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T Nakamoto

Publications and source records attributed to T Nakamoto.

At least 55 records · Page 3Linked to original sources
PubMed1998

Integrin-mediated signal transduction in cells lacking focal adhesion kinase p125FAK.

We have previously shown that integrin-dependent tyrosine phosphorylation of p130Cas (Cas) could be induced in a mouse fibroblast cell line that does not express focal adhesion kinase p125FAK (FAK). By analyzing FAK-deficient (FAK-/-) cells transiently expressing Cas mutant proteins, we demonstrate here that the Src homology 3 (SH3) domain of Cas is indispensable for adhesion-mediated Cas phosphorylation in this mutant cell line. While the FAK directly binds to Cas-SH3, our findings imply that SH3-binding molecule(s) other than FAK might regulate Cas phosphorylation, at least in FAK-/- cells. In this regard, we observed that FAK-/- cells expressed cell adhesion kinase beta (CAKbeta), a protein tyrosine kinase of the FAK subfamily. CAKbeta expressed by FAK-/- cells was associated in vivo with Cas in a Cas-SH3-dependent manner. Moreover, integrin stimulation induces tyrosine phosphorylation of CAKbeta in FAK-/- cells. Thus, our results suggest that CAKbeta contributes to integrin-mediated signal transduction in place of FAK in FAK-deficient cells.

Animals↗
PubMed1998

c-Cbl is tyrosine-phosphorylated by interleukin-4 and enhances mitogenic and survival signals of interleukin-4 receptor by linking with the phosphatidylinositol 3'-kinase pathway.

Interleukin-4 (IL-4) is a cytokine that induces both proliferation and differentiation and suppresses apoptosis of B cells. Although IL-4 has been shown to activate the phosphatidylinositol 3' (PI3)-kinase pathway, the role of PI3 kinase in the IL-4 receptor (IL-4R) signaling remains unclear. In this study, we demonstrated that c-Cbl proto-oncogene product is inducibly phosphorylated on tyrosine residues and is associated with the p85 subunit of PI3-kinase by IL-4 stimulation. Overexpression of c-Cbl enhances the PI3-kinase activity and, at the same time, mitogenic activity and survival of cells in the presence of IL-4. However, these effects of c-Cbl were abolished by wortmannin, a specific inhibitor for the PI3 kinase pathway, or by a point mutation at tyrosine 731 of c-Cbl, which is a major binding site for p85. These results indicate that c-Cbl plays a role in linking IL-4R with the PI3 kinase pathway and thus enhancing the mitogenic and survival signals.

Androstadienes↗
PubMed1998

Cardiovascular anomaly, impaired actin bundling and resistance to Src-induced transformation in mice lacking p130Cas.

p130Cas (Cas), the protein encoded by the Crkas gene (also known as Cas), is an adaptor molecule with a unique structure that contains a Src homology (SH)-3 domain followed by multiple YXXP motifs and a proline-rich region. Cas was originally cloned as a highly tyrosine-phosphorylated protein in cells transformed by v-Src (refs 2,3) or v-Crk (ref. 4) and has subsequently been implicated in a variety of biological processes including cell adhesion, cell migration, growth factor stimulation, cytokine receptor engagement and bacterial infection. To determine its role in vivo, we generated mice lacking Cas. Cas-deficient embryos died in utero showing marked systemic congestion and growth retardation. Histologically, the heart was poorly developed and blood vessels were prominently dilated. Electron microscopic analysis of the heart revealed disorganization of myofibrils and disruption of Z-disks. In addition, actin stress fiber formation was severely impaired in Cas-deficient primary fibroblasts. Moreover, expression of activated Src in Cas-deficient primary fibroblasts did not induce a fully transformed phenotype, possibly owing to insufficient accumulation of actin cytoskeleton in podosomes. These findings have defined Cas function in cardiovascular development, actin filament assembly and Src-induced transformation.

Actin Cytoskeleton↗
PubMed1998

The role of thromboxane A2 in regulating porcine basilar arterial tone.

