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Biomedical subjects

M Hamaguchi

Publications and source records attributed to M Hamaguchi.

At least 181 records · Page 10Linked to original sources
PubMed1991

Comparison of protein tyrosine phosphorylation and morphological changes induced by IL-2 and IL-3.

We constructed cell lines which can proliferate in response to IL-2 or IL-3 by introducing a wild-type and mutant forms of cDNAs encoding the human IL-2R p75 chain into an IL-3 dependent hematopoietic cell line which expresses the p55 chain of the IL-2R. We compared early events that were induced in these cells by IL-2 and IL-3. Analysis of protein tyrosine phosphorylation showed that two common protein bands, pp95 and pp90, were phosphorylated by stimulation of either IL-2 or IL-3, suggesting the possible sharing of part of a signal transduction pathway between IL-2R and IL-3R. Comparison of protein tyrosine phosphorylation profiles induced by IL-2 and IL-3 among a variety of cell lines revealed that the pp90 band is the common tyrosine phosphorylation substrate in the cell lines examined, although the general tyrosine phosphorylation pattern differed in each cell line. Mutant p75 molecules incapable of inducing tyrosine phosphorylation could bind and internalize IL-2, but could not support cell growth. We also found that swift changes of cytoskeletal protein organization are one of the early events caused by signal transduction through either IL-2R and IL-3R. Reorganization of cytoskeletal proteins seems to be associated with protein phosphorylation, as a significant portion of pp90 was found in a detergent-soluble fraction in IL-2 or IL-3 treated cells.

Base Sequence↗
PubMed1991

Aberrant elevation of tyrosine-specific phosphorylation in human gastric cancer cells.

Phosphotyrosine-containing proteins in various human cancer cell lines were studied by immunoblotting with anti-phosphotyrosine antibody. Of 29 cell lines derived from oral epidermoid cancer, esophageal cancer, gastric cancer, colon cancer, pancreatic cancer, hepatocellular carcinoma and malignant melanoma, 3 of the 6 gastric cancer cells showed aberrant elevation of tyrosine-specific phosphorylation. On the other hand, both esophageal cancer cells and colon cancer cells, which were reported to have amplified epidermal growth factor receptor and activated p60v-src kinase, respectively, showed no apparent elevation of tyrosine-specific phosphorylation, and their profiles of phosphorylation were similar to that of normal human fibroblasts. Two gastric cancer cells, NUGC-4 and MKN-45, showed similar profiles of phosphorylation but their responses to growth factors differed from each other. Tyrosine phosphorylation in NUGC-4 was strongly activated by treatment with epidermal growth factor and quickly reduced by the acid treatment which is effective in removing growth factors from cellular surface receptors. On the contrary, phosphorylation in MKN-45 did not respond to either growth factor or acid treatment. These results suggest that NUGC-4 and MKN-45 have tyrosine kinases which are activated by different mechanisms but share similar substrates.

ErbB Receptors↗
PubMed1991

Effect of carvedilol on venous return: a mechanism of reduction in blood pressure.

We studied the effect of carvedilol, a beta-blocker with a potent alpha-blocking activity, on venous return in anesthetized dogs. Though 3 micrograms/kg of carvedilol caused a slight increase in blood pressure and total peripheral resistance, these changes disappeared by further doses of the agent. On the other hand, each dose of carvedilol (3-100 micrograms/kg) decreased venous return significantly and dose-dependently. These results indicate that the hypotensive effect of carvedilol mainly depends on the decrease in venous return rather than arterial vasodilation.

Adrenergic beta-Antagonists↗
PubMed1990

An endoprotease homologous to the blood clotting factor X as a determinant of viral tropism in chick embryo.

Host cell proteases responsible for activation of viral fusion glycoproteins are an important determinant for spread and tropism of various animal viruses. Exemplifying such proteases for the first time, we isolated an endoprotease from chick embryo, that activates para- and orthomyxovirus fusion glycoproteins by cleaving their precursor proteins at a specific, single arginine site. The protease is a calcium dependent serine protease consisting of two subunits, the 33 kd catalytic chain and the 23 kd chain possibly required for Ca2+ binding, and was found to be highly homologous, if not identical, to the blood clotting factor X(FX), a member of the prothrombin family. Its high efficiency and specificity in cleavage reactions was attributable to the properties characteristic of FX. Its role in vivo was strongly supported by cleavage inhibition in ovo highly selective for this virus group with a specific peptide inhibitor against FX.

