Biomedical subjects

M Hamaguchi

Publications and source records attributed to M Hamaguchi.

At least 127 records · Page 7Linked to original sources

PubMed1995

Augmentation of metalloproteinase (gelatinase) activity secreted from Rous sarcoma virus-infected cells correlates with transforming activity of src.

To search for the biochemical properties of cells relevant to transformation by p60v-src, we examined the activities and amounts of metalloproteinase (gelatinase) released from chicken embryonic fibroblasts infected with various mutants of Rous sarcoma virus by zymography and immunoblotting. While nontransforming Src proteins, including cellular p60c-src and its nonmyristylated form, had no stimulatory effect, wild-type p60v-src and the transforming mutant of cellular p60c-src stimulated the secretion and proteolytic activation of metalloproteinases. Moreover, the activation of metalloproteinase secretion strongly correlated with the invasiveness of cells assayed by the modified Boyden Chamber method. Chimeric mutants between v-src and c-src, which are transforming but produce less distinct morphological changes in infected cells, also stimulated the secretion of metalloproteinases as well as wild-type p60v-src. Deletion mutants of v-Src in which varying portions of the NH2-terminal half of p60v-src are deleted stimulated secretion to a level similar to that of wild-type regardless of the degree of morphological change they induce. Together with Src protein, other oncogene products including Yes, Fps, ErbB and Crk were also found to stimulate the secretion of metalloproteinases. Thus, these results suggest that transformation of cells with src and other oncogenes is closely associated with the enhanced secretion of metalloproteinases that may play an important role in tumor invasion and metastasis.

Animals↗

PubMed1995

Evidence of redox-linked signaling for producing a giant signal complex.

Previously we showed that a thiol-reactive heavy metal, HgCl2, crosslinked multiple cell surface receptors through a ligand-independent pathway, which produced massive aggregates of phosphotyrosine (PTYR)-containing proteins beneath plasma membrane [Nakashima et al. (1994): J Immunol 152: 1064-1071]. In this study we characterized these unique aggregates at the molecular level. The lysates in Brij 96 of thymocytes treated with HgCl2 were separated into the supernatant and pellet fractions by simple centrifugation. Selected PTYR-containing proteins and p56lck appeared in the pellet fraction as quickly as 5 s after exposure to HgCl2, and were further increased in amount by 5 min. Although the mechanism of triggering these events was redox-linked, the majority of proteins in the Brij 96-insoluble aggregates were dissociated in SDS-PAGE under nonreducing condition. This suggested that PTYR-containing proteins and p56lck themselves do not form dimer or polymer directly by thiol-mediated bond. The pellet fraction was further found to include some other signal delivery elements, such as GTPase activating protein, phosphatidylinositol 3 kinase, and mitogen-activated protein kinase. Finally, all of these signal elements and selected PTYR-containing proteins were collected in the same fraction by the sucrose density gradient centrifugation. These results suggest a unique redox-linked pathway of formation of a giant signal complex.

Animals↗

PubMed1995

Multiphasic modulation of signal transduction into T lymphocytes by monoiodoacetic acid as a sulfhydryl reagent.

Actions of monoiodoacetic acid (MIA) as a sulfhydryl reagent on the different stages of the T cell receptor (TCR)-mediated signal transduction were examined. MIA (1 mM) prevented anti-TCR (CD3) monoclonal antibody (mAb)-induced energy-dependent receptor capping but at the same time promoted the anti-CD3 mAb/mitogen-induced tyrosine phosphorylation of the T cell activation-linked cellular proteins of 120, 80, 70, 56, and 40 kDa. Relatively low concentration (0.01 mM) of MIA further promoted anti-CD3 mAb-induced transcription of c-fos, production of IL-2, and cell surface expression of IL-2 receptors. The MIA-promoted TCR-mediated IL-2 production actually required signal transduction that could be inhibited by cyclosporin A, genistein, or H-7. In contrast, the same concentration of MIA as promoted the signal transduction for cell activation severely inhibited the anti-CD3 mAb-triggered signal delivery for cell proliferation, selectively at its early stage. We conclude from these results that MIA differentially affects various steps of signaling into T lymphocytes, suggesting that there exist multiple sites of MIA-sensitive or redox-linked control in the signal cascade.

Animals↗

PubMed1995

Antiviral action of interferon-beta on Newcastle disease virus: selectivity to the hemagglutinin-neuraminidase gene expression.

