Biomedical subjects

M Hamaguchi

Publications and source records attributed to M Hamaguchi.

At least 55 records · Page 3Linked to original sources

PubMed2000

v-Src suppresses SHPS-1 expression via the Ras-MAP kinase pathway to promote the oncogenic growth of cells.

We investigated the effect of cell transformation by v-src on the expression and tyrosine phosphorylation of SHPS-1, a putative docking protein for SHP-1 and SHP-2. We found that transformation by v-src virtually inhibited the SHPS-1 expression at mRNA level. While nontransforming Src kinases including c-Src, nonmyristoylated forms of v-Src had no inhibitory effect on SHPS-1 expression, transforming Src kinases including wild-type v-Src and chimeric mutant of c-Src bearing v-Src SH3 substantially suppressed the SHPS-1 expression. In cells expressing temperature sensitive mutant of v-Src, suppression of the SHPS-1 expression was temperature-dependent. In contrast, tyrosine phosphorylation of SHPS-1 was rather activated in cells expressing c-Src or nonmyristoylated forms of v-Src. SHPS-1 expression in SR3Y1 was restored by treatment with herbimycin A, a potent inhibitor of tyrosine kinase, or by the expression of dominant negative form of Ras. Contrary, active form of Mekl markedly suppressed SHPS-1 expression. Finally, overexpression of SHPS-1 in SR3Y1 led to the drastic reduction of anchorage independent growth of the cells. Taken together, our results suggest that the suppression of SHPS-1 expression is a pivotal event for cell transformation by v-src, and the Ras-MAP kinase cascade plays a critical role in the suppression.

3T3 Cells↗

PubMed2000

Molecular cloning and characterization of a novel human gene (HERNA) which encodes a putative RNA-helicase.

A full-length cDNA encoding a novel protein was isolated and sequenced from a human hepatocellular cDNA library. This cDNA consists of 7037 base pairs and has a predicted open reading frame encoding 1924 amino acids. It possesses an RNA-helicase motif containing a DEXH-box in its amino-terminus and an RNase motif in the carboxy-terminus. From a striking homology to Caenorhabditis elegans K12H4.8, it might be a human homolog of the K12H4.8. PCR-based mapping with both a monochromosomal hybrid panel and radiation hybrid cell panels placed the gene to human chromosome 14q31 near the marker D14S605.

Amino Acid Sequence↗

PubMed2000

Clustered cysteine residues in the kinase domain of v-Src: critical role for protein stability, cell transformation and sensitivity to herbimycin A.

We have previously reported the activation of Src by mercuric chloride based on the sulfhydryl modification. To evaluate the significance of cysteine residues in v-Src, we replaced each cysteine to alanine by oligonucleotide-directed mutagenesis and examined its effect on cell transformation. Of ten cysteine residues scattered over v-Src, four cysteines clustered in kinase domain, Cys483, Cys487, Cys496 and Cys498, were important for protein stability and cell transformation, whereas those in SH2 domain were dispensable. A single mutation in Cys498 yielded suppression of kinase activity and a temperature-sensitivity in anchorage independent growth. Double mutation either in Cys483/Cys487 or in Cys496/Cys498 yielded clear temperature-sensitivity in cell transformation and in stability of Src protein. Instability of Src protein was magnified by quadruple mutation in the cysteines, which decreased the half-life of Src to be less than one quarter of that of wild-type. In addition, both Cys483/Cyr487 and Cys496/Cys498 kinases became resistant to in vitro inactivation by herbimycin A, which directly inactivates v-Src in addition to its effect on HSP90. Taken together, our results strongly suggest that the cysteine clustered motif of v-Src are critical for protein stability, cell transformation and in vitro inactivation by herbimycin A.

Amino Acid Sequence↗

PubMed2000

Constitutive activation of MAP kinase kinase (MEK1) is critical and sufficient for the activation of MMP-2.

