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Biomedical subjects

Y Hashimoto

Publications and source records attributed to Y Hashimoto.

At least 397 records · Page 22Linked to original sources
PubMed1998

Assembly of flammutoxin, a cytolytic protein from the edible mushroom Flammulina velutipes, into a pore-forming ring-shaped oligomer on the target cell.

Flammutoxin has been previously isolated as a cardiotoxic and cytolytic polypeptide of 22 or 32 kDa from the fruiting bodies of the edible mushroom Flammulina velutipes. In the present study, we purified flammutoxin as a single haemolytic protein of 31 kDa and studied the mode of its cytolytic action. (1) Flammutoxin caused efflux of potassium ions from human erythrocytes and swelling of the cells before haemolysis. (2) Flammutoxin did not lyse human erythrocytes in the presence of non-electrolytes with hydrodynamic diameters of >5.0 nm, although it caused leakage of potassium ions and swelling of the cells under the same conditions. (3) Experiments including solubilization of cell-bound toxin with 2% (w/v) SDS at 20 degrees C and subsequent Western immunoblots showed that flammutoxin formed a band corresponding to 180 kDa under the conditions where it lysed erythrocytes. (4) Electron microscopy of flammutoxin-treated human erythrocytes revealed the presence of a ring-shaped structure with outer and inner diameters of 10 and 5 nm, respectively, on the cells. (5) A ring-shaped toxin oligomer of the same dimensions was solubilized from the toxin-treated human erythrocytes with 2% (w/v) SDS at 20 degrees C and isolated by a sucrose-gradient ultracentrifugation. These data indicated that flammutoxin assembles into a ring-shaped oligomer possessing a hydrophilic pore of 4-5 nm on target cells.

Basidiomycota↗
PubMed1998

Critical role for the 310 helix region of p57(Kip2) in cyclin-dependent kinase 2 inhibition and growth suppression.

Although crystal structural analysis of cyclin A/cyclin-dependent kinase 2 (Cdk2)/p27 (Russo, A. A., Jeffrey, P. D., Pattern, A. K., Massague, J., and Pavletich, N. P. (1996) Nature 382, 325-331) has suggested that the 310 helix region in Cdk inhibitors of the Cip/Kip family may be involved in the inhibition of cyclin/Cdk activities, there is no biochemical evidence supporting this hypothesis. In the present study, we demonstrated that cyclin and Cdk binding domains of p57 were necessary but were not sufficient in themselves for the inhibition of cyclin A/Cdk2 and cyclin E/Cdk2, and that the 3(10) helix region of this protein is indispensable for the inhibition of these complexes. In contrast, the 3(10) helix regions of p21 and p27 were not required, and cyclin- and Cdk-binding domains alone were sufficient for the inhibition of all cyclin/Cdk complexes examined. Site-directed mutagenesis identified phenylalanine 79 and tyrosine 80 within the 3(10) helix region of p57 as crucial residues for kinase inhibition, supporting the structural evidence that the 3(10) helix binds deep inside the catalytic cleft of Cdk2, mimicking ATP. Mutations within the 3(10) helix region of the p57 molecule completely abolished the ability to arrest the cell cycle at G1 in vivo. These results indicate that this region is specifically utilized by p57 in selectively inhibiting cyclin A or E/Cdk2+ activities. Thus the 3(10) helix motif may confer a specific regulatory mechanism by which p57 differentially regulates Cdk2 and Cdk4 activities.

Adenosine Triphosphate↗
PubMed1998

Down-regulation of c-myc and bcl-2 gene expression in PU.1-induced apoptosis in murine erythroleukemia cells.

