Biomedical subjects

H Yamamura

Publications and source records attributed to H Yamamura.

At least 163 records · Page 9Linked to original sources

PubMed1995

Scanning microscopic evaluation on the development of the cerebral cortex in embryonic mouse subjected to gamma-irradiation.

Morphological events occurring in the developing cerebral hemispheres of mice exposed to a single dose of 60Co gamma-irradiation 1.5 Gy on embryonic day 13 (E13) were evaluated by scanning microscope. Twenty-four hr after the exposure, both cell debris and surviving cells had poured out into the ventricular lumen. Radial glial fibers were more crumpled than in the controls. By day E15, proliferating cells in different stages of the cell cycle appeared in the ventricular zone. The glial fibers formed a network through the brain mantle. By E17 many migrating cells attached to the disorderly glial fibers appeared in the different layers of the thin cerebral mantle. These findings suggest that development of the glial fibers was interrupted as early as 24 hr after the single exposure, meaning that irradiation on the developing brain may disrupt neuronal migration.

Abnormalities, Radiation-Induced↗

PubMed1994

Translocation of p72syk to the cytoskeleton in thrombin-stimulated platelets.

Thrombin stimulation induces a dramatic increase in the activity of p72syk in platelets. We have found that activated p72syk, which is phosphorylated on tyrosine residue(s), translocates from the Triton X-100-soluble fraction to the Triton X-100-insoluble, cytoskeleton-rich fraction after thrombin stimulation. In addition, the redistribution of p72syk from the 100,000 x g Triton X-soluble fraction and the membrane skeleton was found to correlate with an increased level of p72syk in the cytoskeleton. Furthermore, the early phase of p72syk translocation (within 60 s) was significantly inhibited with cytochalasin D, whereas the late phase of p72syk translocation (after 90 s) was completely inhibited with RGDS tetrapeptide treatment. These results suggest that translocation of the activated p72syk to the cytoskeleton correlates with different phases of the platelet activation process through actin polymerization and glycoprotein IIb/IIIa-fibrinogen-mediated aggregation of platelets and, hence, may have a regulatory role in tyrosine phosphorylation of platelets.

Animals↗

PubMed1994

Involvement of protein-tyrosine kinase p72syk in collagen-induced signal transduction in platelets.

Previous studies have demonstrated that activation of platelets by collagen results in a dramatic increase in tyrosine phosphorylation of several cellular proteins, including pp125FAK, through the interaction of collagen with integrin alpha 2 beta 1 (GP Ia-IIa). In this study, we report that p72syk is a potential candidate for the protein-tyrosine phosphorylation event following collagen stimulation in porcine platelets. Washed platelets were stimulated with collagen and the activation of p72syk was assessed in an immunoprecipitation kinase assay. The activity of p72syk increased within 1 min, reached a maximum at 5 min after stimulation by collagen, and the phosphorylation at tyrosine residues of p72syk in platelets also occurred in the same time course as the activation of p72syk. Prior treatment of platelets with cytochalasin D to inhibit actin polymerization, or with aspirin and apyrase to inhibit the secondary reaction, or EGTA and the acetoxymethyl ester of 5,5'-dimethyl-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid to chelate both extracellular and intracellular Ca2+, did not affect the activation of p72syk induced by collagen. Furthermore, herbimycin A, a potent protein-tyrosine-kinase inhibitor, was capable of reducing collagen-evoked p72syk activation, Ca2+ mobilization and platelet aggregation. These results suggest that upon stimulation by collagen p72syk is physically activated by a process that is independent of the effects of Ca2+, ADP, and actin polymerization, and may participate in the regulation of Ca2+ mobilization mediated by collagen in platelets.

Animals↗

PubMed1994

Endothelin-1, one of the most potent histamine releasers in mouse peritoneal mast cells.

Whether endothelin-1 or -3 is capable of inducing histamine release from the peritoneal mast cells of BALB/c mice was investigated in vitro and compared to the release induced by compound 48/80. In contrast to the mouse bone marrow-derived mast cells, which originated from the same strain and have been reported upon previously, the (both crude and purified) peritoneal mast cells potently secreted histamine in response to either endothelin-1 or endothelin-3 in a concentration-dependent fashion. Even at a concentration as low as 10 nM, endothelin-1 induced a histamine release of more than 50% from the peritoneal mast cells. Cyclo(D-Asp-Pro-D-Val-Leu-D-Trp) (BQ-123), an endothelin ETA receptor antagonist, markedly suppressed not only the histamine released induced by endothelin-1 but also that due to endothelin-3 at a similarly low concentration range. Treatment with islet-activating protein (IAP) for 3 h, an inactivator of guanosine triphosphate (GTP) (Gi)-protein, markedly reduced the histamine release induced by endothelin-1. Neomycin at 0.1 and 1 mM or ethylenediaminetetraacetic acid (EDTA) at 0.1 mM in the absence of Ca2+, neither of which affected the histamine release induced by endothelin-1, substantially reduced histamine release caused by compound 48/80. On the other hand, treatment with O-O'-bis(2-aminophenyl)ethyleneglycol-N,N,N',N'-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM), an intracellular calcium chelating agent, completely inhibited the release induced by both endothelin-1 and compound 48/80. These results indicate that endothelin-1 is one of the most potent histamine releasers in mouse peritoneal mast cells discovered so far.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of Variance↗