The aim of the present study was to clarify the participation of endogenous arachidonic acid (AA) metabolites in regulating porcine basilar, coronary, pulmonary and mesenteric arterial tones in vitro. A cyclooxygenase inhibitor, indomethacin, relaxed basilar artery but not other arteries examined. Quinacrine (a phospholipase A2 inhibitor), OKY-046 (a thromboxane (TX) A2 synthetase inhibitor) and ONO-3708 (a TXA2/prostaglandin H2 receptor antagonist) produced relaxation in basilar arteries with intact endothelium. Nordihydroguaiaretic acid (a lipoxygenase inhibitor) had no effect on the tone. The amount of TXB2 (a stable metabolite of TXA2) spontaneously released from porcine basilar arteries was 6-10 fold more than those from other arteries. Indomethacin and OKY-046 mostly inhibited the production of TXB2. Endothelial denudation decreased indomethacin-induced relaxation and the amount of TXB2. These results suggest that a vasoconstricting substance(s) is released from endothelial cells and possibly smooth muscle cells in porcine basilar arteries in vitro. The main constricting substance is proposed to be TXA2. On the other hand, several arteries from peripheral vascular beds did not release this vasoconstricting substance.

Animals↗
PubMed1998

[Evaluation of the FREEDOM O2, DC Concentrator, a portable oxygen concentrator capable of operating under car battery power].

UNLABELLED: The FREEDOM O2 DC Concentrator, an oxygen concentrator which can be powered by a car battery, was evaluated. The oxygen concentrator was used by a 67 year-old man with sequelae of pulmonary tuberculosis who was receiving long-term oxygen therapy at home (HOT), and whose work required automobile trips over long distances. The equipment used was an adsorption-type oxygen concentrator capable of operating on a DC 12 V power supply, and which can be powered from a residential power outlet (AC 100 V) using a dedicated voltage converter. The trunk-shaped equipment measured 21/584 x 42 cm and weighed 17 kg. The noise level of the equipment was 58.2 +/- 2.5 dB (at 1 meter), and the flow rate can be set to 0.25, 0.50, 0.75, 1.0, 1.5, and 2.0 l/min. RESULTS: 1) The O2 concentration which can be generated by this equipment is 93 +/- 3% (0.25 to 1.5 l/min) or 90 +/- 2.8% (2.0 l/min). 2) Using this equipment, the patient was capable of driving himself in comfort for two hours or longer. Further, it was possible to stay in a hotel during a trip, inhaling oxygen generated by the equipment. Hereafter, this equipment should enable or facilitate long-distance driving, travel and lodging for HOT patients.

Aged↗
PubMed1997

Activation of pp60c-src depending on cell density in PC12h cells.

The Src family tyrosine kinases and their substrates are involved in cell-cell and cell-matrix interactions. We found that in PC12h cells, an increase of cell density enhanced the tyrosine phosphorylation levels of several intracellular proteins including p130(cas). Because it is a possible substrate for Src family kinases, we measured pp60(c-src) activity and found that it was higher in high density cultures than in low density cultures. This phenomenon was also observed in PC12 (the parental cell line of the PC12h subclone), Balb/c 3T3, Swiss 3T3, and Hela cells. One of the possible mechanisms regulating the kinase activity of pp60(c-src) is the phosphorylation and dephosphorylation of its negative regulatory site located at its C terminus. However, the tyrosine phosphorylation level of the regulatory site did not change depending on cell density. Subcellular fractionation showed that in high density culture, pp60(c-src) was translocated from detergent-soluble to detergent-insoluble fractions. These results suggest that cell-cell interaction might induce the activation of pp60(c-src) without changing its tyrosine phosphorylation levels.

Animals↗
PubMed1997

The phosphatidylinositol 3' kinase pathway is required for the survival signal of leukocyte tyrosine kinase.

Leukocyte tyrosine kinase (LTK) is a receptor tyrosine kinase which belongs to the insulin receptor superfamily and is mainly expressed in pre-B lymphocytes and neuronal tissues. Recently, we demonstrated that LTK utilizes Shc and IRS-1 as two major substrates and while both equally activate the Ras pathway, only IRS-1 suppresses apoptosis of hematopoietic cells, suggesting the existence of another unidentified signaling pathway downstream of IRS-1, which is relevant to the anti-apoptotic activity. In the present study, we found that wortmannin, a specific inhibitor of phosphatidylinositol 3' (PI3)-kinase, abolished the survival effects of LTK. Although c-Cbl is found to be phosphorylated by LTK and therefore is a second candidate linking LTK with the PI3-kinase pathway along with IRS-1, we found that the p85 subunit of PI3 kinase directly binds to tyrosine 753 of LTK, which is located within a YXXM motif, a consensus binding amino acid sequence for the SH2 domain of p85, but fails to bind to IRS-1 or c-Cbl. Ba/F3 cells which stably express the EGF receptor-LTK chimeric receptor carrying a mutation at tyrosine 753 fell into apoptotic death even in the presence of EGF, indicating that the PI3 kinase pathway is required for the survival effects of LTK.

