The structure and function of nonreceptor tyrosine kinase p72syk expressed in hematopoietic cells.
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Amino Acid Sequence↗
Biomedical subjects
S Yanagi
Publications and source records attributed to S Yanagi.
Syk and Lyn are involved in radiation-induced signaling, but inactivation of Syk or Lyn alone is not sufficient to prevent radiation-induced apoptosis.
Radiation-induced biochemical events that mediate the intracellular signal transduction leading to cell apoptosis are largely unknown. Limited evidence suggests the possible involvement of one or more protein-tyrosine kinases (PTKs) in radiation-induced cellular responses, including apoptosis. However, so far, a PTK(s) responsible for the radiation-induced tyrosine phosphorylation of cellular substrates has not been identified and the role of the PTK(s) in the radiation-induced apoptosis remains unclear. To examine the roles of Syk and Lyn in radiation-induced signal transduction and radiation-induced apoptosis, we analyzed Syk-deficient or Lyn-deficient DT40 B cells along with wild-type cells following radiation. When DT40 B cells were exposed to radiation, the activity of Syk kinase dramatically increased and reached a maximum with 0.25 Grays (Gy) (15 s), and then decreased, whereas Lyn kinase activity increased and reached a maximum with a dose of 1.00 Gy (1 min). However, an apparent difference was not observed in radiation-induced apoptosis among wild-type, Syk-deficient, and Lyn-deficient DT40 B cells. These results indicate that Syk and Lyn kinases are involved in radiation-induced signal transduction, with different kinetics. In addition, our results revealed that functional inactivation of Syk or Lyn alone is not sufficient to prevent radiation-induced apoptosis. Thus, it is suggested that the activation of Syk or Lyn kinase alone may be sufficient to mediate the radiation-induced apoptosis in DT40 B cells, or both kinases may not be required for this biological process.
Animals↗
A new technique for bilateral single stoma loop cutaneous ureterostomy.
PURPOSE: We present a new technique of cutaneous ureterostomy. The results of application of this method and its advantages over prior methods are discussed. MATERIALS AND METHODS: We applied this method in 15 patients with tumors in the pelvic region and 3 with spinal cord injury between January 1992 and January 1994. RESULTS: A tubeless state was achieved in more than 90% of the patients. CONCLUSIONS: The technique described has a wider range of indication than prior techniques and decreases the risk of stomal stenosis arising from postoperative ureteral tension.
Adult↗
Potentiating effects of beta-eudesmol-related cyclohexylidene derivatives on succinylcholine-induced neuromuscular block in isolated phrenic nerve-diaphragm muscles of normal and alloxan-diabetic mice.
beta-Eudesmol, a sesquiterpenoid alcohol contained in Atractylodes lancea, potentiates succinylcholine (SuCh)-induced neuromuscular blockade. The potentiating effect is greater in diabetic muscles than in normal ones. As a ligand for affinity chromatography to study the potentiating mechanism, we designed and synthesized newly beta-eudesmol-related cyclohexylidene derivatives (2-(3-hydroxy-3-methylbutyl)cyclohexylidene; KTE-13, 2-(3-hydroxy-3-methylbutyl)-4-cyclohexylidene carboxylic acid; KTE-32 and 4-tert-butoxycarbonyl-2-(3-hydroxy-3-methylbutyl) cyclohexylidene; KTE-33). We examined the potentiating effects of those compounds in phrenic nerve-diaphragm muscle preparations of normal and alloxan-diabetic mice. KTE-33 (100 microM) potentiated more greatly SuCh-induced neuromuscular blockade in diabetic muscles than in normal ones (the potentiating ratios in normal and diabetic muscles were 6.7 and 10.6, respectively), while KTE-13 (100 microM) and -32 (200 microM) potentiated weakly. These results suggest that the ester group in KTE-33 rather than a carboxyl group in KTE-32 is important in inducing the potentiation of SuCh-induced neuromuscular blockade in diabetic state.
