Search PubMed⌕ Search

Biomedical subjects

S Kume

Publications and source records attributed to S Kume.

At least 19 recordsLinked to original sources
PubMed2000

Molecular cloning and expression profile of Xenopus calcineurin A subunit(1).

We have cloned a cDNA encoding a catalytic subunit of calcineurin (CnA) expressed in Xenopus oocytes. The deduced amino acid sequence indicates 96.3% and 96.8% identities with the mouse and human CnAalpha isoforms, respectively. Xenopus CnA (XCnA) RNA and protein are expressed as maternal and throughout development. Recombinant XCnA protein interacted with calmodulin in the presence of Ca(2+). Deletion of calmodulin binding domain and auto-inhibitory domain revealed calcium independent phosphatase activity, thereby showing that XCnA is likely to be modulated by both calmodulin and calcium.

Amino Acid Sequence↗
PubMed2000

Sphingosine 1-phosphate as a major bioactive lysophospholipid that is released from platelets and interacts with endothelial cells.

The serum-borne lysophospholipid mediators sphingosine 1-phosphate (Sph-1-P) and lysophosphatidic acid (LPA) have been shown to be released from activated platelets and to act on endothelial cells. In this study, we employed the repeated lipid extraction (under alkaline and acidic conditions), capable of detecting Sph-1-P, LPA, and possibly structurally similar lysophospholipids, whereby a marked formation of [(32)P]Sph-1-P, but not [(32)P]LPA, was observed in [(32)P]orthophosphate-labeled platelets. Platelet Sph-1-P release, possibly mediated by protein kinase C, was greatly enhanced in the presence of albumin, which formed a complex with Sph-1-P. This finding suggests that platelet Sph-1-P may become accessible to depletion by albumin when its transbilayer movement (flipping) across the plasma membrane is enhanced by protein kinase C. Although human umbilical vein endothelial cells expressed receptors for both Sph-1-P and LPA, Sph-1-P acted much more potently than LPA on the cells in terms of intracellular Ca(++) mobilization, cytoskeletal reorganization, and migration. The results suggest that Sph-1-P, rather than LPA, is a major bioactive lysophospholipid that is released from platelets and interacts with endothelial cells, under the conditions in which critical platelet-endothelial interactions (including thrombosis, angiogenesis, and atherosclerosis) occur. Furthermore, albumin-bound Sph-1-P may account for at least some of the serum biological activities on endothelial cells, which have been ascribed to the effects of albumin-bound LPA, based on the similarities between LPA and serum effects.

Biological Transport↗
PubMed2000

Galphas family G proteins activate IP(3)-Ca(2+) signaling via gbetagamma and transduce ventralizing signals in Xenopus.

During early embryonic development, IP(3)-Ca(2+) signaling transduces ventral signaling at the time of dorsoventral axis formation. To identify molecules functioning upstream in this signal pathway, we examined effects of a panel of inhibitory antibodies against Galphaq/11, Galphas/olf, or Galphai/o/t/z. While all these antibodies showed direct inhibition of their targets, their effects on redirection of the ventral mesoderm to a dorsal fate varied. Anti-Galphas/olf antibody showed strong induction of dorsal fate, anti-Galphai/o/t/z antibody did so weakly, and anti-Galphaq/11 antibody was without effect. Injection of betaARK, a Gbetagamma inhibitor, mimicked the dorsalizing effect of anti-Galphas/olf antibody, whereas injection of adenylyl cyclase inhibitors at a concentration which inhibited Galphas-coupled cAMP increase did not do so. The activation of Galphas-coupled receptor gave rise to Ca(2+) transients. All these results suggest that activation of the Galphas-coupled receptor relays dorsoventral signal to Gbetagamma, which then stimulates PLCbeta and then the IP(3)-Ca(2+) system. This signaling pathway may play a crucial role in transducing ventral signals.

Amino Acid Sequence↗
PubMed2000

Desensitization of IP3-induced Ca2+ release by overexpression of a constitutively active Gqalpha protein converts ventral to dorsal fate in Xenopus early embryos.