PubMed1994

Radioadaptive response in primary mouse spermatocytes revealed by analysis of synaptonemal complexes.

This study was aimed at estimating the damage to the synaptonemal complex (SC) of male mice caused by radiation exposure at the ultimate pre-meiotic interphase. Four experimental groups were formed: group 1 was an untreated control, group 2 received a low dose (50 mGy) of radiation, group 3 a high dose (4 Gy) of radiation, group 4 was first treated with a low dose (50 mGy) and 4 h later with a high dose (4 Gy). Mice were killed 4 days after the treatment. Early pachytene cells were selected for the electron microscopic analysis of SCs. These cells were supposed to be at pre-meiotic interphase at the time of irradiation. Treatment with a low dose of radiation did not produce any substantial increase in the frequency of SC aberrations, while exposure to the high dose resulted in various types of damage. Group 4 demonstrated significantly lower frequencies of axial breaks/fragments and multiaxial configurations than group 3. At the same time, these two groups did not differ in the frequencies of inter- and intrachromosomal exchanges such as translocations, inversions, and deletions. We suppose that the number of breaks produced by a high dose of radiation was the same in the both groups. However, a proportion of the breaks that remained unrepaired until pachytene and were expressed as gaps, fragments, and multiaxial configurations was reduced by pretreatment with the low dose of radiation.

Adaptation, Physiological↗

PubMed1994

Translocation, activation and association of protein-tyrosine kinase (p72syk) with phosphatidylinositol 3-kinase are early events during platelet activation.

We have previously reported that a non-receptor-type protein-tyrosine kinase p72syk, exists in both membrane and cytosolic fractions in porcine platelets and is activated after thrombin stimulation. To facilitate the understanding of the function of p72syk, we have investigated the topological features, kinase activities and the interaction with another signal-transducing molecule, namely phosphatidylinositol 3-kinase, during platelet activation. Membrane and cytosolic fractions were separated from thrombin-treated porcine platelets, and the amount of p72syk was quantified by the immunoblot technique or the kinase activity of each fraction was determined by an immunoprecipitation kinase assay. After stimulation by thrombin, cytosolic p72syk rapidly translocated to the membrane fraction within 10 s and there was also a significant increase in the amount of p72syk in the cytoskeletal fraction. The autophosphorylation activity of membrane-associated p72syk significantly increased approximately tenfold and reached a maximum at 10 s; the activity subsequently decreased to almost the basal level within 120 s. For similar time courses, association of p72syk with phosphatidylinositol 3-kinase and tyrosine phosphorylation of p72syk were observed. These results suggest that translocation, activation, and association of p72syk with transducing molecules such as phosphatidylinositol 3-kinase, events which occur during platelet activation, may participate in early signal-transduction events.

Animals↗

PubMed1994

Interleukin 2 mediates p72syk activation in peripheral blood lymphocytes.

The ability of interleukin 2 (IL-2) to stimulate p72syk activity in intact porcine peripheral blood lymphocytes was examined. We demonstrated that IL-2 activated p72syk in a time- and dose-dependent manner, for which its peak time and maximum responsive dose were 5 min and 100 U/ml, respectively. This activation was observed only in cytosolic fractions and not in membrane ones. However, IL-2 failed to induced calcium mobilization. Moreover, IL-2-inducible p72synk activation was not affected when extra- intracellular calcium was depleted. These data suggest that the IL-2 signaling pathway through p72syk in peripheral blood lymphocytes is different, at least in part, from other agonists, such as concanavalin A in polymorphonuclear neutrophils which can trigger both the activation of p72syk and intracellular calcium mobilization.

Animals↗

PubMed1994

Endothelin-1 induces release of histamine and leukotriene C4 from mouse bone marrow-derived mast cells.