Interferon-beta (IFN-beta) strongly inhibited the expression of the hemagglutinin-neuraminidase (HN) gene of Newcastle disease virus (NDV), a paramyxovirus, in HeLa cells under the conditions where it did not affect the expression of the four upstream genes encoding the nucleocapsid protein, phosphoprotein, membrane protein and fusion protein. Even the downstream gene, encoding the large protein as well as the genome replication, appeared to be less susceptible to IFN-beta than the HN gene. This selective action of IFN-beta did not appear to be attributable to its well characterized antiviral mechanisms such as acceleration of RNA decay and translation inhibition. No similar down-regulation of a particular gene expression was found with another paramyxovirus, Sendai virus, or with a rhabdovirus, vesicular stomatitis virus, or seems to have been reported previously with any negative-strand RNA viruses. This new effect of IFN-beta thus suggests gene expression mechanism unique to NDV and may further lead to the discovery of a novel biochemical effect of IFN-beta.

Animals↗

PubMed1995

Suppression of gelatinase production with decreased invasiveness of choriocarcinoma cells by human recombinant interferon beta.

OBJECTIVE: Choriocarcinoma is a highly invasive gynecologic tumor, and hematogenous metastases frequently develop. To establish a molecular basis for antiinvasion therapy of choriocarcinoma, we examined the effects of human recombinant interferons on gelatinase production and invasion by choriocarcinoma cells. STUDY DESIGN: Using five choriocarcinoma cell lines, we measured gelatinase activity by gelatin zymography. The effects of recombinant interferons (rIFN-alpha, rIFN-beta, and rIFN-gamma) were then analyzed by Western blot analysis and chemoinvasion assay. RESULTS: High levels of 72 kd gelatinase activity were detected in the highly invasive choriocarcinoma cell lines, two of which also contained an active form of 72 kd gelatinase with an apparent molecular mass of 68 kd. Gelatinase production was decreased by incubation with rIFN-beta. In the chemoinvasion assay, only rIFN-beta had an inhibitory effect on the invasiveness of tumor cells without a cytotoxic effect. CONCLUSION: Choriocarcinoma cells showed high 72 kd gelatinase activity, which suggested a role for the enzyme in vascular metastasis. Studies on the use of rIFN-beta to inhibit metastasis of choriocarcinoma via suppression of gelatinase production are warranted.

Choriocarcinoma↗

PubMed1995

Contribution of activation of K+ channels to glyceryl trinitrate-induced relaxation of rabbit aorta.

1. Possible contribution of K+ channel opening to the relaxation by glyceryl trinitrate (GTN) was examined using isolated rabbit aorta. 2. While glibenclamide and apamin failed to affect relaxation by GTN, both charybdotoxin (ChTx) and iberiotoxin (IbTx) effectively attenuated GTN-induced relaxation. 3. The increase in cGMP produced by GTN was not attenuated by ChTx and IbTx. 4. The inhibitory effect of ChTx on GTN-induced relaxation was not reduced in the presence of zaprinast, indicating that cGMP but not GMP was responsible for activation of the K+ channel. 5. Okadaic acid, a selective inhibitor of protein phosphatase 2A, had no effect on the relaxation by GTN. These results indicate that, though small in degree, activation of a ChTx-sensitive K+ channel (large conductance Ca(2+)-activated K+ channel) is involved in the GTN-induced relaxation in rabbit aorta.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed1995

Use of dextran-primed extracorporeal circuit in the canine model for venous return: with special references to the effects of two dihydropyridine-type Ca2+ channel blockers.

1. A successful canine "venous return (VR)" model is described, in which all hemodynamic parameters were stable for about an hour, by minimizing extracorporeal circuit primed with dextran (DX) so that the dilution of the blood was kept about 33%. 2. Increases in VR by norepinephrine (1 micrograms/kg) and dose-dependent decreases in VR by glyceryl trinitrate (1-30 micrograms/kg) were clearly observed. 3. While 1 microgram/kg of nifedipine produced a small, but significant decrease in VR, only a tendency for decrease was observed with higher doses which produced a definite fall in blood pressure. 4. Nisoldipine (1 microgram/kg) caused a small decrease in VR. However, higher doses that produced a large and persistent hypotension produced an increase in VR due probably to intense reflex translocation of the blood. The increase in VR was reversed with carvedilol, a beta-blocker with alpha-blocking activity.

Animals↗

PubMed1995

Identification of a novel phospholipase C family gene at chromosome 2q33 that is homozygously deleted in human small cell lung carcinoma.

