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Biomedical subjects

Y Hashimoto

Publications and source records attributed to Y Hashimoto.

At least 523 records · Page 29Linked to original sources
PubMed1997

Subunit composition of the pre-T-cell receptor complex analysed by monoclonal antibody against the pre-T-cell receptor alpha chain.

The pre-T-cell receptor (TCR) complex, which consists of a heterodimer of the TCR beta-chain and the pre-TCR alpha-chain, is known to regulate early thymocyte development. The pre-TCR complex contains CD3 subunits as a signal-transducing molecule, but the exact subunit composition of the fully assembled pre-TCR complex remains to be elucidated. In particular, the association of the CD3 zeta-chain with the pre-TCR is controversial. In the present study, we have generated a monoclonal antibody against the cytoplasmic portion of the pre-TCR alpha-chain, and analysed a subunit composition of the pre-TCR complex. We demonstrated that the CD3 zeta-chain is physically associated with the pre-TCR in immature T cells. Thus, the result strongly supports the previous findings that CD3 zeta contributes to signalling mediated through the pre-TCR complex.

Animals↗
PubMed1997

Growth inhibition of Helicobacter pylori by a polyamine synthesis inhibitor, methylglyoxal bis(cyclopentylamidinohydrazone).

The anti-proliferative effect of methylglyoxal bis(cyclopentylamidino-hydrazone) (MGBCP), a multi-enzyme inhibitor of polyamine biosynthesis, on the growth of Helicobacter pylori was investigated. MGBCP inhibited the cell growth of H. pylori in a dose-dependent manner. The inhibition was partially reversed by the addition of spermidine. Synthesis of macromolecules, DNA, RNA and protein, was inhibited in the spermidine-depleted H. pylori cells. These findings suggest that MGBCP exhibits an anti-proliferative effect on H. pylori by suppression of macromolecule synthesis.

Anti-Bacterial Agents↗
PubMed1997

Influence of flavonoids on cell cycle phase as analyzed by flow-cytometry.

Flavonoids were previously reported to have cytostatic activity in vitro and in vivo. In the present study each phase of the cell cycle was observed by flow-cytometry after HCT-15 cells had been cultured with flavonoids for 2 days. In the control group, two peaks were observed on the histogram, the first peak representing double strands of DNA, and the second, tetra strands. Chalcone, which is known to be a precursor of all flavonoids, seemed to block the passage from S to G2 and M phase. Flavonone, which is freely transformed into chalcone in vivo, showed a similar histogram as chalcone. Among the anthocyanins most effective in suppressing the tumor cell growth, cyanin and pelargonidin made a mild rise between the 1st and 2nd peak suggesting block between G1 and S phase. Delphinidin gave a high percent of cells in G1 phase, suggesting prolongation of G1 phase. When quercetin or kaempherol, both of which are flavonols were added to the culture, the second peak rose significantly, indicating a block between G2 and M phases. Thus, the site of these cytostatic agents seems to differ depending on the kind of flavonoid.

Cell Cycle↗
PubMed1997

Effect of allomelanin on tumor growth suppression in vivo and on the cell cycle phase.

Allomelanins are nitrogen-free macromolecular polymers of simple phenols produced by higher plants and fungi. In an earlier work we found that allomelanins were effective in suppressing the growth of cultured tumor cells. In the present study we examined the effect of these polymers on the survival curve of Balb/C mice inoculated i.p. with Meth/A cells (3 x 10(4) cells/mouse), which originated from a malignant lymphoma. Allomelanins were extracted from black sesame seeds and black soybeans, and given p.o. via the drinking water. The dose of allomelanins was approximately 3 mg/mouse/day. The percent of survivals was significantly higher in the experimental groups than in the control. Also, when HCT-15 cells were cultured for 2 days in medium containing 400 micrograms/ml of protein-free allomelanins, histograms obtained from flow-cytometry data showed a significant increase in the fraction between the diploid and tetraploid peaks, indicating blockage of the S phase of the cell cycle. These results suggest that allomelanins suppressed the tumor cell growth in vitro and in vivo, and that the effect was cytostatic, mostly by blockage of the S phase.

Animals↗
PubMed1997

Antitumor effect of anthocyanin fractions extracted from red soybeans and red beans in vitro and in vivo.

Many bioflavonoids extracted from petals of higher plants and from fruit rinds, as well as purified flavonoids, have been reported to have antitumor effects in vitro and in vivo. Bioflavonoids extracted from red soybeans are mostly cyanin conjugated with glucose and rhamnose, whereas bioflavonoids of red beans are cyanin conjugated with rhamnose as revealed by thin-layer chromatogram. Flavonoids extracted from red soybeans were effective in inhibiting the growth of HCT-15 cells in vitro. On the other hand, flavonoids from red beans were not effective, although their hydrolyzed sugar-free forms were growth inhibitory. Sugar-bonded bioflavonoids extracted from both red soybeans and red beans were effective in prolonging the survival of Balb/C mice bearing syngeneic tumor-Meth/A cells, when they were dissolved in drinking water and given at a dose of approximately 500 micrograms/mouse/day.