Amino Acid Sequence↗
PubMed1990

The study of energy metabolism in denervated skeletal muscle with 31P-NMR.

1. The denervated frog sartorius muscle showed a decrease in the energy store more than that in the control. 2. In the caffeine contractures, both the denervated and the innervated muscles showed similar sequential changes in the relative concentration of phosphocreatine (PCr) to beta-adenosine triphosphate (beta-ATP) and inorganic phosphate (Pi) to beta-ATP. Instead, the intracellular pH value of the denervated muscle was lower than that of the control. 3. It is suggested that phosphate metabolism of the denervated muscle during contracture shows little difference from that of the control, nevertheless, the buffering capacity is decreased in the early stage of atrophy.

Animals↗
PubMed1990

Serum independence of transcription from the promoter of an avian retrovirus in v-src-transformed cells is a primary, intracellular effect of increased tyrosine phosphorylation.

We found that transcription from the promoter in the long terminal repeat of Rous sarcoma virus in rat 3Y1 cells is dependent on the presence of serum in the culture. However, this serum dependence of transcription was relieved when 3Y1 cells were transformed by the oncogene v-src. Crossfeeding experiments showed no evidence for the production of a serum-substituting extracellular growth factor by the transformed cells. Using 3Y1 cells transformed with temperature-sensitive Rous sarcoma virus, we showed that the tyrosine kinase activity of pp60v-src was responsible for the serum-sparing effect on the level of RNA expressed from the viral promoter. Sodium orthovanadate, an inhibitor of phosphotyrosine phosphatases that nonspecifically elevates the level of phosphotyrosine-containing proteins in cells, stimulated transcription from the viral promoter. The effects of both pp60v-src and orthovanadate were resistant to cycloheximide. These results suggest that the serum independence of transcription from the viral promoter in v-src-transformed cells was probably due to the constitutive activation of intracellular growth-factor pathways by the tyrosine kinase activity of pp60v-src.

Animals↗
PubMed1990

Production of prolactin by cultures of isolated cells from human first-trimester decidua.

An enriched fraction of first-trimester decidual cells that synthesize and release prolactin (PRL) was obtained by discontinuous Percoll gradient (20-50%) centrifugation of collagenase type I- and deoxyribonuclease I-dispersed cells (3 mg/mL and 50 micrograms/mL, respectively). Centrifugation of the cell suspension yielded three major bands aggregating at the density interfaces. The fraction of the 30-40% Percoll interface contained enlarged decidual cells and constantly secreted significant amounts of PRL into the medium for at least 10 days. The fraction of the 40-50% Percoll interface contained fibroblastic cells and secreted a small amount of PRL into the medium. Cells in the other fractions did not attach to the plastic dishes in 48 hours. Under the influence of progesterone (100 ng/mL), the cultured decidual cells retained their capability of PRL production for at least 10 days because no decline of the secretion rate was observed. The culture system established by the present study is satisfactory for investigating decidual cell functions, including the regulatory mechanisms of PRL production.

Adult↗
PubMed1990

A glycoprotein in the plasma membrane matrix as a major potential substrate of p60v-src.

A potential substrate of p60v-src in Rous sarcoma virus-transformed cells was found to be a 130-kilodalton (kDa) glycoprotein which binds to lectin-Sepharose and can be immunoprecipitated by an anti-phosphotyrosine antibody. This glycoprotein was shown to be distinct from the fibronectin receptor and a cellular protein phosphorylated in p60v-src immune complexes. The protein was a transmembrane protein localized in the plasma membrane and resistant to extraction with Triton X-100. The 130-kDa protein was also highly phosphorylated in cells transformed by Fujinami sarcoma virus or Y73 but not in cells infected with Rous sarcoma virus mutants that encode p60v-src lacking myristoylated N termini. Phosphorylation of this glycoprotein was temperature dependent in cells infected with temperature-sensitive mutants. The good correlation between its phosphorylation and morphological transformation, together with its relative abundance among phosphorylated proteins and its subcellular localization, suggests that phosphorylation of the 130-kDa glycoprotein is one of the primary events important for cell transformation by p60v-src and related oncogene products.

Animals↗
PubMed1990

Oncogenicity of the ret transforming gene in MMTV/ret transgenic mice.