Whether specific binding sites for endothelin-1 and endothelin-3 exist in mouse bone marrow-derived mast cells (BMMC) and if these endothelins are capable of stimulating chemical mediator release from the cells was investigated. A single component of binding sites for endothelin-1 was found in the cells, but no binding sites for endothelin-3 were observed. Endothelin-1 at 1-100 nM concentration dependently induced release of histamine and immunoreactive leukotriene C4 from BMMC, while endothelin-3 at up to 100 nM did not stimulate the release of either mediator. Time course experiments revealed that the release of histamine and immunoreactive leukotriene C4 induced by endothelin-1 occurred rapidly, reaching near maximal levels within 20 s and 2 min, respectively, after the stimulation, while histamine release induced by antigen, at the concentration which induced an extent of release similar to that induced by 100 nM endothelin-1, required comparatively prolonged incubation (approximately 10 min for submaximal levels). Cyclo(D-Asp-Pro-D-Val-Leu-D-Trp) (BQ-123), a selective antagonist of endothelin ETA receptors, not only dissociated [125I]endothelin-1 specifically bound to BMMC but also inhibited the release of both mediators from endothelin-1-induced cells. These results suggested strongly that BMMC have endothelin ETA receptors on their cell membrane, stimulation of which leads to chemical mediator release, probably via a mechanism different from that involved in the antigen-induced release.

Animals↗

PubMed1994

CPTK71, a cytosolic protein-tyrosine kinase previously purified from bovine platelets, is identical with p72syk.

In the previous study, we reported purification and characterization of cytosolic protein-tyrosine kinase, CPTK71, from bovine platelets (Nakamura, S., Yanagi, S. and Yamamura, H. (1988) Eur. J. Biochem. 174, 471-477). In the present study, we have investigated the relationship between CPTK71 and p72syk which was purified from cytosolic fraction of porcine spleen and sequenced from porcine cDNA library. The elution patterns of CPTK71 activity in each purification step closely corresponded with the pattern of immunoreactivity with p72syk. Immunoprecipitation study revealed that specific antibodies against p72syk precipitated the activity of both phosphorylating [Val5]angiotensin II and autophosphorylation of highly purified CPTK71. In addition, the same result was obtained using another anti-p72syk antibody which recognized different amino acid sequence of 72syk. From these results, we conclude that CPTK71 is identical or closely related to p72syk.

Animals↗

PubMed1994

Syk activation by the Src-family tyrosine kinase in the B cell receptor signaling.

Signaling through the B cell antigen receptor (BCR) results in rapid increases in tyrosine phosphorylation on a number of proteins. The BCR associates with two classes of tyrosine kinase: Src-family kinase (Src-protein-tyrosine kinase [PTK]; Lyn, Fyn, Blk, or Lck) and Syk kinase. We have investigated the interaction between the Src-PTK and the Syk kinase in the BCR signaling. In contrast to wild-type B cells, BCR-mediated tyrosine phosphorylation of Syk and activation of its in vitro kinase activity were profoundly reduced in lyn-negative cells. The requirement of the Src-PTK to induce tyrosine phosphorylation and activation of Syk was also demonstrated by cotransfection of syk and src-PTK cDNAs into COS cells. These results suggest that the Src-PTK associated with BCR phosphorylates the tyrosine residue(s) of Syk upon receptor stimulation, enhancing the activity of Syk.

Animals↗

PubMed1994

Purification and characterization of a protein-tyrosine kinase p72syk from porcine spleen.

We have succeeded in purifying p72syk, a non-receptor-type protein-tyrosine kinase carrying high susceptibility to proteolysis [Taniguchi, T., Kobayashi, T., Kondo, J., Takahashi, K., Nakamura, H., Suzuki, J., Nagai, K., Yamada, T., Nakamura, S. and Yamamura, H. (1991) J. Biol. Chem. 266, 15790-15796] from porcine spleen. The purification procedure involves a sequential column chromatography, following extraction with 0.5 M NaCl from spleen homogenate, on phosphocellulose, Sephacryl S-200, heparin-Sepharose CL-6B, Mono Q and Mono S. SDS/PAGE of the final purified sample revealed a 72-kDa protein band with about 95% purity and immunodepletion analysis showed immunological cross-reactivity with anti-p72syk antibody which does not recognize ZAP-70. It was purified approximately 3000-fold with an overall yield of 0.54% according to [Val5]angiotensin II phosphorylation activity and the specific activity of the final sample (30 nmol phosphate.min-1.mg protein-1) was relatively lower than that of the 40-kDa kinase, a catalytic fragment of p72syk which lacks two src homology regions 2 domains. The p72syk had an autophosphorylation activity that was performed by intramolecular catalysis accompanied by a phosphate exchange reaction, and could efficiently phosphorylate tubulin, myelin basic protein and H2B histone. Employing [Val5]angiotensin II as a substrate, the apparent Km value for the peptide was 0.91 mM and that for ATP was 0.48 microM. Mn2+, Mg2+ and Co2+ were effective divalent cations and optimum pH was around 8.0-8.5 for the expression of the activity. These results suggest that the purified p72syk may exist as a less active form compared with the 40-kDa kinase and that the part of p72syk containing two src homology region 2 domains may participate in the regulation of its activity though the enzymic character is quite similar to that of the 40-kDa kinase.