Animals↗
PubMed1997

Direct tumor growth suppressive effect of melanoidin extracted from immunomodulator-PSK.

Melanoidin, which belongs to the melanin group of molecules, was extracted from the polysaccharide biological response modifier PSK. Melanoidin was cultured together with HCT-15 cells derived from human colon cancer and with AGS cells derived from human gastric carcinoma. After four days of culture, cell count was compared with that of the control cells. Significant suppression was observed, that is, 50% suppression was shown at concentrations of melanoidin between 200 and 100 micrograms/ml. A histogram generated by flow cytometry showed elevation of the tetraploid peak and of that between diploid and tetraploid peaks, suggesting blockage of S phase and G2 to M phase of the cell cycle. Thus, melanoidins contained in the immunomodulator PSK revealed to have a direct tumor cell growth inhibitory effect.

Adjuvants, Immunologic↗
PubMed1997

Tumor cell growth-inhibiting effect of melanoidins extracted from miso and soy sauce

Melanoidins, a group of plant-produced melanins, were extracted from miso and shoyu (soy sauce), which are products of soybeans and widely consumed as food in oriental countries. The molecular weight of these melanoidins was approximately 5600 as revealed by Sepharose CL-4B column chromatography. The melanoidins were cultured together with HCT-15 cells derived from human colon carcinoma and AGS cells derived from human gastric carcinoma. Significant cell growth suppression was observed by incubating the cells with these melanoidins. The 50% growth suppression dose ranged between 100 micrograms/ml and 25 micrograms/ml, that is, between 2 x 10(-5) mM/ml and 0.5 x 10(-5) mM/ml. Flow cytometric analysis suggested that these melanoidins blocked the S phase and G2 to M phase of the cell cycle.

Journal Article↗
PubMed1997

Tumor cell growth suppression by chalcone (1,3-diphenyl-2-propen-1-one).

Chalcone or 1,3-diphenyl-2-propen-1-one which is widely distributed in higher plants and is considered to be a precursor of all flavonoids, was studies in terms of its anti-tumor growth activity in vitro and in vivo. Mice given the chalcone, which was suspended in the drinking water, showed a statistically significant rise in percent survival: 66% at the 35th day for the chalcone-treated animals vs. 30% for the control ones. The chalcone also gave significant suppression of growth of HCT-15 and Meth/A cells in vitro. The dose of 50% reduction in growth was between 2.5 micrograms/ml and 0.6 microgram/ml for the HCT-15 cells and less than 10 micrograms/ml for the Meth/A cells. Histograms obtained by flow cytometric examination showed an elevated region between the diploid and the tetra-ploid nucleoidal peaks. A third peak heavier than the tetra-ploid peak was also observed, which peak was not observed in the control. The percentage of cells in the S phase was significantly raised. Microscopically, large nucleated cells were observed following addition of the chalcone. These results suggest that the chalcone induced abnormal DNA synthesis and mitosis in the cultured cells.

Animals↗
PubMed1997

A simple improvement in expression cloning.

Expression cloning is an effective approach for isolating genes encoding proteins that associate with a target species. Several molecules have been isolated by expression cloning, including CRE-BP1 associating with Jun (Macgregor et al., 1990); Grb1, identical to p85 PI3-kinase, with the EGF receptor (Skolnik et al., 1991); and Max with Myc (Blackwood and Eisenman, 1991). Expression cloning involves induction of proteins from a lambda gt11 cDNA expression library and screening the proteins on nitrocellulose membranes using a peptide probe (Macgregor et al., 1990). With this method, we previously isolated an Lck tyrosine kinase-associated protein, LckBP1, which is identical to HS1 (Kitamura et al., 1989, 1995; Takemoto et al., 1995). In those experiments, we used a glutathione S-transferase (GST)-Lck SH3 domain fusion protein as a probe, followed by detection of the complex with anti-GST polyclonal antibody. Whereas the ease of obtaining the fusion construct and high-titer anti-GST polyclonal antibody represented clear advantages, the system suffered from high background and low sensitivity. Here we show that pretreatment of nitrocellulose filters with NaDodSO4 reduces background and, in turn, increases sensitivity.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Expression plasmid vectors with convenient subcloning sites in lambda gt11 that efficiently produce detectable tagged proteins.

We have generated cDNA expression vectors that efficiently produce tagged proteins. The newly introduced cloning site of this plasmid facilitates subcloning of cDNA in the lambda gt11 phage into the plasmid vector. Because the cDNA is inserted next to the motifs of the tagged DNA sequence, the protein produced by the tag sequence-coupled cDNA is easily detected by Western blot analysis or immunoprecipitation using commercially available antibodies. The double-tagged protein significantly enhances the efficiency of Western blot and immunoprecipitation detection as compared with the single-tagged protein.