Animals↗
Myocardial infarct size by serum troponin T and myosin light chain 1 concentration.
The late troponin T (TnT) peak concentration, which is known to be independent of reperfusion of the infarcted zone in acute myocardial infarction (MI), has been suggested to be correlated with clinical estimates of cardiac function and myocardial infarct size. To refine the clinical application of the late TnT peak in infarct sizing, and to examine differences in this estimation in different infarct sites, we measured the serum concentrations of TnT and myosin light chain 1 (MLC1), and compared these values with left ventricular ejection fraction (LVEF) obtained from left ventriculography, and extent score (ES) and severity score (SS) obtained from 201Tl scintigraphy in patients with anterior and inferior myocardial infarction. The late TnT peak concentration was strongly correlated with the MLC1 peak value in patients with anterior MI (r = 0.67, p < 0.05) and in those with inferior MI (r = 0.92, p < 0.0005). Furthermore, there were strong linear correlations between the late TnT peak values and all of the clinical data (LVEF; r = -0.79, p < 0.01, ES; r = 0.75, p < 0.05, SS; r = 0.75, p < 0.05, respectively) in patients with anterior MI. However, these correlations were weak in patients with inferior MI. Similar correlations were observed between MLC1 and the clinical data. Thus, TnT and MLC1 have similar kinetics in the serum at the late phase and can be used to estimate the size of anterior infarct.
Aged↗
Relationship between insufficient redistribution in exercise thallium-201 myocardial single-photon emission computed tomography and reverse redistribution at rest.
It is widely accepted that perfusion defects in 3 to 4-h delayed images in exercise thallium-201 (201Tl) myocardial scintigraphy underestimate the viability of myocardium in the infarct region. In the present study, to examine the contribution of the condition of myocardium which demonstrates reverse redistribution in resting scintigraphy to the insufficiency of redistribution in the 4-h delayed image in exercise scintigraphy, we performed exercise and resting 201Tl myocardial single-photon emission computed tomography in 58 patients with acute myocardial infarction and a single diseased coronary artery. Twenty eight patients demonstrated reverse redistribution (group RR) and 28 showed a fixed defect (group FD) in resting scintigraphy. Redistribution in the 4-h delayed image in exercise scintigraphy was significantly more insufficient in group RR than in group FD (p < 0.01), and the degree of the insufficiency of redistribution in exercise scintigraphy closely correlated with the degree of reverse redistribution in resting scintigraphy (r = 0.79, p < 0.001). We conclude that in patients with acute myocardial infarction, the condition of myocardium which demonstrates reverse redistribution in resting myocardial scintigraphy is related to the insufficiency of redistribution in the delayed image in exercise scintigraphy.
Adult↗
Angiotensin II type-1 receptor antagonist as well as angiotensin converying enzyme inhibitor attenuates the development of heart failure in aortocaval fistula rats.