Amino Acids↗

PubMed1994

Activation of p72syk by thrombin in a cell-free system.

Thrombin dramatically activated p72syk in a time- and dose- dependent fashion in extracts of resting porcine platelets in the presence of EDTA. Separation analysis using Sephacryl S-300 column chromatography has demonstrated that p72syk may exist as large (complex) and small (monomer) forms in resting platelets, and activation of p72syk was only observed in the fraction of large form. Pretreatment with ATP scavenger, GDP beta S and protein phosphatase inhibitors had no effect on this activation. Furthermore, washed immuno-precipitates of large form p72syk were also activated by thrombin or fibrinogen. These results suggest that p72syk may associate with thrombin receptor or other agonist receptors and there may be a novel activation mechanism of non-receptor type protein-tyrosine kinase, which does not require the modification by other protein kinases, protein phosphatases and GTP binding proteins.

Animals↗

PubMed1994

Tyrosine kinases Lyn and Syk regulate B cell receptor-coupled Ca2+ mobilization through distinct pathways.

Stimulation of B lymphocytes through their antigen receptor (BCR) results in rapid increases in tyrosine phosphorylation on a number of proteins and induces both an increase of phosphatidylinositol and mobilization of cytoplasmic free calcium. The BCR associates with two classes of tyrosine kinase: Src-family kinase (Lyn, Fyn, Blk or Lck) and Syk kinase. To dissect the functional roles of these two types of kinase in BCR signaling, lyn-negative and syk-negative B cell lines were established. Syk-deficient B cells abolished the tyrosine phosphorylation of phospholipase C-gamma 2, resulting in the loss of both inositol 1,4,5-trisphosphate (IP3) generation and calcium mobilization upon receptor stimulation. Crosslinking of BCR on Lyn-deficient cells evoked a delayed and slow Ca2+ mobilization, despite the normal kinetics of IP3 turnover. These results demonstrate that Syk mediates IP3 generation, whereas Lyn regulates Ca2+ mobilization through a process independent of IP3 generation.

Animals↗

PubMed1994

Immunocytochemical detection of Ca(2+)-dependent subspecies of protein kinase C in mouse embryos before and during compaction.

To confirm the possibility that protein kinase C is involved in compaction of mouse embryos, the presence and distribution pattern of Ca(2+)-dependent subspecies of this enzyme in mouse embryos, before and during compaction, were examined immunocytochemically with three different monoclonal antibodies. These were MC-1a, MC-2a and MC-3a, which selectively interact with the subspecies of the enzyme known as types I, II and III, respectively. Only when embryos were incubated with MC-3a, was immunofluorescence clearly detected in all cells of embryos before and during compaction. This result demonstrates the presence of type III protein kinase C in embryos before and during compaction and suggests the possibility that the type III enzyme may be involved in compaction. No marked differences were found in the distribution pattern of the type III enzyme between embryos examined before and during compaction.

Animals↗

PubMed1994

Membrane structure of parallel-fibre synaptic terminals in the cerebellum of the jaundiced Gunn rat: freeze-fracture and E-PTA study.

Parallel-fibre synaptic membranes were examined by freeze-fracture and ethanolic-phosphotungstic acid methods in the cerebellum of homozygous (j/j) Gunn rats with hereditary jaundice. Parallel-fibre synapses with dendritic spines of Purkinje cell were severely affected since many Purkinje cells degenerated during the early postnatal period. Some parallel-fibre synaptic terminals lacked their postsynaptic partners and faced astrocytic processes from 18 days of age to the adult stage. These parallel-fibre terminals contained clusters of synaptic vesicles adjacent to synaptic membranes, and synaptic membranes and synaptic cleft materials were identical to those of parallel fibres with postsynaptic partners, as visualized by conventional electron microscopy with osmium tetroxide postfixation and staining of sections with uranyl acetate and lead citrate. In freeze-fractured specimens, the presynaptic membrane of parallel fibres had diffusely distributed large particles and tiny pits on the P-face and protuberances on the E-face, together representing synaptic vesicle attachment sites. Such vesicle attachment sites were present on the presynaptic membranes of parallel fibres without postsynaptic partners from day 18 to the adult stage. After ethanolic-phosphotungstic acid staining, parallel-fibre terminals displayed presynaptic dense projections, intercleft materials and postsynaptic thickening, but some parallel fibres lacked postsynaptic thickening. These observations suggest that the presynaptic membrane structure of the parallel fibre is preserved, even in the absence of a postsynaptic partner, in j/j cerebella. A mechanism for persistence of presynaptic membrane structures without postsynaptic partners in j/j cerebella is discussed.