The constitutively active Gqalpha mutant construct (GqalphaQ-L) in Xenopus early embryos was overexpressed and the effects on dorsoventral patterning examined. It was found that prolonged stimulation of inositol 1,4,5-trisphosphate (IP3)-Ca2+ signaling by overexpression of GqalphaQ-L led to desensitization of IP3-induced Ca2+ release (IICR). Desensitization of IICR on the ventral side specifically induced an ectopic dorsal axis due to the conversion of ventral marginal mesoderm to adopt a dorsal fate. This effect of desensitization resembles that of inhibitory antibodies against the IP3 receptor, as reported previously. These results strengthen the earlier finding that active IP3-Ca2+ signaling functions in ventral signaling during the early embryonic development of Xenopus. Furthermore, the nature of downregulation of the Xenopus IP3 receptor through continuous stimulation of IP3-Ca2+ signaling might play a role in regulating endogenous IP3-Ca2+ signaling in Xenopus early development.

Animals↗
PubMed1999

Role of the inositol 1,4,5-trisphosphate receptor in early embryonic development.

There is now considerable literature on the importance of phosphatidylinositol cycle activation in transducing information of various types across the plasma membrane. Though much of the data derives from studies on somatic cells, there is increasing evidence for crucial events related to development, including fertilization, cell cycle progression and dorsoventral axis formation. In this review, focus is directed mainly to the molecular basis of the inositol 1,4,5-triphosphate receptor expressed in oocytes and early embryos of Xenopus. Recent progress in studies concerning the role of this receptor in early embryonic development is discussed.

Animals↗
PubMed1999

[A case of advanced gastric cancer successfully treated by neoadjuvant chemotherapy with CPT-11, CDDP and 5-FU].

A patient with advanced gastric cancer was treated with combined administration of CPT-11 CDDP and 5-FU before operation. CPT-11 was given intravenously at a dose of 30 mg/m2/day on day 1 and day 8. At the same time, 5 mg/m2/day CDDP and 350 mg/m2/day 5-FU were infused for 2 weeks. The patient experienced no other adverse reaction than a mild degree of nausea. Histological examination of the resected specimen revealed complete disappearance of cancer cells both in the stomach and the regional lymphnodes.

Adult↗
PubMed1998

A cross-sectional evaluation of spontaneous platelet aggregation in relation to complications in patients with type II diabetes mellitus.

To clarify the relationship between platelet function and diabetic complications, we investigated spontaneous platelet aggregation (SPA) and agonist-induced platelet aggregation by a particle counting method using light scattering (LS) and by a conventional light transmission method (LT) in 23 age- and sex-matched control subjects and 74 patients with type II diabetes mellitus. We also observed platelets using the FIC-2 (TOA Medical Electronics, Kobe, Japan) flow cytometer and imaging device. Observation by the FIC-2 device showed microaggregates of platelets in samples with increased SPA-LS. SPA-LS was significantly elevated in patients with type II diabetes mellitus as a whole compared with control subjects. SPA-LS also showed significant differences between control subjects and three diabetic patient subgroups with a varying severity of retinopathy, nephropathy, or neuropathy, and the mean values increased along with the increasing severity of complications. On the other hand, although SPA-LT also showed significant differences between these groups, the absolute values were all less than 10%, which we believe does not warrant quantitative analysis. Adenosine-5'-diphosphate (ADP)-induced platelet aggregation failed to show significant differences between controls and subjects with a varying severity of retinopathy by either LS or LT, which indicates that SPA is more sensitive than agonist-induced platelet aggregation in relation to diabetic complications. We observed significant correlations between SPA-LS and the patients' age, hemoglobin A1c (HbA1c) level, plasma fibrinogen level, or 6-keto-PGF1alpha (6KF) to 11-dehydro-thromboxane B2 (TXB2) ratio. Our study demonstrated a close relationship between platelet hyperaggregability and diabetic complications, and a longitudinal prospective study of SPA-LS in diabetic patients is warranted to clarify cause-and-effect relationships.

Cross-Sectional Studies↗
PubMed1998

Quantification of sphingosine derivatives in human platelets: inducible formation of free sphingosine.

