Search PubMed⌕ Search

Biomedical subjects

M Wakui

Publications and source records attributed to M Wakui.

At least 37 records · Page 2Linked to original sources
PubMed1998

Thrombopoietin induces association of Crkl with STAT5 but not STAT3 in human platelets.

Crkl, a 39-kD SH2, SH3 domain-containing adapter protein, is constitutively tyrosine phosphorylated in hematopoietic cells from chronic myelogenous leukemia (CML) patients. We recently reported that thrombopoietin induces tyrosine phosphorylation of Crkl in normal platelets. In this study, we demonstrate that thrombopoietin induces association of Crkl with a tyrosine phosphorylated 95- to 100-kD protein in platelets and in UT7/TPO cells, a thrombopoietin-dependent megakaryocytic cell line. With specific antibodies against STAT5, we demonstrate that the 95- to 100-kD protein in Crkl immunoprecipitates is STAT5. This coimmunoprecipitation was specific in that Crkl immunoprecipitates do not contain STAT3, although STAT3 becomes tyrosine phosphorylated in thrombopoietin-stimulated platelets. The coimmunoprecipitaion of Crkl with STAT5 was inhibited by the immunizing peptide for Crkl antisera or phenyl phosphate (20 mmol/L). After denaturing of Crkl immunoprecipitates, Crkl was still immunoprecipitated by Crkl antisera. However, coimmunoprecipitation of STAT5 was not observed. Coincident with STAT5 tyrosine phosphorylation, thrombopoietin induces activation of STAT5 DNA-binding activity as demonstrated by electrophoretic mobility shift assays (EMSA). Using a beta-casein promoter STAT5 binding site as a probe, we have also demonstrated that Crkl antisera supershift the STAT5-DNA complex, suggesting that Crkl is a component of the complex in the nucleus. Furthermore, interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and erythropoietin also induce Crkl-STAT5 complex formation in responding cells in a stimulation-dependent manner. In vitro, glutathione S-transferase (GST)-Crkl bound to STAT5 inducibly through its SH2 domain. These results indicate that thrombopoietin, IL-3, GM-CSF, and erythropoietin commonly induce association of STAT5 and Crkl and that the complex translocates to the nucleus and binds to DNA. Interestingly, such association between STAT5 and Crkl was not observed in cytokine-stimulated murine cells, suggesting an intriguing possibility that components of the human STAT5-DNA complex may be different from those of the murine counterpart.

Acute-Phase Proteins↗
PubMed1998

The genomic organization of the human corticotropin-releasing factor type-1 receptor.

We determined the genomic organization of human CRF type-1 receptor (hCRF-R1). The gene coding for hCRF-R1 consists of at least 14 exons and spans over 20 kilobases. hCRF-R1's three reported isoforms originate from the same gene by alternative splicing. The first hCRF-R1, which binds to CRF with the highest affinity and transduces the most sensitive cAMP accumulation in response to CRF, is encoded in a total of 13 exons, the only one excluded being exon 6. The second isoform contains an additional 29-amino acid sequence which corresponds to exon 6. Unlike the first isoform, the third lacks a 40-amino acid sequence, corresponding to exon 3. Exon-intron boundaries are the same as that of the consensus sequence. Locations of introns in the coding sequence are similar to human CRF-R1, rat CRF-R1, human CRF-R2alpha and others belonging to the human glucagon receptor family.

Amino Acid Sequence↗
PubMed1998

Kinetics of Ca2+ release evoked by photolysis of caged InsP3 in rat submandibular cells.