We found that over-expression of PU.1, a member of the ets family of transcription factors, induces apoptotic cell death along with differentiation of DMSO stimulation in murine erythroleukemia (MEL) cells. To elucidate the molecular mechanisms of apoptosis, cell-cycle distribution and expression of several genes encoding apoptosis-promoting and -inhibiting factors were analyzed during the process of PU.1-induced apoptosis. FACS analysis revealed that cells were accumulated in the G0/G1 phase of the cell cycle before apoptosis. Morphological analysis of PI-stained nuclei of the apoptotic cells sorted by a FACScan showed 22.6% in G0/G1, 35.8% in S and 8.5% in G2/M phase by fluorescent microscopy after cell sorting, suggesting that PU.1-induced apoptosis in MEL cells occurs in G0/G1 through S phases. Semi-quantitative RT-PCR revealed that expression of c-myc and bcl-2 genes was reduced during the apoptotic process, while expression of bax and bcl-X(L) genes was not changed. Expression of the p53 gene was reduced rather than enhanced, suggesting that PU.1-induced apoptosis in MEL cells is p53-independent. Apoptosis was inhibited by adding 30% serum in culture, while no reduction of c-myc and bcl-2 gene expression was observed. Forced expression of the c-myc, bcl-2 and bcl-X(L) genes protected MEL cells from apoptosis. Our results suggest that a reduction of at least 2 important apoptosis-inhibiting factors, c-Myc and Bcl-2, is involved in PU.1-induced apoptosis in MEL cells.

Animals↗
PubMed1998

Enhanced potency of perfluorinated thalidomide derivatives for inhibition of LPS-induced tumor necrosis factor-alpha production is associated with a change of mechanism of action.

Perfluorination of phthalimides leads to dramatically increased potency as inhibitors of TNF-alpha production. We examined the enantiodependence for several tetrafluorophthalimides and alpha-methylthalidomide, 3. Only 3 exhibited strikingly enantiodependent activity. The key structural determinant for the enhanced activity is the tetrafluorophthaloyl group, which confers enhanced potency and a change in the mechanism of inhibition.

Cell Line↗
PubMed1998

Ion chromatography on anion exchangers modified with mucopolysaccharides.

Anion exchangers modified with mucopolysaccharides, such as chondroitin sulfates and heparin, were used for the stationary phase in ion chromatography. Unusual retention behavior of anions was observed for the modified stationary phases. A 50-microM concentration of tartaric acid could separate inorganic anions in a reasonable time. The retention of analytes could be changed by changing the eluent composition.

Chromatography, Ion Exchange↗
PubMed1998

Specific detection of phosphatidylinositol 3,4,5-trisphosphate binding proteins by the PIP3 analogue beads: an application for rapid purification of the PIP3 binding proteins.

Phosphatidylinositol (PI) 3-kinase is known as one of the key molecules involved in the various biological events such as vesicle trafficking, cytoskeletal rearrangements and cell survival. T clarify the molecular basis underlying these events, we have tried to identify the proteins that can interact with phosphatidylinositol 3,4,5-trisphosphate (PIP3), the lipid product of PI3-kinase. Using a new PIP3 analogue, PIP3-APB, we synthesized an affinity column for PIP3 binding proteins. This enabled us to purify and identify several PIP3 binding proteins such as Tec tyrosine kinase, Gap1m, and Akt, as the candidates for the downstream molecules of PI3-kinase. All of these proteins contain PH domains, possible binding sites for phospholipids. Studies with various deletion mutants of Tec or Gap1m revealed that their PH domains are indeed the binding sites for PIP3. These results demonstrate that this PIP3-analogue binds various PIP3 binding proteins with high specificity and may be useful to elucidate the downstream mechanisms of PI3-kinases-mediated signaling pathways.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Salmonella typhi rpoS mutant is less cytotoxic than the parent strain but survives inside resting THP-1 macrophages.

Transcription of the stationary-phase sigma factor RpoS of Salmonella typhi increased in the macrophage. A single rpoS mutant of S. typhi was constructed to analyze the role of RpoS in intracellular multiplication of the bacterium and host cell killing. This mutant was sensitive to starvation, low pH and hydrogen peroxide; however, it could still multiply inside resting macrophages and was less cytotoxic than the wild-type strain. Therefore, S. typhi might produce RpoS-dependent factors which could contribute to host cell death.