OBJECTIVES: We investigated the effect of chronic administration of an angiotensin II type-1 receptor antagonist in the development of heart failure due to volume overload in rats. METHODS: Aortocaval fistula (AVF), a model of volume overloaded heart failure, was induced in rats by our newly developed technique using a simple and rapid 18-gauge needle multipuncture. After 3 weeks of oral administration of an angiotensin II receptor antagonist TCV-116, 1 mg/kg per day, we evaluated the hemodynamics, heart weight, and degree of left ventricular dilatation. We also compared the effect of TCV-116 with that of an angiotensin-converting enzyme inhibitor delapril, 1 g/L in drinking water. RESULTS: AVF heart failure produced by our technique exhibited significant increases in the left ventricular end-diastolic pressure (LVEDP) (12 = 1 vs 4 +/- 1 mmHg, p < 0.05), right atrial pressure (RAP) (5.0 +/- 0.6 vs 1.0 +/- 0.4 mmHg, p < 0.05), right ventricular systolic pressure (RVSP) (58 +/- 6 vs 33 +/- 1 mmHg, p < 0.05), left ventricular weight (LVW) (3.00 +/- 0.13 vs 2.09 +/- 0.04 g/kg BW, p < 0.05), right ventricular weight (RVW) (0.93 +/- 0.05 vs 0.59 +/- 0.01 g/kg BW, p < 0.05), and left ventricular end-diastolic volume index (LVEDVI) (2.55 +/- 0.14 vs 0.80 +/- 0.12 ml/kg BW, p < 0.05) as compared with these values in sham-operated rats. There were no differences in shunt ratio between untreated and TCV-116- and delapril-treated AVF groups. TCV-116 improved these hemodynamics, as did delapril (TCV-116 vs delapril: LVEDP 8 +/- 1 vs 8 +/- 1, RAP: 3.8 +/- 0.6 vs 2.3 +/- 1.4, RASP: 50 +/- 2 vs 46 +/- 3, LVW: 2.53 +/- 0.11 vs 2.52 +/- 0.15, RVW: 0.80 +/- 0.04 vs 0.77 +/- 0.06, LVEDVI: 1.67 +/- 0.15 vs 1.70 +/- 0.17). CONCLUSION: These results suggest that AVF rats with volume overload produced by a new multipuncture method exhibit both right- and left-side heart failure. Angiotensin II type-1 receptor antagonist as well as angiotensin converting enzyme inhibitor attenuate the development of this type of heart failure in rats.
Analysis of Variance↗
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↗
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↗
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↗
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↗
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↗
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↗
Cardioprotective effect of the angiotensin II type 1 receptor antagonist TCV-116 on ischemia-reperfusion injury.
We investigated the protective effect of angiotensin II (Ang II) type 1 receptor antagonist on myocardial ischemia-reperfusion injury and the role of exogenous Ang II to this injury in perfused hearts. We orally administered TCV-116 (Ang II type 1 receptor antagonist) and delapril (angiotensin converting enzyme inhibitor) to Wistar rats for 1 week and measured the immunoreactive cardiac Ang II. Immunoreactive cardiac Ang II (pg/gm tissue) was 14.3 +/- 2.0 in control group, 11.8 +/- 0.8 in TCV-116-treated group, and 7.3 +/- 0.6 in delapril-treated group (p < 0.05 compared to TCV-116-treated group; p < 0.01 compared to control group). The 15 hearts (five rats in each group) were perfused by a langendorff method and global ischemia was maintained for 30 min. Both TCV-116 and delapril were found to improve postischemic cardiac function and decrease reperfusion creatine kinase (CK) release. Ang II injection before ischemia worsened postischemic cardiac function and increased reperfusion CK release. Only TCV-116 prevented this injury. These data indicated that TCV-116 Ang II type 1 receptor antagonist was effective against myocardial ischemia-reperfusion injury, and exogenous Ang II accelerated this injury through Ang II type 1 receptor.
Angiotensin II↗
Intracellular calcium dependent activation of p72syk in platelets.
We previously demonstrated that thrombin-induced activation of p72syk was independent of intracellular Ca2+ elevation in platelets. However, our previous studies also demonstrated that activation of platelets by ionophore A23187 results in a dramatic increase in tyrosine phosphorylation of several cellular proteins. In the present study, we investigated the effect of Ca2+ elevation on the activity of p72syk. When washed porcine platelets were stimulated with ionophore A23187 and the activity of p72syk was assessed by means of an immunoprecipitation kinase assay, A23187 caused a time- and dose-dependent increase in the specific activity of p72syk. In addition, pretreatment of platelets with both aspirin and ADP scavengers or chelation of extracellular Ca2+ by EGTA had no effect on the A23187-induced activation of p72syk. These results indicate that A23187-induced activation of p72syk is independent of the formation of endoperoxide/thromboxane A2, released ADP and extracellular Ca2+, suggesting the existence of a novel pathway for activation of p72syk. Furthermore, evidence is presented which indicates a synergistic effect of A23187 and thrombin on the activation of p72syk, and an inhibitory effect of pretreatment with phorbol 12-myristate 13-acetate, a protein kinase C activator, on the activation of p72syk induced by either A23187 or thrombin.