Quantitative time-resolved measurements of cytosolic Ca2+ release by photolysis of caged InsP3 have been made in single rat submandibular cells using patch clamp whole-cell recording to measure the Ca2+-activated Cl- and K+ currents. Photolytic release of InsP3 from caged InsP3 at 100 Joules caused transient inward (V(H) = 60 mV) and outward (V(H) = 0 mV) currents, which were nearly symmetric in their time course. The inward current was reduced when pipette Cl- concentration was decreased, and the outward current was suppressed by K+ channel blockers, indicating that they were carried by Cl- and K+, respectively. Intracellular pre-loading of the InsP3 receptor antagonist heparin or the Ca2+ chelator EGTA clearly prevented both inward and outward currents, indicating that activation of Ca2+-dependent Cl- and K+ currents underlies the inward and the outward currents. At low flash intensities, InsP3 caused Ca2+ release which normally activated the K+ and Cl- currents in a mono-transient manner. At higher intensities, however, InsP3 induced an additional delayed outward K+ current (I[K,(delay)]). I[K(delay)] was independent of the initial K+ current, independent of extracellular Ca2+, inhibited by TEA, and gradually prolongated by repeated flashes. The photolytic release of Ca2+ from caged Ca2+ did not mimic the I[K(delay)]. It is suggested that Ca2+ releases from the InsP3-sensitive pools in an InsP3 concentration-dependent manner. Low concentrations of InsP3 induce the transient Ca2+-dependent Cl- and K+ currents, which reflects the local Ca2+ release, whereas high concentrations of InsP3 induce a delayed Ca2+-dependent K+ current, which may reflect the Ca2+ wave propagation.

Animals↗
PubMed1998

Intracellular cAMP potentiates voltage-dependent activation of L-type Ca2+ channels in rat islet beta-cells.

Intracellular cAMP-dependent modulation of L-type Ca2+ channel activation in cultured rat islet beta-cells has been investigated using the patch-clamp whole-cell current recording mode. The L-type voltage-dependent Ca2+ current (ICa) showed a fast activation followed by a slow inactivation, and was sensitive to Ca2+ channel blockers, for example nifedipine. Application of a cAMP analogue, dibutyryl cyclic AMP (db-cAMP), increased the magnitude of the peak ICa in a concentration-dependent manner. Values of the half-activation potentials (V1/2), taken from activation curves for ICa, were -16.7 +/- 1.8 and -21.9 +/- 3.4 mV (P < 0.05) before and after application of db-cAMP, respectively, with no change of the slope factor (k) or the reversal potential. Pretreatment with a specific protein kinase A antagonist, Rp-cAMP, prevented the potentiating effect of db-cAMP. These results indicate that in rat islet beta-cells, phosphorylation of cAMP-dependent kinase potentiates the voltage-dependent activation of L-type Ca2+ channels.

Animals↗
PubMed1998

Trichomonas foetus meningoencephalitis after allogeneic peripheral blood stem cell transplantation.

A 34-year-old man with refractory acute myelogenous leukemia underwent allogeneic peripheral blood stem cell transplantation (PBSCT) from his HLA-matched sibling. Engraftment was prompt and no acute GVHD developed. However, high fever persisted even after engraftment, and the patient developed headache, diplopia, vertigo and nuchal rigidity on day 20 posttransplant. Cerebrospinal fluid (CSF) showed pleocytosis with no detectable microorganisms. Despite therapy with broad-spectrum antibiotics, antifungal agents and antituberculous drugs, he developed rapid mental deterioration with seizures and died on day 40. Just prior to his death, trichomonads were isolated from both CSF and urine. Scanning electron microscopic examination identified the trichomonad as Trichomonas foetus. At autopsy, trichomonads were detected histopathologically in an area involving meningoencephalitis. To our knowledge, this is the first case of T. foetus meningoencephalitis in a recipient of allogeneic PBSCT and, more importantly, the first human case of T. foetus infection.

Adult↗
PubMed1998

Clinical factors influencing posttransfusion platelet increment in patients undergoing hematopoietic progenitor cell transplantation--a prospective analysis.