Since a considerably high incidence of allelic loss on chromosome 2q was detected in lung carcinoma and a homozygous deletion at chromosome 2q33 was detected in a small cell lung carcinoma cell line, NCI-H82, a novel tumor suppressor gene has been suggested to be present in this chromosomal region. In the present study, we constructed a cosmid contig map covering the homozygous deleted region, which was estimated as being 220 kbp in size, and identified a gene from the deleted region. All of the coding exons of this gene were homozygously deleted in this cell line, while a 5'-non-coding exons was retained. Since the gene encodes a protein with striking similarity to several members of a family of phospholipase C, we designated this gene as PLC-L (phospholipase C-deleted in lung carcinoma). The PLC-L gene was expressed in a variety of fetal and adult organs including the lung. However, its expression was greatly reduced in seven of 13 (53.8%) of small cell lung carcinoma and 13 of 15 (86.7%) of non-small cell lung carcinoma cell lines. Since its homology to phospholipase C genes suggests the involvement of the PLC-L gene in inositol phospholipid-based intracellular signaling cascade, it is possible that aberrant expression of the PLC-L gene contributes to the genesis or progression of human lung carcinoma.

Amino Acid Sequence↗

PubMed1995

Herbimycin A inhibits phorbol ester-induced morphologic changes, adhesion, and megakaryocytic differentiation of the leukemia cell line, MEG-01.

12-O-Tetradecanoylphorbol 13-acetate (TPA) induces rapid changes in the morphology of the human megakaryoblastic leukemia cell line, MEG-01, as well as changes in adhesion and megakaryocytic differentiation. To investigate the signal transduction pathway of these three phenomena, we studied the effect of herbimycin A, an inhibitor of tyrosine kinase (TK) and the effects of calphostin C, a specific inhibitor protein kinase C (PKC) on TPA treated MEG-01 cells. Both herbimycin A and calphostin C inhibited all three TPA-induced phenomena, suggesting that both pathways are required for these phenomena. Herbimycin A but not calphostin C blocked the tyrosine phosphorylation of cellular proteins. Immunohistochemical staining of PKC using an anti-PKC monoclonal antibody showed that herbimycin A did not interfere with the translocation and subsequent down regulation of PKC induced by TPA, suggesting that the TPA-induced effect on PKC (translocation and probably its activation) is not dependent on TK. Induction of c-fos and c-jun expression by TPA was inhibited by both herbimycin A and calphostin C, suggesting that both PKC and TK pathways are necessary for the induction of the TPA-induced transcription factor AP1, which is a known TPA-inducible early immediate gene product. Taken together, our results show that the tyrosine kinase signal transduction system as well as the PKC pathway is indispensable for the TPA-induced phenomena of morphologic change, cell attachment, early immediate gene expression, and lineage-specific phenotypic expression in the MEG-01 cell line.

Benzoquinones↗

PubMed1995

[The structure and function of cadherins and their pathophysiological roles in diseases].

Cadherins are a family of Ca(2+)-dependent cell adhesion molecules that mediate cell adhesion by homophilic interaction and play critical role in multicellular organ formation. Recent progress revealed the three-dimensional structure of extracellular cadherin repeats that made great progress in the study of cell adhesion by cadherins. The perturbation of cadherin function in transformed cells results in inhibition of cell-cell adhesion, that appears to promote metastasis and invasiveness of tumor cells. Expression as well as modifications of catenins, which link cadherins to cytoskeletons, have crucial effects on this purturbation mechanisms. Tyrosine phosphorylation of cadherin catenin complex may be the important regulatory mechanism.

Cadherins↗

PubMed1995

Diagnosis of hemophilia B carriers using two novel dinucleotide polymorphisms and Hha I RFLP of the factor IX gene in Japanese subjects.

We identified two novel dinucleotide polymorphisms in intron A at nucleotide 192 (FIX192) and in the 5' flanking region at nucleotide -793 (FIX-793) of the factor IX gene, which are present in normal Japanese. The Hha I restriction fragment length polymorphism (FIX-HhaI) located 8 kb 3' to the factor IX gene was also found to be an efficient marker for detecting carriers in a Japanese family with hemophilia B. Each of these polymorphisms was able to be rapidly ascertained by the polymerase chain reaction (PCR) technique. In 22 Japanese families with hemophilia B, 18 families (81.8%) were heterozygous for at least one of these polymorphisms, whereas 11 (50%) were informative for the extragenic DXS99/Sac I RFLP which was previously reported as a useful gene marker for Japanese hemophilia B. Using all 4 polymorphisms together, the informative rate improved to 86.4%. Carrier detection and, possibly, the prenatal diagnosis of hemophilia B can be achieved effectively and rapidly in Japanese with these polymorphisms.