We investigated the role of MEK1 signaling in MMP-2 activation by use of constitutive active/dominant negative forms of MEK1 and MEK1-specific inhibitor. We found that cell transformation with active forms of MEK1 dramatically increased secretion and proteolytic activation of MMP-2 and subsequently stimulated invasiveness of cells. Contrary, expression of dominant negative form of MEK1 in v-src-transformed cells or in Con A-activated cells resulted in the suppression of the augmented secretion and proteolytic activation of MMP-2. In addition, treatment of v-src-transformed cells with PD98059, a MEK1-specific inhibitor, strongly suppressed the secretion and activation of MMP-2, whereas treatment with wortmannin, a PI3 kinase inhibitor, showed no clear effect on MMP-2 secretion. Taken together, these results strongly suggest that MEK-MAP kinase signaling, but not PI3 kinase signaling, plays a critical role in the activation of MMP-2 secretion and, subsequently, in the invasiveness of v-src-transformed cells.

Animals↗

PubMed2000

Molecular cloning and characterization of human MAWD, a novel protein containing WD-40 repeats frequently overexpressed in breast cancer.

A full-length cDNA clone encoding a novel protein containing WD-40 repeats, which were frequently involved in protein-protein interactions, was isolated and sequenced. This clone had a predicted open reading frame (ORF) encoding 350 amino acids possessing six repeats of WD-40 motif. It was most closely homologous to TRIP-1, a phosphorylation substrate of the transforming growth factor-beta type II receptor. In the process of characterizing the function of the new gene product, we found that overexpression of the gene seemed to activate mitogen-activated protein kinase and to promote anchorage-independent growth of the cells. Moreover, the gene product was frequently overexpressed in human tumor breast tissues compared with their normal breast tissues, suggesting that the gene might be involved in the tumor progression. Radiation hybrid mapping placed the gene into human chromosome 12q11-12 near the marker D12S1593.

Amino Acid Sequence↗

PubMed2000

Molecular cloning of a novel human gene (SIRP-B2) which encodes a new member of the SIRP/SHPS-1 protein family.

A full-length cDNA encoding a novel protein was isolated and sequenced from a human placental cDNA library. This cDNA consists of 1,735 base pairs and has a predicted open reading frame (ORF) encoding 354 amino acids. It possesses a putative signal sequence, a long extracellular domain, a transmembrane region, a short intracellular domain, and no catalytic domain, which is highly homologous to signal-regulatory protein (SIRP)-beta suggesting that it seems to be a new member of the SIRP family. Polymerase chain reaction (PCR)-based mapping with both a monochromosomal hybrid panel and radiation hybrid cell panels placed the gene to human chromosome 20p13 near the marker D20S906.

Amino Acid Sequence↗

PubMed2000

Fibronectin activates matrix metalloproteinase-9 secretion via the MEK1-MAPK and the PI3K-Akt pathways in ovarian cancer cells.

Cell adhesion to the extracellular matrix appears to trigger a cascade of intracellular signalings. We have previously shown that treatment of ovarian cancer cells, NOM1, with fibronectin (FN) stimulated matrix metalloproteinase (MMP)-9 secretion and thereby activated the invasiveness of cells via the FAK/Ras signaling pathway. By use of chemical inhibitors, we investigated the downstream effectors critical for FN-dependent secretion of MMP-9. Treatment of cells with MEK1 inhibitors, U0126 and PD98059, dramatically suppressed the secretion of MMP-9 activated by FN. Similarly, P1-3 kinase inhibitors, Wortmannin and LY294002, strongly suppressed the FN-dependent secretion of MMP-9 together with the inhibition of Akt activation. In contrast, a specific PKC inhibitor (GF109203X) showed no inhibitory effect on the FN-dependent MMP-9 secretion. Moreover, we found that both the MEK1 inhibitor and the P13-K inhibitor, but not the PKC inhibitor, strongly suppressed the invasiveness of NOM1 cells. Taken together, our results suggest that activation of dual signaling pathways, MEKI-MAPK and P13K-Akt, is required for the FN-dependent activation of MMP-9 secretion. Our results suggest the importance of these signaling molecules as a chemotherapeutic target for cancer.

Androstadienes↗

PubMed2000

Successful ribavirin therapy for severe adenovirus hemorrhagic cystitis after allogeneic marrow transplant from close HLA donors rather than distant donors.