BACKGROUND: Platelet transfusion refractoriness remains problematic in the management of patients who have undergone hematopoietic progenitor cell transplantation. Bone marrow transplantation itself is reported to be a relevant factor hampering efficient platelet transfusions. However, a prospective analysis assessing factors affecting platelet transfusion efficacy in the setting of hematopoietic progenitor cell transplantation has yet to be conducted. STUDY DESIGN AND METHODS: To identify factors independently influencing platelet transfusion efficacy after hematopoietic progenitor cell transplantation, a prospective study was performed to determine the effectiveness of platelet transfusions by estimating posttransfusion (16-hour) corrected count increments (CCI) in 42 consecutive patients (26 who received allogeneic transplants and 16 who received autologous transplants) with 439 available platelet transfusions. RESULTS: The mean CCI and percentage of CCI <4500 for all transfusions were 6161.1 +/- 7775.2 per microL and 42.1 percent, respectively. Multiple linear regression analyses revealed high total bilirubin, total body irradiation, high serum tacrolimus, and high serum cyclosporin A to be major factors independently predicting a lower CCI. HLA antibodies with restricted specificity and platelet antibodies were detected transiently in 17 and 14 percent of the patients, respectively. The presence of these antibodies was not, however, associated with a poor response to platelet transfusions. CONCLUSION: Platelet transfusion efficacy in hematopoietic progenitor cell transplant recipients is markedly influenced by clinical factors specific to the procedure as well as those already recognized in other settings. Alloimmunization is not, however, a major factor associated with a poor response to platelet transfusions after this procedure.

Adolescent↗
PubMed1998

Potassium currents in freshly dissociated uterine myocytes from nonpregnant and late-pregnant rats.

In freshly dissociated uterine myocytes, the outward current is carried by K+ through channels highly selective for K+. Typically, nonpregnant myocytes have rather noisy K+ currents; half of them also have a fast-inactivating transient outward current (ITO). In contrast, the current records are not noisy in late pregnant myocytes, and ITO densities are low. The whole-cell IK of nonpregnant myocytes respond strongly to changes in [Ca2+]o or changes in [Ca2+]i caused by photolysis of caged Ca2+ compounds, nitr 5 or DM-nitrophene, but that of late-pregnant myocytes respond weakly or not at all. The Ca2+ insensitivity of the latter is present before any exposure to dissociating enzymes. By holding at -80, -40, or 0 mV and digital subtractions, the whole-cell IK of each type of myocyte can be separated into one noninactivating and two inactivating components with half-inactivation at approximately -61 and -22 mV. The noninactivating components, which consist mainly of iberiotoxin-susceptible large-conductance Ca2+-activated K+ currents, are half-activated at 39 mV in nonpregnant myocytes, but at 63 mV in late-pregnant myocytes. In detached membrane patches from the latter, identified 139 pS, Ca2+-sensitive K+ channels also have a half-open probability at 68 mV, and are less sensitive to Ca2+ than similar channels in taenia coli myocytes. Ca2+-activated K+ currents, susceptible to tetraethylammonium, charybdotoxin, and iberiotoxin contribute 30-35% of the total IK in nonpregnant myocytes, but <20% in late-pregnant myocytes. Dendrotoxin-susceptible, small-conductance delayed rectifier currents are not seen in nonpregnant myocytes, but contribute approximately 20% of total IK in late-pregnant myocytes. Thus, in late-pregnancy, myometrial excitability is increased by changes in K+ currents that include a suppression of the ITO, a redistribution of IK expression from large-conductance Ca2+-activated channels to smaller-conductance delayed rectifier channels, a lowered Ca2+ sensitivity, and a positive shift of the activation of some large-conductance Ca2+-activated channels.

4-Aminopyridine↗
PubMed1998

Regulation of spontaneous rhythmic contractions in rat pregnant myometrium by corticotrophin-releasing factor.

To elucidate the role of corticotrophin-releasing factor (CRF) in the spontaneous rhythmic contractions of pregnant myometrium, isometric contractions of longitudinal myometrial preparations taken from Sprague-Dawley rats (18-21 days gestation) were measured. CRF (0.03-30 nM) significantly increased the amplitude (117 +/- 4% of control at 30 nM, mean +/- S.E.M.) and significantly decreased the frequency (74 +/- 5%) in a concentration-dependent manner. The area under the tension curve did not increase (89 +/- 8%). Such effects of CRF were not observed in preparations pretreated with the RP diastereomer of adenosine cyclic 3',5'-phosphorothioate (Rp-cAMPS, 10 microM), an inhibitor of protein kinase A. In preparations pretreated with indomethacin (1 microM), CRF caused little change in the amplitude but significantly decreased the frequency of the contractions. Intracellular cAMP levels in the preparations, measured by enzyme immunoassay, rose when CRF was applied at 30 nM. These results suggest that the slight positive inotropic action of CRF is due to activation of both cAMP and prostaglandin pathways, whereas the negative chronotropic action is due solely to the cAMP signalling pathway.