Animals↗

PubMed1994

Asynchronous volume changes of the two ventricles after Fontan operation in patients with a biventricular heart.

Coordinated contraction of the ventricle is an important determinant of pump function, which seems to be particularly important in Fontan circulation with one pumping ventricle. We analyzed the synchronism of contraction of the two ventricles in 11 patients with a biventricular heart who had undergone Fontan operation. Curves representing ventricular volume changes in a cardiac cycle measured on angiograms were smoothed and divided into 20 segments. We calculated the number of segments of the same directional volume changes (synchronous changes) between the two ventricles (synchronous ratio). We also calculated the total volume of the two ventricles (the two as one whole ventricle) by adding their volumes in each segment and calculated the ratio (stroke volume ratio) of the aortic stroke volume from the whole ventricle to the sum of stroke volumes of the morphological right and left ventricles. If the two ventricles ejected the blood in a completely synchronous manner, these ratios should be 1.0. In seven patients with synchronous ratios of 0.75 or greater and a stroke volume ratio of greater than 0.95, the cardiac index was 3.2 +/- 0.3 l/min/m2, the maximum total volume (corresponding to end-diastolic volume) was 106 +/- 45% normal, and the ejection fraction was 0.44 +/- 0.10. In four patients with ratios of less than 0.70 and 0.95, respectively, the parameters were 2.4 +/- 0.5 (P < 0.05), 193 +/- 92%, and 0.33 +/- 0.08, respectively. The synchronous ratio was inversely correlated with cardiac output. In conclusion, synchronism of the cardiac cycle of the two ventricles affects Fontan circulation in patients with a biventricular heart.

Adolescent↗

PubMed1994

Inhibitory effect of ONO-1078 on specific binding of peptide leukotrienes to human lung crude membrane.

We investigated the effects of ONO-1078, a newly synthesized peptide leukotriene (p-LT antagonist, on the specific binding of radiolabelled [3H]-LTC4, [3H]-LTD4 and [3H]-LTE4 to a human lung crude membrane fraction (HLMF). The binding assay was performed under conditions in which [3H]-LTC4 and [3H]-LTD4 were not metabolized by HLMF; that is, the metabolism of LTC4 to LTD4 or LTE4 was almost completely prevented by pretreating HLMF with 5 mM acivicin at 37 degrees C for 180 min, and metabolism of LTD4 to LTE4 was inhibited by including 5 mM L-cysteine and 5 mM glycine in the assay. [3H]-LTD4 specific binding was potently and concentration-dependently dissociated by ONO-1078. Its potency was 180-fold stronger than that of FPL 55712, a standardized p-LT antagonist, whereas high concentrations of ONO-1078 similar to those of FPL 55712 were required to inhibit [3H]-LTC4 specific binding. The rank order of the inhibitory potencies of p-LT agonists and antagonists for [3H]-LTD4 specific binding was LTD4 > ONO-1078 > LTE4 > LTC4 > FPI 55712. On the other hand, not only high concentrations of ONO-1078 and FPL 55712 but also more than a 100-fold excess of unlabelled LTE4 was required to inhibit [3H]-LTE4 specific binding, indicating that the binding sites do not appear to be receptors of LTE4. From these results, it is suggested that ONO-1078 is a highly potent LTD4 antagonist which is expected to be very effective on bronchial asthma.

Cell Membrane↗

PubMed1994

Pulmonary neuroendocrine carcinoma in a four-month-old dog.

A 4-month-old male Siberian Husky dog had a history of coughing, high fever and anorexia. Thoracic radiographs revealed increased radiographic density in the cranial and middle lobes of the right lung, with pleural effusion. Cytological examination of the pleural fluid suggested carcinomatous pleuritis. Right-side thoracotomy and resection of the cranial and middle lobes were performed. Histopathological examination of the resected tissue revealed an anaplastic large cell carcinoma. The tumour cells were positive for neuron specific enolase and also contained neuroendocrine granules. A particularly unusual feature of this case of pulmonary neuroendocrine carcinoma was the young age of the affected animal.

Animals↗