Base Sequence↗

PubMed1995

A quantitative protein S deficiency associated with a novel nonsense mutation and markedly reduced levels of mutated mRNA.

A 50-year-old Japanese man who had experienced recurrent episodes of venous thrombosis was found to have a hereditary protein S deficiency. The amount of total protein S antigen in plasma was reduced by approximately 50% in the patient and his two sons. DNA sequence analysis revealed a novel nonsense mutation, TAG for Gln 522 (CAG), in exon 14 of the protein S gene. Family studies performed by mutagenic PCR followed by restriction enzyme digestion showed that the proband and his two sons were heterozygous for this mutation. An mRNA-based analysis indicated that transcripts of the mutated allele were markedly reduced in the platelets of the affected individuals. Immunoblot analysis failed to detect the truncated mutant of protein S in the plasma or platelets of affected members. Our results demonstrated that this novel nonsense mutation was responsible for the quantitative deficiency of protein S.

Amino Acid Sequence↗

PubMed1995

[Carrier detection and prenatal diagnosis for hemophilia A using the inversion analysis of the factor VIII gene].

Hemophilia A is an X-linked hemorrhagic disorder caused by heterogeneous mutations in the factor VIII gene. Recently, it was reported that approximately 50% of the cases of severe hemophilia A may be caused by a common inversion in the factor VIII gene. In this study, we analyzed 33 Japanese patients with severe hemophilia A for the presence of this inversion mutation using the non-RI Southern blotting, and detected inversion mutations of the factor VIII gene in 12 patients (36.4%). We also showed that the inversion analysis of the factor VIII gene was useful for carrier detection and prenatal diagnosis in a hemophilia A family, which we had not been able to diagnose by the analysis of restriction fragment length polymorphisms (RFLPs) of the factor VIII gene. The detection of inversion mutations in the factor VIII gene using non-RI Southern blotting analysis appears to be very useful for the genetic counseling for severe hemophilia A.

Adult↗

PubMed1994

Plasmic degradation of fibrin rapidly decreases platelet adhesion and spreading.

Plasmin cleaves fibrin at or near sites involved in platelet recognition and may modulate platelet adhesion and spreading. Using an in vitro system, we have characterized the effects of limited plasmic degradation of polymerized fibrin on platelet adhesion and spreading. As shown by scanning electron microscopy, exposure to plasmin changed the tight fibrillar fibrin surface to a less dense structure with irregular and broken fibers. There was a gradient of proteolytic degradation through the fibrin clot as shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis with the most extensive degradation at the surface. Plasmic degradation resulted in a rapid and progressive decrease in platelet adhesion. Plasmin exposure for 5 minutes resulted in only 6% solubilization of the fibrin but a 56% decrease in platelet adhesion. After 30 minutes of plasmin exposure, spreading of adherent platelets on fibrin also decreased sharply to a minimum of 35% of baseline. Inhibition experiments with specific monoclonal antibodies (MoAbs) indicated that platelet adhesion to undergraded fibrin involved residues within the sequence 566 through 580 of the alpha chain (including the RGDS site), the carboxyl terminal dodecapeptide of the gamma chain, and the amino terminus of the beta chain. MoAb 7E3, reactive with alpha IIb beta 3, inhibited platelet adhesion to fibrinogen by 90% +/- 5%, and to desA fibrin, prepared with Reptilase (American Diagnostica, Greenwich, CT), by 94% +/- 6%, whereas inhibition of adhesion to undegraded desAB fibrin was significantly less (48% +/- 8%, P < .01). The addition of 7E3 to MoAb T2G1, reactive with beta 15-21, significantly increased inhibition to desAB fibrin to 69% +/- 6% (P < .025), suggesting that the newly exposed amino terminus of the beta chain contributes to platelet adhesion. The results show that plasmin exposure of fibrin markedly decreases platelet adhesion and spreading, suggesting that plasmin degradation may play a role in modulating cellular responses to fibrin.

Antibodies, Monoclonal↗

PubMed1994

Redox mechanism as alternative to ligand binding for receptor activation delivering disregulated cellular signals.