Bacterial Proteins↗
PubMed1998

Protein refolding by reversed micelles utilizing solid-liquid extraction technique.

This article reports that a reversed micellar solution is useful for refolding proteins directly from a solid source. The solubilization of denatured RNase A, which had been prepared by reprecipitation from the denaturant protein solution, into reversed micelles formulated with sodium di-2-ethylhexyl sulfosuccinate (AOT) has been investigated by a solid-liquid extraction system. This method is an alternative to the ordinary protein extraction in reversed micelles based on the liquid-liquid extraction. The solid-liquid extraction method was found to facilitate the solubilization of denatured proteins more efficiently in the reversed micellar media than the ordinary phase transfer method of liquid extraction. The refolding of denatured RNase A entrapped in reversed micelles was attained by adding a redox reagent (reduced and oxidized glutathion). Enzymatic activity of RNase A was gradually recovered with time in the reversed micelles. The denatured RNase A was completely refolded within 30 h. In addition, the efficiency of protein refolding was enhanced when reversed micelles were applied to denatured RNase A containing a higher protein concentration that, in the case of aqueous media, would lead to protein aggregation. The solid-liquid extraction technique using reversed micelles affords better scale-up advantages in the direct refolding process of insoluble protein aggregates.

Acetone↗
PubMed1998

Expression of the Elm1 gene, a novel gene of the CCN (connective tissue growth factor, Cyr61/Cef10, and neuroblastoma overexpressed gene) family, suppresses In vivo tumor growth and metastasis of K-1735 murine melanoma cells.

We previously isolated a partial cDNA fragment of a novel gene, Elm1 (expressed in low-metastatic cells), that is expressed in low-metastatic but not in high-metastatic K-1735 mouse melanoma cells. Here we determined the full-length cDNA structure of Elm1 and investigated the effect of Elm1 expression on growth and metastatic potential of K-1735 cells. The Elm1 gene encodes a predicted protein of 367 amino acids showing approximately 40% amino acid identity with the CCN (connective tissue growth factor [CTGF], Cyr61/Cef10, neuroblastoma overexpressed gene [Nov]) family proteins, which consist of secreted cysteine-rich proteins with growth regulatory functions. Elm1 is also a cysteine-rich protein and contains a signal peptide and four domains conserved in the CCN family proteins. Elm1 was highly conserved, expressed ubiquitously in diverse organs, and mapped to mouse chromosome 15. High-metastatic K-1735 M-2 cells, which did not express Elm1, were transfected with an Elm1 expression vector, and several stable clones with Elm1 expression were established. The in vivo growth rates of cells expressing a high level of Elm1 were remarkably slower than those of cells expressing a low level of Elm1. Metastatic potential of transfectants was reduced in proportion to the level of Elm1 expression. Thus, Elm1 is a novel gene of CCN family that can suppress the in vivo growth and metastatic potential of K-1735 mouse melanoma cells.

Animals↗
PubMed1998

Construction of a yeast expression system with positive selection for gene insertion in the absence of a specific phenotype.

A positive-selection system of cloned inserts in Escherichia coli has been devised using a streptomycin-resistant (Smr) gene and streptomycin-dependent (Smd) E. coli. A vector, pHA394, based on the yeast expression vector pAM82 has the Smr gene, which inactivates streptomycin (Sm) and invalidates the streptomycin dependence, resulting in a very low transformation efficiency. Replacement of the Smr gene by the recombinant vector allows high-frequency transformation. This system was applied to the lysozyme gene. After the yeast secretion signal was fused to the lysozyme gene using an intermediate vector, pHA474, the Smr gene of pHA394 was replaced by the fusion gene, followed by transformation of Smd E. coli. Analysis of the transformants showed that the plasmid gene contained 100% of the lysozyme gene.

Anti-Bacterial Agents↗
PubMed1998

Resting 123I-BMIPP scintigraphy in diagnosis of effort angina pectoris with reference to subsets of the disease.