Animals↗
Comparison of the effects of dobutamine and isoproterenol in ischemic hearts by phosphorus-31 nuclear magnetic resonance spectroscopy.
We compared the effects of 2 inotropic agents, dobutamine and isoproterenol on regional coronary blood flow, contractile function, hemodynamics and levels of phosphate compounds in the acutely ischemic canine heart. Dogs were instrumented to determine regional coronary blood flow (non-radioactive microsphere method), contractile function (sonomicrometry), and hemodynamics. Myocardial phosphate compounds were measured simultaneously by the phosphate-31 (31P) magnetic resonance spectroscopic technique. Both drugs augmented the global performance of the heart, but produced no significant improvement in regional contractile function in the ischemic region. Isoproterenol significantly increased the ratio of inorganic phosphate to phosphocreatine in the ischemic region, as compared to dobutamine. However, no significant differences were seen in myocardial lactate consumption with inotropic stimulation between the 2 groups. 31P magnetic resonance spectroscopy could be used to differentiate between the effects of dobutamine and isoproterenol on energy metabolism in ischemic myocardium despite the lack of significant differences in regional function and myocardial lactate consumption. The significant tachycardia without an augmentation of systemic blood pressure induced by isoproterenol may account for this unfavorable effect on myocardial energy metabolism.
Animals↗
Protein-tyrosine kinase p72syk is activated by platelet activating factor in platelets.
It has been demonstrated that activation of platelets by platelet-activating factor (PAF) results in a dramatic increase in tyrosine phosphorylation of several cellular proteins. We report here that p72syk is a potential candidate for the protein-tyrosine phosphorylation following PAF stimulation in porcine platelets. Immunoprecipitation kinase assay revealed that PAF stimulation resulted in a rapid activation of p72syk which peaked at 10 s. The level of activation was found to be dose dependent and could be completely inhibited by the PAF receptor antagonist, CV3988. Phosphorylation at the tyrosine residues of p72syk coincided with activation of p72syk. Pretreatment of platelets with aspirin and apyrase did not affect PAF induced activation of p72syk. Furthermore, genistein, a potent protein-tyrosine-kinase inhibitor, diminished PAF-induced p72syk activation and Ca2+ mobilization as well as platelet aggregation. These results suggest that p72syk may play a critical role in PAF-induced aggregation, possibly through regulation of Ca2+ mobilization.
Animals↗
Accumulation of aluminum in cancers of the liver, stomach, duodenum and mammary glands of rats.
The aim of the present investigation was to evaluate abnormal changes in trace element concentrations during carcinogenesis. First, Al, Zn and Cu in the liver tissues of rats were measured by atomic absorption analysis over a half year of hepatocarcinogenesis. Male Wistar rats were given carcinogenic food containing 600 mg/kg of 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB) in a basal diet for several months. After 4 to 6 months of feeding, hepatocarcinomas developed in the rats. Zn and Cu concentrations in the hepatocarcinomas of the 3'-MeDAB group significantly decreased as compared with normal liver tissues of the control groups. On the other hand, the aluminum concentration in the hepatocarcinomas was more than three times that in the normal liver tissues. The Al and Se contents of developed gastric and mammary cancers were measured in Experiment II. Male and female rats were given 1-methyl-3-nitrothoguanidine(MNNG) and 2,7-dimehtylbenz(a)anthracene(DMBA), respectively. After several months, carcinomas developed in over half of the rats. The Al and Se concentrations in cancers, livers and the blood were determined by atomic absorption analysis. It was shown that both gastric and mammary carcinomas contained a high level of aluminum and very little selenium in comparison with normal liver tissues. The present study demonstrated that aluminum accumulated in experimentally induced carcinomas in rats, i.e., cancers of the liver, stomach, duodenum and mammary glands.
9,10-Dimethyl-1,2-benzanthracene↗