Animals↗
PubMed1998

Isoprenaline enhances both voltage-dependent Ba2+ and K+ currents in guinea-pig vas deferens.

In the guinea-pig vas deferens, isoprenaline (ISO) can exert significantly enhanced effects on voltage-dependent Ca2+ channel current carried by Ba2+ (IBa) via beta-adrenoceptor activation. Voltage-dependent Ba2+ (IBa) and K+ currents (I K) were recorded in the preparation voltage-clamped in a Krebs solution replaced Ca2+ by Ba2+ (Ba2+ solution) of 36 degrees C. At a holding potential of -40 mV, ISO markedly increased IBa evoked by depolarizing pulses, whereas it also increased I K at a holding potential of -20 mV so as to inactivate Ca2+ channel. This ISO-induced enhancement of IBa seems to be contradictory to the suppression of contraction by beta-adrenoceptor activation in normal Krebs solution.

Adrenergic beta-Agonists↗
PubMed1998

[Management of invasive carcinoma of bladder and abdominal aortic aneurysm (AAA); technique of radical total cystectomy and simultaneous AAA repair].

BACKGROUND: There is no consensus on the optimal surgical treatment for patients with concomitant invasive carcinoma of bladder and abdominal aortic aneurysm (AAA). We experienced two patients who were treated successfully with simultaneous radical cystectomy and AAA repair. The techniques required for the combined procedure and case reports are discussed. PROCEDURE: The goal of the one-stage operation was to minimize the risk of graft infection without compromising postoperative morbidity and mortality secondary to carcinoma of bladder. Initially pelvic lymph node dissection and radical cystectomy were performed. We preferred retrograde cyctoprostatectomy because most of the cystectomy procedure can be performed without opening the peritoneal cavity and the extent of the retroperitoneal dissection can be minimal. A single-stoma ureterocutaneostomy was preferable urinary diversion. Urinary diversions which utilize intestine such as ileal conduit or ileal urinary reservoir may cause contamination of a graft with bowel content and should be avoided. Before or after urinary diversion, aneurysmal resection and a bifurcated graft replacement were performed. The replaced graft was wrapped with the aneurysmal wall. The major omentum was mobilized and fixed in front of the graft, thereby serving as a protective barrier of the graft. A Dacron graft which was sealed with rifampicin-bonding gelatin was used to further reduce the risk of graft infection. RESULT: Two male patients were treated with the one stage radical cystectomy and AAA repair. Single-stoma ureterocutaneostomy and bilateral ureterocutaneostomy were selected as a urinary diversion. No major postoperative complications, except for paralytic ileus in one case, were observed. CONCLUSION: Our experience and reports of others indicate that radical cystectomy and simultaneous AAA repair can be safely performed with less morbidity than staged operations for the management of concomitant invasive carcinoma of bladder and AAA.

Aged↗
PubMed1998

[Severe hepatic veno-occlusive disease (VOD) which was successfully treated with supportive therapy, but subsequently developed late-recurrence].