Cross-linking with specific ligand is a general requirement for ordered activation of cell surface receptors. In this study we demonstrated a novel pathway for disregulated receptor activation through a redox mechanism. Treatment of murine thymocytes or spleen cells with thiol-reactive HgCl2, a known inducer of autoimmune proliferative lymphocyte disorders in rodents, was found to induce tyrosine phosphorylation of several cellular proteins, which was up to 100 times as extensive as that triggered by stimulation with antireceptor antibody or mitogen. Through the cross-linkage by thiol-reactive bivalent mercury, transmembrane CD4, CD3, and CD45 and glycosylphosphatidylinositol-anchored Thy-1 were aggregated together on thymocytes or T lymphocytes. Along with the aggregation of Thy-1 and CD4, nonreceptor protein tyrosine kinase p56lck was aggregated and activated. These events were linked to extensive protein tyrosine phosphorylation, which was visualized as a well localized spot beneath the membrane. Under appropriate conditions, this novel pathway of multiple receptor aggregation delivered a disregulated signal into T lymphocytes, which cross-talked to the antireceptor antibody-induced signal, for prolonged cell proliferation and IL-2 production. These results suggest a novel mechanism of disregulation of the ligand-dependent receptor function.

Animals↗

PubMed1994

Messenger RNA levels of five genes located at chromosome 11q13 in B-cell tumors with chromosome translocation t(11;14)(q13;q32).

To identify genes activated by chromosome translocation t(11;14)(q13;q32), mRNA levels of five genes (cyclin D1, EXP1, MB38, HST1, and INT2) at chromosome 11q13 were investigated. The cyclin D1 mRNA increased in BCL-1-rearranged B-cell tumor cell lines SP-49, NOP-2, FLAM-76, KMS-12-PE, and KMS-12-BM cells, while it was not detected in cell lines without the translocation, Raji, U266, and HEL cells. A significant amount of the MB38 mRNA was detected irrelevantly to the translocation in all of these cell lines. The mRNAs of EXP1, HST1, and INT2 were undetectable in these cells. The results suggested that the translocation activates cyclin D1 alone, while the mRNA levels of the other four genes are regulated independently of the translocation.

Blotting, Northern↗

PubMed1994

Abundant but inactive-state gp140proto-trk is expressed in neuroblastomas of patients with good prognosis.

Steady-state levels of gp140proto-trk in cell lines and tumor tissues of neuroblastoma were examined by immunoblotting with anti-gp140proto-trk. The level of gp140proto-trk varied but showed good correlations with the stage of the tumor and the age of the patients at the time of diagnosis. Moreover, patients with higher expression of gp140proto-trk clearly had a far better survival rate than those with lower expression, suggesting that suppression of gp140proto-trk strongly correlates with the malignant conversion of the tumor. However, we found that neither autophosphorylation of gp140proto-trk nor tyrosine phosphorylation of cellular proteins was elevated in tumors of the higher expression group. These results suggest that gp140proto-trk does not actively participate in the process of transformation or the suppression of malignant conversion. Rather, the higher level of gp140proto-trk may reflect the greater level of differentiation of tumor cells.

Age Factors↗

PubMed1994

Assignment of disulfide bridges in the fusion glycoprotein of Sendai virus.

The mature fusion (F) glycoprotein of the paramyxovirus family consists of two disulfide-linked subunits, the N-terminal F2 and the C-terminal F1 subunits, and contains 10 cysteine residues which are highly conserved at specific positions. The high level of conservation strongly suggests that they are indeed disulfide linked and play important roles in the folding and functioning of the molecule. However, it has not even been clarified which cysteine residues link the F2 and F1 subunits. This report describes our assignment of the disulfide bridges in purified Sendai virus F glycoprotein by fragmentation of the polypeptide and isolation of cystine-containing peptides and determination of their N-terminal sequences. The data demonstrate that all of the 10 cysteine residues participate in disulfide bridges and that Cys-70, the only cysteine in F2, and Cys-199, the most upstream cysteine in F1, form the interchain bond. Of the remaining eight cysteine residues clustered near the transmembrane domain of F1, the specific bridges identified are Cys-338 to Cys-347 and Cys-362 to Cys-370. Although no exact pairings between the subsequent four residues were defined, it seems likely that the most downstream, Cys-424, is linked to Cys-394, Cys-399, or Cys-401. Thus, we conclude that the cysteine-rich domain indeed contributes to the formation of a bunched structure containing at least two tandem cystine loops.

Amino Acid Sequence↗