We have successfully produced transgenic mice that carry the ret oncogene driven by a mouse mammary tumor virus promoter/enhancer. Mammary and salivary gland adenocarcinomas were developed in a stochastic fashion in these mice. Moreover, premalignant tumors with hyperplastic and dysplastic lesions of Harderian glands and male reproductive tracts frequently occurred at young ages. High expression of the transgene was closely associated with the development of these tumors although the levels of the transgene expression were variable among individuals. In addition, large amounts of phosphotyrosine-containing proteins were detected in cell lysates from mammary and salivary adenocarcinomas by immunoblotting with the anti-phosphotyrosine antibody.

Animals↗
PubMed1990

DNA sequence analysis of three inhibitor-positive hemophilia B patients without gross gene deletion: identification of four novel mutations in factor IX gene.

Three hemophilia B patients with anti-factor IX antibodies who had no detectable gross deletion of the factor IX gene by Southern blotting analysis were investigated at the molecular level. All eight exons, accompanied by their splicing junction sites and presumptive promoter regions of the factor IX gene in these patients (total 5.5 kb in length) were amplified with the use of the polymerase chain reaction, followed by complete nucleotide sequence analysis. Three different types of novel single base substitutions and a 2 base-pair nucleotide deletion were identified. Patient HB-5 had two point mutations in his factor IX gene. One was located at the promoter region at nucleotide -793 and the other (C-to-T transition) was found in exon VI of the gene changing Gln-191 to a stop codon. Patient HB-6 had a point mutation (G-to-A) in the splice acceptor site, which interrupted the normal splicing of the last intron G. A small two-nucleotide deletion in exon III was detected in patient HB-7 and yielded frameshifted amino acids and terminated by a stop codon. These resuslts suggest that not only the gross gene deletion of factor IX gene but also the point mutations or small nucleotide deletion that may cause the interruption of coding informations for mature protein synthesis is predisposed to development of anti-factor IX inhibitors in patients with hemophilia B.

Autoantibodies↗
PubMed1989

Comparison of recombinant tissue-type plasminogen activator (rt-PA) expressed in mouse C127 cells and human vascular plasminogen activator (HV-PA).

Tissue-type plasminogen activator produced by recombinant DNA technology (rt-PA) has now been recognized as a promising clot-selective thrombolytic agent. We have compared the properties of rt-PA expressed in mouse C127 cells with those of naturally occurring human vascular plasminogen activator (HV-PA). The molecular weight of HV-PA and rt-PA was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to be approx. 66,000. HV-PA and rt-PA were labile and rapidly lost their activities at pH values below 5.5. The optimum pH of HV-PA and rt-PA for plasminogen activation was around 8.5. HV-PA and rt-PA appeared to be very similar in amidolytic properties, amino-acid composition and carbohydrate composition. Moreover, the N-terminal amino-acid sequence of HV-PA was in good agreement with that of rt-PA. The purified preparations of HV-PA and rt-PA had specific activities of about 250,000 and 600,000 IU/mg, respectively. Both activators bound to fibrin clots to similar degree. In immunodiffusion as well as in the quenching experiments of the fibrinolytic activities, rt-PA appeared to be immunodiffusion as well as in the quenching experiments of the fibrinolytic activities, rt-PA appeared to be immunologically indistinguishable from HV-PA. All these findings indicate that rt-PA expressed in mouse C127 cells is identical with naturally occurring HV-PA in physical and chemical properties.

Amino Acid Sequence↗
PubMed1989

Dihydropyrimidines: novel calcium antagonists with potent and long-lasting vasodilative and antihypertensive activity.

The novel calcium antagonists 3-N-substituted-3,4-dihydropyrimidines 1 and 9 and 3-N-substituted-dihydro-pyrimidin-2(1H)-ones 8 were regioselectively synthesized in good yields. Compounds 1 [especially 1s [R1 = (CH2)2N(benzyl)(2-naphthylmethyl), R2 = i-Pr, X = 0-NO2] and 1t [R1 = (CH2)2N(benzyl)(3,4-dichlorobenzyl), R2 = i-Pr, X = 0-NO2]] exhibited not only more potent and longer lasting vasodilative action but also a hypotensive activity with slow onset as compared with dihydropyridines. Moreover, some dihydropyrimidines [1q [R1 = (CH2)2N(benzyl)(3-phenylpropyl), R2 = CH2(cyclopropyl), X = 0-NO2], 1s, and 1t] were weaker in blocking atrioventricular conduction in anesthetized open-chest dogs and less toxic than the dihydropyridines.