To elucidate the physiologic role of sphingolipid-derived products as signaling molecules, we analyzed the levels of endogenous sphingosine (Sph) derivatives in human platelets. When the platelets were stimulated with thrombin or 12-O-tetradecanoylphorbol 13-acetate, neither ceramide formation nor sphingomyelin hydrolysis was observed, which suggests that the sphingomyelin cycle may not be an essential part of the signaling pathway under these conditions. In contrast, Sph was found to increase in platelets upon stimulation. The level of Sph 1-phosphate, which is formed from Sph by the action of Sph kinase, was not affected under our conditions. Although it has been established that Sph inhibits protein kinase C, which regulates the functional responses of the platelets, Sph levels which exert an inhibitory effect on protein kinase C cannot be attained under physiological conditions (without exogenous Sph). Considering the stimulation of the synthesis of Sph by the physiological agonist thrombin, we speculate that Sph is a signaling molecule of physiological importance in platelets, but protein kinase C may not be its target.

Adult↗
PubMed1998

Effect of restricted feed intake of dams and heat stress on mineral status of newborn calves.

To clarify the effects of restricted feed intake, heat stress, and parity on the mineral status of cows and heifers around parturition and on the mineral status of their calves during 1 wk of age, data were collected from 66 Holstein cows and heifers and their calves. In Experiment 1, 36 heifers and mature cows that calved during hot or cool weather were fed to meet requirements for total digestible nutrients (TDN), protein, and minerals. In Experiment 2, 15 mature cows that calved during hot or cool weather were fed to meet maintenance requirements for TDN plus requirements for TDN for the last 2 mo of gestation, and 15 heifers were fed to meet TDN requirements. Heat stress increased rectal temperatures of newborn calves. Blood hematocrit and hemoglobin of heifers around parturition were higher than those of mature cows, but blood hematocrit and hemoglobin of calves born from heifers were lower. The restricted feed intake of dams decreased blood hematocrit and hemoglobin as well as plasma Fe of calves in hot and cool weather. Plasma Ca, inorganic P, and alkaline phosphatase as well as colostral Ca, P, Mg, and Zn of heifers were higher than those of mature cows, but plasma Mg of heifers was lower. Plasma Mg of calves and their dams was lower in hot weather than in cool weather, and restricted feed intake accelerated the reduction in plasma Mg of calves and their dams during hot weather. Plasma Na of calves and their dams was higher in hot weather than in cool weather. Heat stress increased plasma K of heifers and their calves. Heat stress increased Ca concentration in meconium of calves born from cows, and the restricted feed intake increased P concentrations in meconium. These results suggest that the maintenance of optimum erythropoiesis and mineral status in heatstressed periparturient cows and heifers and their calves must be met by dietary energy and minerals that are fed at maintenance concentrations plus excess requirements necessary during the gestation period.

Animals↗
PubMed1998

[Immunohistochemical study on keratin no. 8, 18 and 19 expression of carcinoid tumors].

The purpose of this study is to evaluate keratin expression in neuroendocrine cells and carcinoid tumor of the gastrointestinal tract and lung. Neuroendocrine cells in the lung and the gastrointestinal tract reacted with low-molecular keratin strongly, but not with high-molecular keratin. The low-molecular keratin expression of carcinoid tumor in the lung and the gastrointestinal tract, which varied in degree, were identical to that of neuroendocrine cells. Keratin 8 and 18 were found abundantly in neuroendocrine tumor and carcinoid tumor of the gastrointestinal tract, and in a lesser amount in carcinoid tumor of the lung, whereas keratin 19 was found in all cases with variable amounts. Based on these results, the combination of antibodies for several types of keratin is useful for examining keratin expression as a marker of epithelium in carcinoid tumor.

Adult↗
PubMed1998

[Appearance of extrahepatic lesions in the superior lip and orbit of a patient with liver metastases of gastric cancer following partial response by hepatic arterial infusion chemotherapy].