This study was undertaken to assess the diagnostic value of resting 123I-BMIPP scintigraphy in patients with effort angina pectoris. One hundred and four patients underwent scintigraphic and angiographic examinations. The subsets of the patients were stable effort angina pectoris (stable type) in 27 cases, new onset of effort angina pectoris (new onset type) in 21 cases, and worsening effort angina pectoris (worsening type) in 35 cases. The remaining 21 cases were subjects without evidences of coronary artery disease (non-CAD). 123I-BMIPP was injected under resting and pain free condition, then data for single photon emission tomography (SPECT) were acquired. The positive regional 123I-BMIPP defects in three coronary territories were visually judged on the tomographic images. The overall sensitivity to diagnose the patients was 62.6% (52/83) and the overall specificity to exclude non-CAD subjects was 95.2% (20/21). The detection rate in each subset of the disease was 48.1% (13/27) in stable type, 47.6% (10/21) in new onset type and 77.1% (27/35) in worsening type (p < 0.05 versus two other types). For detection of stenosed vessels, the overall sensitivity was 41.4% (56/148) and the overall specificity was 93.8% (152/164). The rate of detection of stenosed vessels was 31.7% (13/41) in stable type, 31.4% (11/35) in new onset type, and 55.6% (40/72) in worsening type (p < 0.05 versus two other types). Vessels with 75% stenosis were more sensitively detected in the worsening type (33.3%; 4/12) compared to the remaining two types (8.3%; 1/12) even though the difference was not significant. The resting 123I-BMIPP scintigraphy was therefore valuable in diagnosing patients with effort angina pectoris and involved coronary arteries especially in the subset of patients with worsening type.

Aged↗
PubMed1998

Cloning and mapping of Np95 gene which encodes a novel nuclear protein associated with cell proliferation.

We previously obtained a monoclonal antibody (Th-10a mAb) that recognizes a single 95-kDa mouse nuclear protein (NP95). Immunostaining analyses revealed that the NP95 was specifically stained in the S phase of normal mouse thymocytes. In contrast, mouse T cell lymphoma cells exhibited a constantly high level of NP95 accumulation irrespective of cell stages during the cell cycle. In the present study, we isolated the cDNA encoding the NP95 from a lambdagt-11 cDNA expression library, using the Th-10a mAb. Sequencing of the whole 3.5-kb cDNA revealed that NP95 is a novel nuclear protein with an open reading frame (ORF) consisting of 782 amino acids. The ORF contains a zinc finger motif, a potential ATP/GTP binding site, a putative cyclin A/E-cdk2 phosphorylation site, and the retinoblastoma protein (RB)-binding motif "IXCXE". The chromosomal location of Np95 gene was determined by fluorescence in situ hybridization. Np95 gene locates on mouse Chromosome (Chr) 17DE1.1. and rat Chr 9q11.2-q12.1. Np95 was strongly expressed in the testis, spleen, thymus, and lung tissues, but not in the brain, liver, or skeletal muscles. These results collectively implicate this novel nuclear protein in cell cycle progression and/or DNA replication.

Amino Acid Sequence↗
PubMed1998

Histopathological assessment of localized proliferation in cases of Bowen's disease using immunostaining and a laser cytometer.

In order to evaluate the localized proliferative activity of intratumor cells in Bowen's disease using tissue sections, skin specimens from ten patients were compared with skin samples from seven normal individuals for their expression of proliferating cell nuclear antigen (PCNA), Ki-67 immunostaining and intranuclear DNA contents, quantitated with a laser cytometer (LCM). In normal epidermis, the largest proportion of PCNA- and Ki-67-positive cells was observed in the basal cell layer, with the amounts decreasing through the suprabasal cell layer towards the prickle cell layer. Examination by LCM also revealed the highest average fluorescence intensity of individual nuclei in the basal cell layer and, as with the immunohistological parameters, reducing towards the upper layer of the epidermis. In the Bowen's disease tissue sections, the largest proportion of PCNA- and Ki-67-positive cells was found in contact with the basement membrane (base of the tumor), with lower amounts in the center of the tumor nest and in the marginal epidermis. The average fluorescence intensities of individual nuclei were in line with these results. These results show that tumor cells distributed in Bowen's disease tumor nests have different proliferative activities depending on their location.

