximodistally, where fluorescence recovered gradually (recovery half-time, 44.2 +/- 11.2 min; n = 36), suggesting that the polymers are stationary but dynamic and that the true moving form could be small oligomers or heterodimers.

Animals↗
PubMed1995

Intrathymically expressed c-kit ligand (stem cell factor) is a major factor driving expansion of very immature thymocytes in vivo.

To investigate the role of the receptor-type tyrosine kinase, c-kit and its ligand, stem cell factor (SCF) in T cell development, we analyzed c-kit (W/W) and SCF (SI/SI) deficient mice. We also engrafted wild-type or SCF-deficient fetal thymi onto wild-type recipient mice and analyzed the rate of proliferation by in vivo bromodeoxyuridine labeling. The results show that the most immature thymocyte compartment defined as CD3-CD4-CD8- is significantly reduced in SI/SI grafts and W/W thymi compared with wild-type counterparts. Also, the expansion rate of these immature thymocytes in SI/SI graft is reduced by -50%. These experiments provide direct evidence for an important role for c-kit-SCF interactions in expansion of very early thymocytes.

Animals↗
PubMed1995

A mutation in the ceruloplasmin gene is associated with systemic hemosiderosis in humans.

We identified a mutation in the ceruloplasmin (Cp) gene in a Japanese family with aceruloplasminemia, some of whose members showed extrapyramidal disorders, cerebellar ataxia, and diabetes mellitus. A post-mortem study of the proband revealed excessive iron deposition mainly in the brain, liver and pancreas. The G to A transition at the splice acceptor site introduces a premature termination codon at the amino acid position 991 by defective splicing, thereby truncating the carboxyl terminus of Cp in affected individuals. We conclude that the mutation in the Cp gene is associated with systemic hemosiderosis in humans.

Alternative Splicing↗
PubMed1995

Synapsin I deficiency results in the structural change in the presynaptic terminals in the murine nervous system.

Synapsin I is one of the major synaptic vesicle-associated proteins. Previous experiments implicated its crucial role in synaptogenesis and transmitter release. To better define the role of synapsin I in vivo, we used gene targeting to disrupt the murine synapsin I gene. Mutant mice lacking synapsin I appeared to develop normally and did not have gross anatomical abnormalities. However, when we examined the presynaptic structure of the hippocampal CA3 field in detail, we found that the sizes of mossy fiber giant terminals were significantly smaller, the number of synaptic vesicles became reduced, and the presynaptic structures altered, although the mossy fiber long-term potentiation remained intact. These results suggest significant contribution of synapsin I to the formation and maintenance of the presynaptic structure.

Animals↗
PubMed1995

Two-phase approach for the expression of high-affinity human anti-human immunodeficiency virus immunoglobulin Fab domains in Escherichia coli.

We describe here a two-phase approach for the development of high-affinity human anti-HIV immunoglobulin Fab domains in a bacterial expression system. The first phase of this technique involves the generation of human hybridoma cell lines producing high-affinity antibodies (MAbs). Anti-HIV-1 human MAbs from peripheral blood lymphocytes (PBLs) were prepared from an HIV-1-seropositive patient and from an HIV-1-seronegative volunteer immunized with HIV-1 rgp160. One MAb (T15G1), derived from the blood of the seropositive donor, was specific for HIV-1 gp41, recognized gp41 on the surface of HIV-1-infected cells and bound this antigen with an apparent dissociation constant of 4 x 10(-10) M. A second MAb (M7B5), developed from the immunized volunteer, was specific for HIV-1 gp120 with a dissociation constant on the order of 8 x 10(-10) M, but was unable to recognize cell surface antigen. In the second phase of this technique the Fab domains of these two MAbs were molecularly cloned into a bacterial expression vector. mRNA was isolated from the M7B5 and T15G1 hybridoma cell lines and used as a template for the production of cDNA. The cDNA was amplified using the polymerase chain reaction (PCR) technique, and then fused, in frame, into a bacterial expression vector. The recombinant Fabs (rFabM7B5 and rFabT15G1) were expressed as dicistronic messages in bacteria using the IPTG-inducible lactose promoter (LacZ). DNA sequencing was used to define the gamma chain isotypes and the VH and VL chain gene usage. The binding specificities of rFabM7B5 and rFabT15G1 were indistinguishable from their respective intact MAbs.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