A 76-year-old man was totally gastrectomized in June, 1996 for advanced gastric cancer. In February, 1997, multiple liver metastases were found by abdominal CT scan, and hepatic arterial infusion chemotherapy was started with 5-FU, EPI and MMC by an implantable reservoir indwelled via the left subclavian artery. This treatment was judged to have led to a partial response because reduced focus size of the liver metastasis was revealed by CT scan and levels of tumor markers decreased significantly. However, metastatic foci were found in the skin of the superior lip and the orbit in December 1997. He was treated with a variety of therapies, but died in January, 1998. Many cases with metastatic hepatic cancer have a poor prognosis because of the appearance of extrahepatic lesions in spite of the fact that a partial response can be obtained by hepatic arterial infusion chemotherapy. The present case was unique since extrahepatic lesions appeared at very rare sites such as the superior lip and the orbit.

Aged↗
PubMed1997

Glycoprotein Ib-von Willebrand factor interactions activate tyrosine kinases in human platelets.

von Willebrand factor (vWF ) in the presence of botrocetin induces p72(syk) activation, assessed as its autophosphorylated level and in vitro kinase assays, the transient association of p72(syk) with p60(c-src), and the translocation of p60(c-src) and p54/58(lyn) to cytoskeletal fractions. Jararaca glycoprotein Ib-binding protein (GPIb-BP), which specifically binds to GPIb, abolished these phenomena, suggesting that they are mediated by the vWF-GPIb interaction. These tyrosine kinase-related events were not inhibited by GRGDS peptide (plus EGTA), indicating that GPIIb/IIIa is not involved in the observed responses. Shc, an adaptor protein, was also tyrosine phosphorylated by the botrocetin-vWF activation. When GPIb was immunoprecipitated with nonfunctional monoclonal antibodies (MoAbs) directed against GPIb, a kinase activity was found to associate with GPIb upon botrocetin-vWF activation. On the other hand, anti-GPIb MoAbs that inhibit the vWF-GPIb interaction did not coprecipitate a kinase activity. Because the recovery of GPIb did not differ significantly, it is suggested that the excessive presence of inhibitory anti-GPIb MoAb dissociated a kinase activity from GPIb. Phosphoamino acid analysis showed that the kinase activity was that of a tyrosine kinase. The identity of the tyrosine kinase and the mode of interaction with the cytoplasmic region of GPIb await to be determined. Our findings suggest that the tyrosine kinase associated with GPIb serves at a most proximal step in the signal transduction pathway involved in the vWF-GPIb-induced platelet activation, which leads to other tyrosine kinase-related intracellular signals.

Blood Platelets↗
PubMed1997

Role of inositol 1,4,5-trisphosphate receptor in ventral signaling in Xenopus embryos.

The inositol 1,4,5-trisphosphate (IP3) receptor is a calcium ion channel involved in the release of free Ca2+ from intracellular stores. For analysis of the role of IP3-induced Ca2+ release (IICR) on patterning of the embryonic body, monoclonal antibodies that inhibit IICR were produced. Injection of these blocking antibodies into the ventral part of early Xenopus embryos induced modest dorsal differentiation. A close correlation between IICR blocking potencies and ectopic dorsal axis induction frequency suggests that an active IP3-Ca2+ signal may participate in the modulation of ventral differentiation.

Activins↗
PubMed1997

N,N-dimethylsphingosine 1-phosphate activates human platelets.

We recently reported that N,N-dimethylsphingosine 1-phosphate (DMS-1-P) can be formed from N,N-dimethylsphingosine (DMS) in activated platelets [Y. Yatomi et al., Biochem. Biophys. Res. Commun. 231 (1997) 848-851]. In this study, we synthesized, for the first time, DMS-1-P and examined the functional effects of DMS-1-P and its related sphingolipids on platelets. Although exogenous DMS was inactive, its phosphorylated derivative, DMS-1-P, induced platelet intracellular Ca2+ mobilization and shape change, but not aggregation or release reactions. Since sphingosine 1-phosphate (Sph-1-P) is structurally related to DMS-1-P and activates platelets more strongly than DMS-1-P, a competitive binding experiment for [3H]Sph-1-P was performed using DMS-1-P. DMS-1-P reduced the binding of [3H]Sph-1-P to platelets almost as much as unlabeled Sph-1-P did. These results suggest that DMS-1-P activates platelets via an interaction with a platelet surface receptor for Sph-1-P.