Bowen's Disease↗
PubMed1998

Characterization of antigenic epitopes in anti-ulcer pectic polysaccharides from Bupleurum falcatum L. using several carbohydrases.

A polyclonal antibody (anti-bupleuran 2IIc/PG-1-IgG) against the "ramified" region (PG-1) of an anti-ulcer pectic polysaccharide was prepared and its antigenic epitopes were analyzed by using several carbohydrases. Enzymatic removal of arabinosyl residues from PG-1 by endo-(1-->5)-alpha-L-arabinanase (from Aspergillus niger) did not reduce the binding ability of anti-bupleuran 2IIc/PG-1-IgG to PG-1. When the endo-(1-->5)-alpha-L-arabinanase-resistant fraction (EA-1) was digested with rhamnogalacturonase A (rRGase A from A. aculeatus), a high-molecular-mass fragment fraction (RA-1) and an oligosaccharide fraction (RA-3) were obtained. RA-3 contained at least four kinds of oligosaccharides liberated from the rhamnogalacturonan core. This partial removal of the rhamnogalacturonan core in EA-1 also did not reduce the binding of the antibody to the polysaccharide. Further digestion of RA-1 with exo-(1-->3)-beta-D-galactanase (from Irpex lacteus), gave a high-molecular-mass fragment (EXG-1) and a trace of oligosaccharides (EXG-3). Methylation and FABMS analyses indicated that EXG-3 contained mono- and di-galactosyl oligosaccharides possessing terminal GlcA or GlcA4Me. Removal of the EXG-3 fraction from RA-1 by exo-(1-->3)-beta-D-galactanase significantly reduced the ability of the binding of the antibody to the polysaccharide. When PG-1 was digested with endo-(1-->6)-beta-D-galactanase (from Trichoderma viride) or beta-D-glucuronidase (from A. niger), the reactivities of both enzyme-resistant fractions to the antibody were decreased in comparison with that of PG-1. Both radish arabinogalactan (containing GlcA4Me) and beta-D-GlcpA-(1-->6)-beta-D-Galp-(1-->6)-D-Galp were shown to inhibit the reactivity of PG-1 to the antibody by competitive ELISA. These results suggest that 6-linked galactosyl chains containing terminal GlcA or GlcA4Me attached to (1-->3)-beta-D-galactosyl chains, are important sugar residues in the antigenic epitopes of the "ramified" region of bupleuran 2IIc.

Bupleurum↗
PubMed1998

Additional recognition sites in the C-terminal heparin-binding domain of fibronectin promote adhesion of PMA-treated U937 cells.

Recently we have shown an evidence that a peptide, corresponding to residues Gln1892 to Gly1910, from the C-terminal heparin-binding domain of fibronectin promotes adhesion of phorbol-12-myristate 13-acetate (PMA)-treated U937 cells and binds to both integrin alpha 4 beta 1 and glycosaminoglycans on U937 cells surface. We present additional adhesion-promoting sites to PMA-treated U937 cells present in the C-terminal heparin-binding domain of fibronectin. Three synthetic peptides (residues Ala1819 to Lys1830, designated E5; Thr1828 to Gly1940, E4; and Lys1946 to Leu1963, D1) were active to inhibit adhesion of PMA-treated U937 cells to the 29-kDa fragment comprising the C-terminal heparin-binding domain of fibronectin. Scrambled versions of these peptides had no inhibitory activity on this adhesion. The IgG-conjugated peptides (IgG-E5, IgG-E4, and IgG-D1) were also active and supported adhesion to an extent comparable to that of the 29-kDa fragment. The adhesion of PMA-treated U937 cells on these three IgG-conjugated peptides was only inhibited by glycosaminoglycans and not by integrin alpha 4 beta 1. These results indicate that additional adhesion-promoting sequences are present in the C-terminal heparin-binding domain of fibronectin and that the activity of these peptides depends on peptide sequence, mainly the result of net charges or net hydropathy indices.