Androstadienes↗
PubMed1997

Purification and molecular cloning of SH2- and SH3-containing inositol polyphosphate-5-phosphatase, which is involved in the signaling pathway of granulocyte-macrophage colony-stimulating factor, erythropoietin, and Bcr-Abl.

Grb2/Ash and Shc are the adapter proteins that link tyrosine-kinase receptors to Ras and make tyrosine-kinase functionally associated with receptors and Ras in fibroblasts and hematopoietic cells. Grb2/Ash and Shc have the SH3, SH2, or phosphotyrosine binding domains. These domains bind to proteins containing proline-rich regions or tyrosine-phosphorylated proteins and contribute to the association of Grb2/Ash and Shc with other signaling molecules. However, there could remain unidentified signaling molecules that physically and functionally interact with these adapter proteins and have biologically important roles in the signaling pathways. By using the GST fusion protein including the full length of Grb2/Ash, we have found that c-Cbl and an unidentified 135-kD protein (pp135) are associated with Grb2/Ash. We have also found that they become tyrosine-phosphorylated by treatment of a human leukemia cell line, UT-7, with granulocyte-macrophage colony-stimulating factor (GM-CSF). We have purified the pp135 by using GST-Grb2/Ash affinity column and have isolated the full-length complementary DNA (cDNA) encoding the pp135 using a cDNA probe, which was obtained by the degenerate polymerase chain reaction based on a peptide sequence of the purified pp135. The cloned cDNA has 3,958 nucleotides that contain a single long open reading frame of 3,567 nucleotides, encoding a 1,189 amino acid protein with a predicted molecular weight of approximately 133 kD. The deduced amino acid sequence reveals that pp135 is a protein that has one SH2, one SH3, and one proline-rich domain. The pp135, which contains two motifs conserved among the inositol polyphosphate-5-phosphatase proteins, was shown to have the inositol polyphosphate-5-phosphatase activity. The pp135 was revealed to associate constitutively with Grb2/Ash and inducibly with Shc using UT-7 cells stimulated with GM-CSF. In the cell lines derived from human chronic myelogenous leukemia, pp135 was constitutively tyrosine-phosphorylated and associated with Shc and Bcr-Abl. These facts suggest that pp135 is a signaling molecule that has a unique enzymatic activity and should play an important role in the signaling pathway triggered by GM-CSF and in the transformation of hematopoietic cells caused by Bcr-Abl.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Characterization of the kinase activity essential for tyrosine phosphorylation of p130Cas in fibroblasts.

The cellular transformation by v-Src or v-Crk induces tyrosine phosphorylation of a common substrate molecule, p130Cas (Cas), which tightly binds these oncoproteins in vivo. From its structure, Cas is deduced to be an ideal substrate for Src family kinases and Abl kinase. The tyrosine kinase activity associated with Cas was analysed using mouse variant fibroblasts lacking at least one of tyrosine kinases. In normal fibroblasts, Cas is associated with a significant level of tyrosine kinase activity which efficiently phosphorylates Cas in vitro. The Cas-associated tyrosine kinase activity was remarkably elevated in Csk-/- cells, which resulted in hyperphosphorylation of cellular Cas. The associated kinase activity was slightly increased in Src-/- cells whereas not significantly changed in Abl-/- nor Fak-/- cells. On the contrary, the Cas-associated kinase activity was remarkably decreased in Fyn-/- cells. At the same time, association of Cas with Fyn kinase in vitro was most obviously detected in normal fibroblasts as well as Csk-/- cells. Transient expression of v-Crk induced elevation of the Cas-associated kinase activity in all of these cell lines except the primary culture of Fyn-/- fibroblasts. These results indicate that Fyn kinase plays an essential role in v-Crk-mediated phosphorylation of Cas.

3T3 Cells↗
PubMed1997

Ligation of the T cell antigen receptor induces tyrosine phosphorylation of p105CasL, a member of the p130Cas-related docking protein family, and its subsequent binding to the Src homology 2 domain of c-Crk.