A 40-year-old man with chronic myelogenous leukemia in chronic phase received an allogeneic marrow graft from his HLA identical brother. He was conditioned with busulfan (16 mg/kg) and cyclophosphamide (120 mg/kg). Graft-versus-host disease (GVHD) prophylaxis was attempted with cyclosporine A (CYA) and methotrexate. On day 30, weight gain, ascites and hepatomegaly developed in addition to an elevation of total bilirubin (TB). He was diagnosed as having veno-occlusive disease (VOD) and treated conservatively. The TB level increased up to 20.1 mg/dl on day 66, then reduced to 2.1 mg/dl on day 129. By that time ascites and hepatomegaly also had completely resolved. However, on day 134. The TB level started to increase again, when the lesions of chronic GVHD were observed in the eye, the mouth, and the skin. CYA was started on day 142, and FK506 was substituted for CYA on day 161. Despite the improvement of oral and skin lesions, TB level continued to rise, and he died of respiratory failure due to ARDS on day 186. Autopsy revealed both acute and old hepatic VOD lesions, suggesting the occurrence of late-onset VOD which probably contributed to the liver dysfunction observed after clinical resolution of the first episode of VOD.

Adult↗
PubMed1997

Na+-dependent release of intracellular Ca2+ induced by purinoceptors in parotid acinar cells of the rat.

In rat parotid acinar cells, ATP caused a transient increase in the intracellular Ca2+ concentration ([Ca2+]i) in the absence of external Ca2+. The ATP-induced Ca2+ response was strongly suppressed by removal of external Na+. The sequence of potency in increasing [Ca2+]i was 3'-o-(4-benzoyl) benzoyl-ATP > ATP > uridine 5'-triphosphate (UTP). Adenosine, AMP, ADP or alpha,beta-metylene ATP did not cause an increase in [Ca2+]i. The 3'-o-(4-benzoyl) benzoyl-ATP-induced increase in [Ca2+]i was abolished by removal of external Na+, but the UTP-induced response was not. The threshold external Na+ concentration required for ATP- or 3'-o-(4-benzoyl) benzoyl-ATP-induced Ca2+ release was 10-20 mM. ATP but not UTP caused a rise in the intracellular Na+ concentration ([Na+]i). Ca2+ release stimulated by caffeine or treatment with ryanodine reduced the Ca2+ release evoked by ATP. These results suggest that ATP, acting through P2Z purinoceptors, causes Na+ entry by opening cation-permeable channels, and thereafter the increase in [Na+]i triggers Ca2+ release from ryanodine-sensitive stores. UTP, acting through P2U purinoceptors, causes Ca2+ release independent of external Na+.

Adenosine Triphosphate↗
PubMed1997

Oxidized LDLs but not native LDLs augment Ba2+ currents through L-type Ca2+ channels of the A7r5 smooth muscle-derived cell line.

The whole-cell patch-clamp method was used on A7r5 smooth muscle-derived cell line, and Ba2+ currents through Ca2+ channels were recorded. The A7r5 cells showed voltage-dependent, long-lasting Ba2+ currents which were markedly inhibited by nifedipine (10 microM). The magnitude of the maximum Ba2+ current (IBa(max)) was augmented by an application of dbcAMP (1 mM), but not affected by TPA (80 nM). Noradrenaline (NA) at 100 microM caused an increase in the IBa(max) by 19.7% in the presence of phentolamine (10 microM). This effect was cancelled by Rp-cAMPs (10 microM). In the presence of propranolol (10 microM), NA tended to reduce the IBa(max). Application of Ox-LDLs at 100 microg protein/ml caused an increase in the IBa(max) by 15.7%, whereas native LDLs did not change the IBa(max). Rp-cAMPs was ineffective to the Ox-LDL action on the IBa(max). In the presence of Ox-LDLs, NA augmented the IBa(max) by 21.4% in the presence of phentolamine. These results suggest that Ox-LDLs activate L-type Ca2+ channels of A7r5 cells by a mechanism independent of cAMP/PKA signalling.

Aorta↗
PubMed1997

GLP-I(7-36) amide augments Ba2+ current through L-type Ca2+ channel of rat pancreatic beta-cell in a cAMP-dependent manner.