3T3 Cells↗
PubMed1997

In vivo assessment of droperidol-induced bronchial relaxation in dogs using a superfine fibreoptic bronchoscope.

Droperidol has been reported to cause bronchodilatation but its mechanism(s) of action is unknown. We have evaluated the spasmolytic effect of droperidol on histamine- and serotonin (5-HT)-induced bronchoconstriction in dogs. Bronchial cross-sectional area was assessed with a superfine fibreoptic bronchoscope. Twenty-eight mongrel dogs were allocated randomly to one of two groups (histamine and 5-HT) to receive either histamine or 5-HT to induce bronchoconstriction. Changes in bronchial cross-sectional area were presented as percentage of basal bronchial cross-sectional area. Continuous i.v. infusion of histamine 500 micrograms kg-1 h-1 or 5-HT 500 micrograms kg-1 h-1 decreased percentage bronchial cross-sectional area by 46.4 (14.3)% or 68.9 (13.7)%, respectively. In both groups, droperidol reversed bronchoconstriction in a dose-dependent manner. In the histamine but not in the 5-HT group, plasma adrenaline and noradrenaline concentrations increased significantly after i.v. droperidol. In addition, propranolol antagonized droperidol-induced relaxation in the histamine but not in the 5-HT group. Our data indicate that the spasmolytic effect of droperidol on canine airway was caused, at least in part, by both catecholamine releases and 5-HT receptor antagonism.

Animals↗
PubMed1997

Measurement of bronchodilatation using a superfine fibreoptic bronchoscope.

In this study, we report the development and accuracy of a direct technique to measure airway calibre using a superfine fibreoptic bronchoscope. Ten mongrel dogs were anaesthetized with pentobarbitone and the trachea intubated with a tracheal tube; the small lumen of the tube allowed passage of a superfine fibreoptic bronchoscope (od 2.2 mm). Bronchial cross-sectional area and airway pressure were recorded continuously and dynamic pulmonary compliance and airway resistance calculated. The dogs were allocated to one of two groups. In the first group (six dogs), bronchoconstriction was induced with histamine 10 micrograms kg-1 i.v. and 500 micrograms kg-1 h-1 c.i.v. Thirty minutes later, adrenaline 0-0.4 mg kg-1 was given i.v. Bronchial cross-sectional area, dynamic pulmonary compliance and airway resistance were assessed simultaneously. In the second group, 0.9% saline was given 30 min after placement of the superfine fibreoptic bronchoscope and 10 min later atropine 0.1 microgram kg-1 was administered. In the first group, histamine decreased mean percentage bronchial cross-sectional area by 49.2 (SD 11.5) %, reduced dynamic pulmonary compliance from 32.1 (12.6) to 22.3 (5.2) ml cm H2O-1 and increased airway resistance from 39.1 (11.6) to 57.2 (10.2) cm H2O litre-1 s-1. Adrenaline produced a dose-dependent increase in percentage bronchial cross-sectional area and dynamic pulmonary compliance to 119.4 (31.3)% and 27.4 (5.5) ml cm H2O-1, respectively, and a decrease in airway resistance to 43.9 (7.2) cm H2O litre-1 s-1. There were significant correlations between percentage bronchial cross-sectional area and dynamic pulmonary compliance (r = 0.720, P < 0.0001) and airway resistance (r = 0.727, P < 0.0001). Atropine 0.1 mg kg-1 increased basal bronchial cross-sectional area to 137.5 (16.9) %. These data indicate that adrenaline reversed histamine- and pentobarbitone-induced bronchoconstriction.

Airway Resistance↗
PubMed1997

Analysis of the stabilization of hen lysozyme by helix macrodipole and charged side chain interaction.

In the N-terminal region of the alpha-helix of the c-type lysozymes, two Asx residues exist at the 18th and 27th positions. Hen lysozyme has Asp18/Asn27 (18D/27N), and we prepared three mutant lysozymes, Asn18/Asn27 (18N/27N), Asn18/Asp27 (18N/27D), and Asp18/Asp27 (18D/27D). The stability of the wild-type (18D/27N) lysozyme supported the existence of a hydrogen bond between the side chain of Asp18 and the amide group at the N1 position in the alpha-helix, while the stability of the 18N/27D lysozyme supported the presence of the capping box between the Ser24 (N-cap) and Asp27 residues. Although electrostatic repulsion was observed between Asp18 and Asp27 residues in 18D/27D lysozyme, the dissociation of each residue contributed to stabilizing the B-helix in 18D/27D lysozyme through hydrogen bonding and charge-helix macrodipole interaction. This is the first evidence that two neighboring negative charges at the N-terminus of the helix both increased the stability of the protein.