Blood Platelets↗
PubMed1997

Activation of the G protein Gq/11 through tyrosine phosphorylation of the alpha subunit.

Various receptors coupled to the heterotrimeric guanine nucleotide-binding protein Gq/11 stimulate formation of inositol-1,4,5-trisphosphate (IP3). Activation of these receptors also induces protein tyrosine phosphorylation. Formation of IP3 in response to stimulated receptors that couple to Gq/11 was blocked by protein tyrosine kinase inhibitors. These inhibitors appeared to act before activation of Gq/11. Moreover, stimulation of receptors coupled to Gq/11 induced phosphorylation on a tyrosine residue (Tyr356) of the Galphaq/11 subunit, and this tyrosine phosphorylation event was essential for Gq/11 activation. Tyrosine phosphorylation of Galphaq/11 induced changes in its interaction with receptors. Therefore, tyrosine phosphorylation of Galphaq/11 appears to regulate the activation of Gq/11 protein.

Animals↗
PubMed1997

Tyrosine phosphorylation and p72syk activation by an anti-glycoprotein Ib monoclonal antibody.

NNKY5-5, an IgG monoclonal antibody directed against the von Willebrand factor-binding domain of glycoprotein (GP) Ib alpha, induced weak but irreversible aggregation (or association) of platelets in citrate-anticoagulated platelet-rich plasma. This phenomenon was defined as small aggregate formation (SAF). Platelets in hirudin-anticoagulated plasma or washed platelets showed little response to NNKY5-5 alone, but the antibody potentiated aggregation induced by low concentrations of adenosine diphosphate or platelet-activating factor. NNKY5-5 did not induce granule release or intracellular Ca2+ mobilization. However, NNKY5-5 caused tyrosine phosphorylation of a 64-kD protein and activation of a tyrosine kinase, p72syk. An anti-Fc gamma II receptor antibody had no effect on SAF, suggesting that NNKY5-5 activated platelets by interacting with glycoprotein Ib. Fab' fragments of NNKY5-5 did not induce SAF, but potentiated aggregation induced by other agonists. The Fab' fragment of NNKY5-5 induced the activation of p72syk, suggesting that such activation was independent of the Fc gamma II receptor. Cross-linking of the receptor-bound Fab' fragment of NNKY5-5 with a secondary antibody induced SAF. GRGDS peptide, chelation of extracellular Ca2+, and an anti-GPIIb/IIIa antibody inhibited NNKY5-5-induced SAF, but had no effect on 64-kD protein tyrosine phosphorylation or p72syk activations. Various inhibitors, including aspirin and protein kinase C, had no effect on SAF, protein tyrosine phosphorylation, or p72syk activation. In contrast, tyrphostin 47, a potent tyrosine kinase inhibitor, inhibited NNKY5-5-induced SAF as well as tyrosine phosphorylation and p72syk activation. Our findings suggest that binding of NNKY5-5 to GPIb potentiates platelet aggregation by facilitating the interaction between fibrinogen and GPIIb/IIIa through a mechanism associated with p72syk activation and tyrosine phosphorylation of a 64-kD protein.

Antibodies, Monoclonal↗
PubMed1997

N,N-dimethylsphingosine phosphorylation in human platelets.

Metabolism of sphingosine (Sph) derivatives in human platelets was examined. [3H]Sph was rapidly and heavily phosphorylated into sphingosine 1-phosphate, similarly in resting and stimulated platelets. [14C]N,N-dimethylsphingosine was stable in resting platelets, while it was converted into N,N-dimethylsphingosine 1-phosphate (DMS-1-P), although weakly, in platelets stimulated with thrombin or 12-O-tetradecanoylphorbol 13-acetate. This DMS-1-P formation was inhibited by staurosporine, a potent protein kinase inhibitor. [3H]C2-ceramide was unchanged both in resting and stimulated platelets. Our report is the first to describe production of DMS-1-P in a biological system.

Blood Platelets↗