p105CasL (CasL) is a recently identified signaling molecule closely related to the p130Cas (Crk-associated substrate) docking protein. CasL has a single Src homology (SH) 3 domain in its N-terminal portion followed by multiple consensus motifs for binding to SH2 domains. Like original p130Cas, CasL undergoes tyrosine phosphorylation upon integrin-mediated cell adhesion. In the present report, we provide direct evidence that CasL is also involved in T cell antigen receptor (TcR)-mediated signal transduction. In binding studies in vitro using glutathione-S-transferase fusion proteins, we have identified 105- and 120-kDa phosphotyrosyl proteins (pp105 and pp120, respectively) tightly bound to the SH2 domain of the Crk adapter protein in the H9 human T cell line after stimulation through the CD3/TcR complex. pp120, but not pp105, also bound to the SH3 of another adapter protein, Ash/Grb2. Immunoblotting with specific antibodies revealed that pp120 and pp105 were identical to the c-cbl proto-oncogene product (p120cbl) and CasL, respectively. Association between Crk and tyrosine-phosphorylated CasL after TcR stimulation was also confirmed in vivo. CasL phosphorylation induced by TcR ligation reached maximal levels within 2 min and rapidly declined thereafter, whereas the integrin-dependent response occurred slowly and was more prolonged. Finally, we demonstrated that Crk/CasL association occurred in peripheral blood T lymphocytes in response to TcR engagement. Our findings suggest that CasL is involved in T cell activation signals and resides at a point where two distinct receptor-mediated signaling pathways converge. This provides one mechanism by which integrins may mediate T cell co-stimulation.

Adaptor Proteins, Signal Transducing↗
PubMed1997

I.v. fentanyl decreases the clearance of midazolam.

We have examined the effect of fentanyl on the pharmacokinetics of midazolam in patients undergoing orthopaedic surgery. Thirty patients were allocated randomly to receive fentanyl 200 micrograms and midazolam 0.2 mg kg-1 (fentanyl group, n = 15) or placebo and midazolam 0.2 mg kg-1 (placebo group, n = 15) in a double-blind manner for induction of anaesthesia. Anaesthesia was maintained with nitrous oxide and isoflurane. Systemic clearance of midazolam was decreased by 30% (P = 0.002) and elimination half-time was prolonged by 50% (P = 0.04) in the fentanyl group compared with the placebo group. There were no differences in the distribution half-time or volume of distribution at steady state between the two groups. These findings indicate that elimination of midazolam was inhibited by fentanyl during general anaesthesia.

Adolescent↗
PubMed1997

Inhalation of interleukin-2 combined with subcutaneous administration of interferon for the treatment of pulmonary metastases from renal cell carcinoma.

BACKGROUND: Interleukin-2 is the most promising antitumor agent for advanced renal cell carcinoma, but systemic immunotherapy with interleukin-2 might be limited because of inadequate efficacy and severe adverse effects. In this study, we treated 7 patients with lung metastases from renal cell carcinoma with topical application of interleukin-2 by inhalation. METHODS: Patients received 100,000 IU of interleukin-2 by inhalation 4 times a day and 9,000,000 IU of interferon-alfa-2a subcutaneously for 5 consecutive days per week. They also received, by oral administration, 800 mg of cimetidine and 50 mg of indomethacin per day. After informed consent was obtained, the treatment started and the absence of any intolerable adverse effects was confirmed in a hospital. Then the treatment continued in an outpatient clinic for at least 3 months. RESULTS: Of 6 assessable patients, 5 responded to this treatment; 2 patients developed a partial response (33%) and 3 remained stable (67%). Disease progressed in the remaining patient. Therapy was discontinued in 1 patient because of his poor general condition. No severe adverse effects were observed, but pulmonary fibrosis probably associated with this treatment occurred in 1 patient. CONCLUSION: Although more cases and further evaluation are necessary to assess the significance and the safety of the inhalation of interleukin-2, this treatment is anticipated to be an option for selected patients with lung metastases from renal cell carcinoma.

Administration, Inhalation↗
PubMed1997

Requirements for localization of p130cas to focal adhesions.

p130cas (Cas) is an adapter protein that has an SH3 domain followed by multiple SH2 binding motifs in the substrate domain. It also contains a tyrosine residue and a proline-rich sequence near the C terminus, which are the binding sites for the SH2 and SH3 domains of Src kinase, respectively. Cas was originally identified as a major tyrosine-phosphorylated protein in v-Crk- and v-Src-transformed cells. Subsequently, Cas was shown to be inducibly tyrosine phosphorylated upon integrin stimulation; it is therefore regarded as one of the focal adhesion proteins. Using an immunofluorescence study, we examined the subcellular localization of Cas and determined the regions required for its localization to focal adhesions. In nontransformed cells, Cas was localized predominantly to the cytoplasm and partially to focal adhesions. However, in 527F-c-Src-transformed cells, Cas was localized mainly to podosomes, where the focal adhesion proteins are assembled. The localization of Cas to focal adhesions was also observed in cells expressing the kinase-negative 527F/295M-c-Src. A series of analyses with deletion mutants expressed in various cells revealed that the SH3 domain of Cas is necessary for its localization to focal adhesions in nontransformed cells while both the SH3 domain and the C-terminal Src binding domain of Cas are required in 527F-c-Src-transformed cells and fibronectin-stimulated cells. In addition, the localization of Cas to focal adhesions was abolished in Src-negative cells. These results demonstrate that the SH3 domain of Cas and the association of Cas with Src kinase play a pivotal role in the localization of Cas to focal adhesions.