Intravenous ribavirin was given to nine patients who had developed severe adenovirus-induced hemorrhagic cystitis (AD-HC) which was resistant to conventional therapy or where there was involvement of other organs after allogeneic BMT. Three patients recovered completely from AD-HC, two of whom had been resistant to vidarabine. All three had received sibling BMTs (2 HLA matched, 1 HLA mismatched). Five patients who received BMTs from related (2 HLA mismatched) or unrelated (1 HLA matched, 2 HLA mismatched) showed an improvement in symptoms but had recurrent AD-HC after discontinuation of ribavirin. Improvement in clinical symptoms and termination of virus excretion were well correlated. The last patient who received a mismatched unrelated BMT died during ribavirin therapy. Ribavirin was notably more effective among patients receiving BMTs from siblings in contrast to patients receiving BMTs from alternative donors (<0.05). One patient experienced severe pancytopenia during the second treatment with ribavirin after HC recurrence and recovered after ceasing ribavirin. Thus, ribavirin seems to be very effective for severe AD-HC for some recipients who receive transplants from a genetically close donor. Bone Marrow Transplantation (2000) 25, 545-548.

Adenoviridae Infections↗

PubMed2000

A case of purpura fulminans is caused by homozygous delta8857 mutation (protein C-nagoya) and successfully treated with activated protein C concentrate.

We report a Japanese patient who developed purpura fulminans and disseminated intravascular coagulation (DIC) shortly after birth. The patient was diagnosed to be homozygous for protein C deficiency and was treated with an activated protein C (APC) concentrate. Intravenous infusions of APC markedly improved the necrotic skin lesions and the anticoagulation by APC enabled successful DIC control. The identified mutation (Delta8857) results in impaired intracellular transport and protein maturation and would be the cause of the complete protein C deficiency. This is the seventh case of the mutation that has been exclusively reported in Japan, but is the first report of a homozygous case. Our findings propose new therapeutic and diagnostic tools for the management of this fatal thrombotic disease.

Disseminated Intravascular Coagulation↗

PubMed2000

Combined effects of inhaled nitric oxide and positive end-expiratory pressure during mechanical ventilation in acute respiratory distress syndrome.

We studied the combined effects of inhaled nitric oxide (INO) and positive end expiratory pressure (PEEP) during mechanical ventilation in patients with acute respiratory distress syndrome (ARDS). Eleven patients received 0 and 4 parts per million of INO in random order for 30 min at PEEP levels of 0, 5, and 10 cm H2O. Respiratory and cardiovascular parameters were measured. The addition of INO and PEEP significantly improved arterial oxygenation (p < 0.005 and p < 0.0001, respectively). The combined effect of INO and PEEP on arterial oxygenation was remarkable during 10 cm H2O PEEP. There was synergistic effect on arterial oxygenation by combining INO and 10 cm H2O PEEP. The present study showed that the combination of INO and 10 cm H2O PEEP enhanced arterial oxygenation in patients with ARDS.

Administration, Inhalation↗

PubMed2000

Clinical significance of major and minor bcr/abl chimeric transcripts in essential thrombocythemia.

BACKGROUND: Contradictory results have been reported in terms of detecting bcr/abl transcripts in patients with essential thrombocythemia. The aim of this study was to investigate whether the bcr/abl transcript could be found in patients with essential thrombocythemia (ET). METHODS: The bcr/abl transcript was amplified by the RT-nested PCR method using RNA extract from leukocytes taken from 14 essential thrombocythemia patients. The amplified DNAs were electrophoresed in 1% agarose and visualized with ethidium bromide. The DNA bands associated with the bcr/abl transcript were then extracted and followed by DNA sequence analysis. RESULTS: Major bcr/abl transcripts of the b3a2 type and minor ones of the e1a2 type were found in one and two ET patients, respectively. The incidence of bcr/abl transcripts was 21.4% (three of 14 patients). CONCLUSION: Our experiments confirmed that bcr/abl transcripts are present in some patients with essential thrombocythemia.

Adult↗

PubMed2000

Rap2 as a slowly responding molecular switch in the Rap1 signaling cascade.