Amino Acid Sequence↗
PubMed1998

Constitutive association of EGF receptor with the CrkII-23 mutant that inhibits transformation of NRK cells by EGF and TGF-beta.

Crk belongs to the adapter proteins that participate in many signalling pathways from cell surface receptors. We have characterised the CrkII-23 mutant that inhibits the transformation of NRK cells induced by epidermal growth factor (EGF) and transforming growth factor (TGF)-beta. To study the biochemical difference, cDNAs of the wild-type CrkII and the CrkII-23 mutant were introduced stably into NIH 3T3 cells expressing EGF receptor (EGFR). Both CrkII and CrkII-23 were phosphorylated on tyrosine upon EGF simulation with similar time course and dose dependency. Whereas the wild-type CrkII bound to EGFR only after EGF stimulation, CrkII-23 bound to EGFR from before stimulation. Mutation in the Src homology (SH) 2 or amino-terminal SH3 domain did not abolish the binding of CrkII-23 to EGFR in the quiescent cells, suggesting that the binding is mediated by a novel mechanism. These CrkII-23-derived mutants, however, did not suppress transformation of NRK cells by EGF and TGF-beta. Hence, both the SH2 and amino-terminal SH3 domains are required to inhibit transformation of NRK cells. These results suggest that persistent signalling from CrkII-23 bound to EGFR suppresses transformation by EGF and TGF-beta in NRK23 cells.

3T3 Cells↗
PubMed1998

Interferon-gamma-dependent induction of manganese superoxide dismutase activity of SV40-transformed human keratinocytes by anti-Fas antibody and by TNF-alpha.

It has been reported that cellular oxidative stress induces apoptosis, that may be inhibited by scavengers of reactive oxygen intermediates (ROIs). Superoxide dismutase (SOD) is among the most active scavengers of ROIs, providing defense against the cellular oxidative stress. Fas antigen and tumor necrosis factor (TNF) receptor are the cell surface proteins, stimulation of which induces apoptosis of keratinocytes. Using SV40-transformed human keratinocytes (SVHK cells), we investigated the effects of anti-Fas antibody and TNF-alpha on the SOD activity. Treatment of SVHK cells with anti-Fas antibody or TNF-alpha in the presence of interferon-gamma (IFN-gamma) resulted in an increase in Mn-SOD activity, Cu,Zn-SOD activity was not affected. In the absence of IFN-gamma, no increase in Mn-SOD activity was detected. The induction of IFN-gamma-dependent Mn-SOD activity by anti-Fas antibody or TNF-alpha was concentration-dependent; the maximal effect was observed at 1-10 micrograms/ml and 5-10 ng/ml, respectively. The increase in Mn-SOD activity was observed at 6 h following the treatment and remained for at least 48 h. Northern blot analyses showed that Mn-SOD mRNA increased within 3 h without a significant change in Cu,Zn-SOD mRNA. The addition of both anti-Fas antibody and TNF-alpha in the presence of IFN-gamma resulted in an additive increase in Mn-SOD activity. Although the addition of 12-o-tetradecanoylphorbol-13-acetate (TPA) singly to the incubation medium had no effect on either Mn-, or Cu,Zn-SOD activity, it significantly augmented the IFN-gamma-dependent induction of Mn-SOD activity by anti-Fas antibody or by TNF-alpha. The protein kinase C inhibitor, 1-(5-isoquinoline-sulfonyl)-2-methyl piperazine dihydrochloride (H-7), significantly inhibited the TPA-dependent increase in Mn-SOD activity. These results indicate that the stimulation of Fas antigen or TNF receptor increases Mn-SOD activity of SVHK cells in the presence of IFN-gamma and that TPA augments the process through the activation of protein kinase C.

Antibodies↗