Animals↗
PubMed1989

In vivo phosphorylation states and kinase activities of transforming p60c-src mutants.

To further investigate regulation of p60src by tyrosine phosphorylation, the in vivo phosphorylation states and kinase activities of transforming mutants derived from p60c-src were examined. One set of six in vitro recombinants encoded chimeric proteins containing the carboxyl terminus of chicken p60c-src (including Tyr 527) in combination with various isolated amino acid substitutions present in p60v-src. Another set of seven viral isolates were randomly selected from soft-agar colonies infected with replicating retroviruses that originally encoded normal p60c-src. Results show that mutations at multiple sites throughout amino- and carboxy-terminal domains are sufficient to alter the in vivo phosphorylation state and kinase activity of p60c-src. Moreover, all of the transforming p60c-src mutants exhibited elevated in vivo kinase activities, as assayed by immune blot analysis of cellular proteins using antibodies to phosphotyrosine, which correlated well with enhanced auto-phosphorylation at Tyr 416. By contrast, Tyr 527 phosphorylation in the transforming mutants varied from low to high levels that were correlated to a limited extent with levels of in vivo kinase activities. Taken together, results suggest that, in addition to complete dephosphorylation of Tyr 527, other mechanisms contribute to p60c-src activation.

Animals↗
PubMed1989

Localization of major potential substrates of p60v-src kinase in the plasma membrane matrix fraction.

Subcellular localization of potential substrates of a tyrosine-protein kinase, p60v-src, was analyzed by cell fractionation in combination with immunoblotting with antiphosphotyrosine antibody. In cells transformed by wild type Rous sarcoma virus, most phosphotyrosine-containing proteins were found both in plasma membranes and in a cytoplasmic matrix structure associated with plasma membranes and resistant to nonionic detergent extraction (plasma membrane matrix). A similar localization of phosphotyrosine-containing proteins was obtained in cells transformed by PRCII, Y73, or Fujinami sarcoma virus. On the other hand, in cells infected with Rous sarcoma virus mutants that encode nonmyristylated p60v-src, tyrosine phosphorylation was found mostly in proteins which were different from those identified in wild type-infected cells and were distributed to both plasma membrane and cytosolic fractions. These results suggest that most cellular substrate proteins, phosphorylation of some of which may be critical for the initiation of transformation, are present primarily in the plasma membrane-matrix structure.

Animals↗
PubMed1989

[A carcinoma of the right breast arising after a mastectomy in primary malignant lymphoma of the left breast].

In 1987, a 51-year-old woman visited our hospital, having noticed a painless 1.5 x 1.5 cm tumor of the left breast. On biopsy, it was pathologically diagnosed as being a primary malignant lymphoma of the breast. Thus we performed a modified radical mastectomy (Patey's method), in combination with chemotherapy (Cyclophosphamide, Vincristine, Prednisolone). Two years after the operation, a tumor of the right breast, 1.0 x 1.0 cm in size, was noticed. At this time, since an excised specimen was pathologically determined as being a scirrhous carcinoma, we performed a modified radical mastectomy (Auchincloss' method), followed by chemotherapy (Tegafur).

Adenocarcinoma, Scirrhous↗
PubMed1989

Extragenic factor IX gene RFLP is useful for detecting carriers of Japanese hemophilia B.

Seventy-eight X chromosomes from 25 normal Japanese subjects and 22 family members with hemophilia B (coagulation factor IX deficiency) were examined with an extragenic factor IX DNA probe, pX58dIIIc at DXS99 locus. In contrast to the previously described nonpolymorphic RFLPs in the factor IX gene, DXS99 locus RFLP produced by SacI digestion was detected among those Japanese subjects with allelic frequencies of 0.48 and 0.52. The estimated heterozygosity rate of this extragenic RFLP among Japanese females was about 50%. The study of hemophilia B family members showed that DXS99 locus RFLP was informative in 9 out of 13 families tested (69.2%). No recombination events between the factor IX gene locus (F9) and DXS99 locus have been noted among nine families analyzed. DXS99 SacI RFLP is a useful gene indicator of carrier-ship of hemophilia B.

Factor IX↗