Rap2 is a member of the Ras family of GTPases and exhibits 60% identity to Rap1, but the function and regulation of Rap2 remain obscure. We found that, unlike the other Ras family proteins, the GTP-bound active form exceeded 50% of total Rap2 protein in adherent cells. Guanine nucleotide exchange factors (GEFs) for Rap1, C3G, Epac (or cyclic AMP [cAMP]-GEF), CalDAG-GEFI, PDZ-GEF1, and GFR efficiently increased the level of GTP-Rap2 both in 293T cells and in vitro. GTPase-activating proteins (GAPs) for Rap1, rap1GAPII and SPA-1, stimulated Rap2 GTPase, but with low efficiency. The half-life of GTP-Rap2 was significantly longer than that of GTP-Rap1 in 293T cells, indicating that low sensitivity to GAPs caused a high GTP/GDP ratio on Rap2. Rap2 bound to the Ras-binding domain of Raf and inhibited Ras-dependent activation of Elk1 transcription factor, as did Rap1. The level of GTP-Rap2 in rat 3Y1 fibroblasts was decreased by the expression of v-Src, and expression of a GTPase-deficient Rap2 mutant inhibited v-Src-dependent transformation of 3Y1 cells. Altogether, Rap2 is regulated by a similar set of GEFs and GAPs as Rap1 and functions as a slowly responding molecular switch in the Rap1 signaling cascade.

3T3 Cells↗

PubMed2000

Intercept-PCR, an improvement for elevating performance to find a new member of a certain gene family.

We have established a method by which the performance of reverse transcriptase coupled polymerase chain reaction (RT-PCR) for seeking a new gene is improved. The actual procedure is quite easy: it is only to add several specific oligonucleotides into the reaction mixture of the usual RT-PCR. To verify the effectiveness of this method is also easy: it is only to detect the PCR products in the preliminary experiment. The finding in the present study provides valuable information for gene cloning tactics.

Base Sequence↗

PubMed2000

Velopharyngeal closure pattern exerted by contraction of the pharyngeal constrictor muscle in dogs.

OBJECTIVE: This study was performed to investigate aspects of velopharyngeal closure demonstrated by movements of the parapharyngeal wall under various physiological conditions and especially the stimulation of several intrapneumonal receptors. MATERIALS AND METHODS: Fifteen mongrel dogs were anesthetized and a nasopharyngeal fiberscope was inserted from the oronasal fistula at the center of the hard palate, with the tip fixed around the choana. Mechanical and chemical stimulation was applied, and ongoing velopharyngeal movements were observed. In each case examined, the muscle nerve located under the bilateral levator veli palatini (LVP) was severed. RESULTS: Two different closure patterns were recognized between rebreathing and deglutition. Chemical stimulation of the pulmonary irritant receptor and type-J receptor induced a sphincteric tight closure of the velopharynx but only a weak inward movement of the lateral pharyngeal wall at lung inflation. CONCLUSION: The movements of the parapharyngeal wall, mainly represented by the pharyngeal constrictor muscle's contraction, are facilitated as a sphincteric velopharyngeal closure under reflexive movements such as deglutition or gagging.

Animals↗

PubMed2000

C-Cbl protein in human cancer tissues is frequently tyrosine phosphorylated in a tumor-specific manner.

To search for the signaling pathway relevant to the tumorigenesis in human, we examined the expression and tyrosine phosphorylation of c-cbl proto-oncogene product, c-Cbl, in various human cancer cell lines and surgical specimens. In cells derived from human cancer, we found constitutive tyrosine phosphorylation of c-Cbl protein, whereas its phosphorylation was undetectable in control ECV304 cells. Expression and tyrosine phosphorylation of c-Cbl was also examined in various surgical specimens. Thirty-six surgical specimens obtained from human tumor tissues were studied: 9 gastric carcinomas, 10 colon carcinomas, 6 renal carcinomas, 2 hepatomas, 2 brain tumors, 2 uterus tumors, 1 breast carcinoma, 1 thyroid tumor, 1 bladder tumor and 2 lung carcinomas. We found tyrosine phosphorylation of c-Cbl protein in 12 cases (33%) of these tumor tissues in a tumor-specific manner. These results suggest the importance of c-Cbl signaling in tumorigenesis in human.