The whole-cell patch-clamp method was used to examine the effect of glucagon-like peptide I (GLP-I)(7-36) amide on the activation process of L-type Ca2+ channels of rat pancreatic beta-cells. After depolarization, GLP-I (1-100 nmol/l) caused action potentials in cells exposed to a glucose-free solution for 10 min. The percentage of cells producing action potential depended on the concentration of GLP-I. In some cells, GLP-I caused action potentials without the prior depolarization of the membrane. In cells exposed to the glucose-free solution for longer than 30 min, or in cells that were deprived of ATP by a means of the conventional whole-cell configuration, GLP-I (20 nmol/l) did not cause the electrical excitation. Application of GLP-I augmented the maximum Ba2+ current (IBa) through L-type Ca2+ channels and shifted the current voltage curve to the left. Values of changes in the maximum IBa depended on GLP-I concentration. Application of dibutyryl cAMP (dbcAMP, 1 mmol/l) also augmented IBa. In cells pretreated with Rp-cAMP, dbcAMP did not change the magnitude of IBa. Also in cells pretreated with Rp-cAMP, GLP-I failed to augment IBa. These results suggest that in pancreatic beta-cells, GLP-I, by a cAMP-dependent mechanism, increases opening of L-type Ca2+ channels. cAMP-dependent augmentation of Ca2+ entry as well as cAMP production itself by GLP-I plays a crucial role in controlling insulin secretion.

Action Potentials↗
PubMed1996

Crkl is constitutively tyrosine phosphorylated in platelets from chronic myelogenous leukemia patients and inducibly phosphorylated in normal platelets stimulated by thrombopoietin.

Platelet functions such as aggregation and clot retraction are often abnormal in chronic mylogenous leukemia (CML) patients. However, the molecular mechanisms of these altered functions are unknown. As expression of the p210bcr-abl oncogene product, a constitutively active tyrosine kinase, is known to have an essential role in the pathogenesis of CML and tyrosine phosphorylation is intimately involved in various aspects of platelet activation, we examined the pattern of protein tyrosine phosphorylation in platelets from 15 CML patients by immunoblotting with a monoclonal antiphosphotyrosine antibody (4G10). Before and after stimulation with thrombin, the only consistent difference between normal and CML platelets was the presence of a tyrosine phosphorylated protein with a relative molecular weight of 39 kD. This tyrosine phosphorylated protein was identified as crid, an SH2, SH3 containing adapter protein. Thus, as previously demonstrated for neutrophils from CML patients, tyrosine phosphorylation of p39crkl persists in mature platelets. No tyrosine phosphorylation of crid was detected following stimulation with thrombin in normal platelets. However, crkl became incorporated into the Triton X-100 insoluble residue following thrombin stimulation in a manner dependent on platelet aggregation. Further, we found that crkl is an endogenous substrate for calpain, a protease that may be involved in postaggregation signaling processes. This suggests that crkl may be involved in the reorganization of the cytoskeleton during normal platelet aggregation and its tyrosine phosphorylation in CML platelets may contribute to the abnormal platelet function in CML patients. Finally, we found that thrombopoietin induces tyrosine phosphorylation of crk1 in normal platelets and FDCP cells genetically engineered to express human c-Mpl. This suggests that crk1 can be phosphorylated by a kinase other than p210bcr-abl and that crk1 may have a role in signaling by thrombopoietin.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Thimerosal modulates the agonist-specific cytosolic Ca2+ oscillatory patterns in single pancreatic acinar cells of mouse.

Modulation of the agonist-specific cytosolic Ca2+ oscillatory pattern by thimerosal has been investigated in single pancreatic acinar cells using patch-clamp perforated whole-cell recording to measure the calcium-dependent chloride current (I(C1)(Ca2+)). 1 microM thimerosal, which fails to evoke Ca2+ oscillation alone, clearly changed the pattern of Ca2+ oscillation from pulsatile spikes (evoked by low concentrations of activators) to sinusoidal or transient oscillations. The mimetic action of thimerosal was independent of extracellular Ca2+, was blocked by extracellular application of dithiothreitol or 10 mM caffeine, as well as by internal perfusion with heparin; but was unaffected by ruthenium red. We conclude that thimerosal modulates the agonist-specific cytosolic Ca2+ oscillatory patterns mediated by sensitizing the InsP3-induced Ca2+ release.

Acetylcholine↗