Amino Acid Sequence↗
PubMed1997

Influence of mutations of the N-cap residue, Gly4, on stability and structure of hen lysozyme.

Hen lysozyme, with three alpha-helices (A, B, and C), is a c-type lysozyme. In these lysozymes, Ser24 and Asp88 located at the N-cap position in the B- and C-helix, respectively, are mostly conserved, but residue 4 at the N-cap position in A-helix is variable. To investigate the effect of mutation at position 4 on the stability of hen lysozyme, we prepared five mutant lysozymes and examined their stabilities and structures. Gly4Pro lysozyme (G4P), in which Gly4 was replaced by Pro, was less stable by 8.8 kJ/mol than the wild-type lysozyme, possibly because the side chain at position 7 is shifted away from the A-helix. The other mutant lysozymes were of almost equal stability to the wild-type lysozyme, although the hydrogen bonds of the amide groups at positions N1-N3 in the A-helix were absent or altered. These results indicated that various mutations at the N-cap position in the A-helix would be allowed as long as the negative charge of Glu7 at the N-terminus stabilized the A-helix.

Amino Acid Sequence↗
PubMed1997

Improvement of the refolding yield and solubility of hen egg-white lysozyme by altering the Met residue attached to its N-terminus to Ser.

When hen egg-white lysozyme was produced in Escherichia coli, it possessed an extra methionine residue at the N-terminus (Met(-1)-lysozyme). The Met(-1)-lysozyme showed a decreased refolding yield and solubility compared with the native hen egg-white lysozyme, as the methionine is a hydrophobic amino acid. A Met(-2)Pro(-1) or Met(-2)Ser(-1) sequence was introduced at the N-terminus of hen egg-white lysozyme. The methionine residue in these hen egg-white lysozymes was completely removed by methionine aminopeptidase, as expected, since the penultimate residue was proline or serine. From the analyses of solubility, stability and refolding yield, it was found that an extra Ser residue attached to the N-terminus of hen egg-white lysozyme (Ser(-1)-lysozyme) showed closer characteristics to the native hen egg-white lysozyme than did Met(-1) or an extra Pro residue attached to the N-terminus of hen egg-white lysozyme (Pro(-1)-lysozyme). Moreover, the tertiary conformation of Ser(-1)-lysozyme examined by NMR spectroscopy and its activity were almost identical with those of native hen egg-white lysozyme.

Animals↗
PubMed1997

The pharmacologic profile of 606A, a novel angiotensin II receptor antagonist.

The pharmacologic profile of a novel angiotensin I (AT1) receptor antagonist 606A was studied in various in vitro and in vivo preparations. The 606A showed a high affinity at AT1 receptors [inhibition constant (Ki), 12.8 +/- 0.4 nM] in rabbit adrenal cortical membrane and a low affinity to AT2 receptors (Ki, > 1 mM) in bovine cerebellar membrane, indicating potent and selective AT1 properties. In the guinea pig aorta, 606A reduced the maximal angiotensin II-induced contraction (pD'2, 9.06 +/- 0.04), whereas EXP3174 showed suppression of the maximum response and a shift to the right of the concentration-response curve at lower and higher concentrations, respectively (conventionally calculated pd'2, 8.61 +/- 0.23). The 606A had no effects on KC1-, norepinephrine-, serotonin-, and endothelin-1-induced contractions or any agonist activities. In anesthetized dogs, 606A inhibited the angiotensin II-induced pressor response 35 times more potently than losartan. In renal hypertensive rats and spontaneously hypertensive rats (SHRs), 606A decreased systolic blood pressure 10 and 3 times more potently than losartan, respectively, without any chronotropic effects. By repeated administration of 606A to SHRs for 2 weeks, an augmentation of the hypotensive effect was observed No rebound phenomena occurred after discontinuation. These results indicate the 606A is a potent AT1-selective insurmountable angiotensin II receptor antagonist having more potent angiotensin II receptor antagonistic and hypotensive effects than losartan in in vivo models. 606A is suggested to be a useful agent for the treatment of patients with hypertension.

Angiotensin II↗
PubMed1997

A verrucous lesion on skin grafted after necrotizing fasciitis in a diabetic patient successfully treated with combined topical 5-FU and tacalcitol.

Many complications of diabetes mellitus involve the feet. These include infections, neuropathy, vasculopathy, and poor wound healing. Neuropathy causes chronic pressure or friction on an area of sensory loss and occasionally causes verrucous skin lesions. We describe a diabetic patient, complicated by necrotizing fasciitis, who developed a verrucous skin lesion on a skin graft site. The verrucous skin lesion was treated successfully with combined topical 5-fluorouracil and vitamin D3 application.

Administration, Topical↗