Carcinoma↗

PubMed2000

Prognostic impact of telomerase activity in patients with neuroblastoma.

Neuroblastoma is one of the most common malignant neoplasms occurring among children. The prognosis for this disease is strongly associated with age, disease stage, histology, and some biologic features. It has been reported that telomerase, a ribonucleoprotein enzyme, which maintains the telomere length in immortal cells, is related to disease stage and other biologic features. The purpose of this study was to evaluate the prognostic value of telomerase activity compared to TrkA expression in 65 patients with neuroblastoma. Telomerase activity and TrkA expression were examined in tissue samples collected between 1980 and 1994 from 65 patients by polymerase chain reaction-based telomerase activity. TrkA expression was examined by immunoblotting using a rabbit anti-gp140 proto-trk polyclonal antibody. Low telomerase activity was found in 22 of 30 (73.3%) patients with Stages 1, 2, or 4S neuroblastomas; 7 of 13 (53.8%) with Stage 3; and 8 of 22 (36.3%) with stage 4; no telomerase activity was detected in 7 of 22 (31.8%) patients with Stage 4 neuroblastoma. The 5-year event-free survival (EFS) rate was 86.5% for patients with low telomerase activity, while it was 53.8% for patients with high telomerase activity. By the combination of telomerase activity and TrkA expression, the 5-year EFS rate was highest among patients with a high TrkA expression and a low or non-existent telomerase activity (91.7%), and it was lowest among patients with a low TrkA expression and a high telomerase activity (29.6%). Thus, it appears that telomerase activity would be a useful prognostic factor for neuroblastoma, especially when used in combination with the TrkA expression.

Adolescent↗

PubMed2000

Combination of inhaled nitric oxide therapy and inverse ratio ventilation in patients with sepsis-associated acute respiratory distress syndrome.

Inverse ratio ventilation (IRV) is a ventilatory technique that uses an inspiratory to expiratory ratio (I:E) greater than 1:1. We studied the effects of mechanical ventilation with an I:E of 1:3, 1:1, and 2:1 on arterial oxygenation in 10 patients with sepsis-associated acute respiratory distress syndrome (ARDS). At each I:E, patients received 0 and 4 ppm of inhaled nitric oxide (INO) in random order for 30 min. Respiratory and cardiovascular parameters were measured. Of the 10 patients studied, 7 responded to IRV and 3 did not. An increase in the I:E and the addition of INO significantly improved arterial oxygenation in the responders (p < 0. 0001 and p < 0.006, respectively). The combination of an increase in the I:E and INO had an additive effect on arterial oxygenation. The combined use of IRV and INO is a more effective method of avoiding hypoxemia than either INO or IRV alone.

Administration, Inhalation↗

PubMed2000

No significant association between HA-1 incompatibility and incidence of acute graft-versus-host disease after HLA-identical sibling bone marrow transplantation in Japanese patients.

We retrospectively examined HA-1 typing with polymerase chain reaction using sequence-specific primers in 120 samples from 60 HLA-A2-positive Japanese bone marrow transplantation recipients who received short-term methotrexate and cyclosporin A for graft-versus-host disease (GVHD) prophylaxis and their HLA-identical sibling donors. HA-1-incompatible pairs were observed in 22% of the samples. The probability of developing acute GVHD (grade II to IV) in HA-1-incompatible and -compatible patients was 0% and 19%, respectively (P = .10). In a comparison between HA-1-incompatible and -compatible patients with standard-risk leukemia, in whom age, patient/donor sex, and use of a total body irradiation-containing regimen were equivalent, the probability of developing acute GVHD (grade II to IV) was 0% and 10%, respectively (P = .38). No evidence of recurrent leukemia was observed in the HA-1-incompatible patients with standard-risk leukemia, compared with 37% in HA-1-compatible patients (P = .11). In conclusion, HA-1 incompatibility may not be a risk factor for grade II to IV acute GVHD in Japanese patients who receive methotrexate and cyclosporin A and undergo bone marrow transplantation from HLA-identical sibling donors.

Adolescent↗