3T3 Cells↗
PubMed1997

Postnatal caffeine effects on copper, zinc, and iron concentrations in mammary gland, milk, and plasma of lactating dams and their offspring.

Beginning on the day of delivery and until the 15th or 22nd day of lactation, one group of dams received a 20% protein diet as a basal diet and one group received the basal diet supplemented with caffeine (4 mg/100 g body weight). A correlation between caffeine concentrations in the dams' plasma and milk was observed. In the caffeine group, levels of Fe and Cu in the dams' mammary glands at day 22 were decreased. Copper levels in the milk at day 15 and Fe and Zn levels in the milk at day 22 showed a significant decrease. Copper concentration in the plasma of 15-day-old pups showed a significant decrease also, but Zn and Fe concentration showed no difference between caffeine and noncaffeine control groups. The present study shows that the dams' consumption of a caffeine-containing diet influences trace elements of mammary glands, milk, and pups' plasma.

Animals↗
PubMed1997

[Long-term effects of a selective alpha 1-adrenergic inhibitor on right and left ventricular masses in patients with chronic pulmonary disease].

We examined the effect of one year of treatment with the selective alpha 1-adrenergic inhibitor doxazosin on right ventricular mass (RV mass), left ventricular mass (LV mass), and arterial blood gases. The subjects were 24 outpatients (18 men and 6 women, mean age 68.3 +/- 9.4 years) with chronic pulmonary disease complicated by hypertension who were clinically stable. One year of drug therapy was associated with significant decreases both in systolic pressure (159 +/- 15.2 vs 125.8 +/- 14.1 mmHg, p < 0.05, n = 24), and in LV mass index (101.0 +/- 13.4 vs 97.6 +/- 11.8 gm-2, p < 0.05), n = 24). We obtained the RV mass index by multiplying the thallium score RV/LV count and the LV mass index obtained by echocardiography. One year of drug therapy was associated with a significant increase in RV mass index (42.9 +/- 31.2 vs 53.6 +/- 30.5 gm-2, p < 0.05, n = 8). Vital capacity decreased (2.18 +/- 1.95 vs 1.95 +/- 0.57 l, p < 0.05, n = 24), but PaO2 improved (77.3 +/- 17.2 vs 82.2 +/- 2.4 mmHg, p < 0.05, n = 24). These data indicate that doxazosin can decrease blood pressure and can depress the left ventricle with no adverse effect on oxygenation in patients with chronic pulmonary disease complicated by hypertension. The worsening of RV hypertrophy may have been caused by a mechanism different from the one that caused LV hypertrophy, and by an increase in the work load on the right ventricles secondary to lung deterioration.

Adrenergic alpha-Antagonists↗
PubMed1997

[Simultaneous ambulatory electrocardiography and pulse oximetry].

We developed a system for 24-hour ambulatory recording of blood oxygenation (SpO2) and electrocardiography (ECG). Using this system, we studied 10 healthy volunteers and 7 patients with chronic pulmonary diseases. The system incorporated a portable pulse oximeter (SM50) manufactured by Fukuda Denshi KK; the first and second channels were used to record ECG data and the third was used to record SpO2 data. An SpO2 sensor (Dispo-sensor D-25; Nellcor Inc.) was applied to the fourth of fifth finger. The SpO2 data (MicrO2; Siemens AG) were digitized and stored in the ambulatory recording device; the ECG was recorded simultaneously. The data were analyzed with a model DMW-9000H analyzer (Fukuda Denshi KK). A custom-designed program was also used, to remove noise errors. In the healty volunteers, SpO2 was at least 90% for the entire 24 hours. In all the patients, SpO2 fell below 90% at rest during the night or after a 15-minute walk. Transient atrial tachycardia was observed in 3 patients, and during the tachycardia the SpO2 was low. The number of extra ventricular beats divided by the total number of beats increased more in the patients than in the healthy volunteers (1.21 +/- 0.89 vs 0.6 +/- 0.3%, p < 0.05). SpO2 did not change significantly in the patients. In outpatients and in patients receiving home health care, the present system facilitates simultaneous diagnosis of respiratory failure an arrhythmias. In patients with chronic pulmonary diseases desaturation may cause transient atrial tachycardia.

Adult↗