Biomedical subjects

H Yamamura

Publications and source records attributed to H Yamamura.

At least 235 records · Page 13Linked to original sources

PubMed1991

Identification of the phosphorylation sites of H2B histone by a catalytic fragment of p72syk from porcine spleen.

Phosphorylated sites of calf thymus H2B histone were investigated with a catalytic fragment of 72 kDa protein-tyrosine kinase (p72syk). Three of five tyrosine residues in H2B histone can be phosphorylated by this kinase. In this analysis, H2B histone was thoroughly phosphorylated in vitro with [gamma-32P]ATP and the kinase, and then digested with a lysylendopeptidase. The resulting radioactive phosphopeptides were separated by a reverse-phase column on high performance liquid chromatography. Subsequent sequential Edman degradation of the purified phosphopeptides revealed that 40Y, 83Y and 121Y were phosphorylated. 121Y is the major phosphorylated residue in H2B histone. No phosphorylation was detected in 37Y and 42Y. Although the consensus sequence was not defined from these analyses, our data suggest that higher-order structure(s) in addition to primary one may participate in recognition of H2B histone by this protein kinase.

Amino Acid Sequence↗

PubMed1991

The lectin wheat germ agglutinin stimulates a protein-tyrosine kinase activity of p72syk in porcine splenocytes.

We previously reported a molecular cloning of porcine gene syk encoding a non-receptor type 72-kDa protein-tyrosine kinase and a generation of anti-CPTK40 antibodies which could immunoprecipitate the activity of p72syk (Taniguchi et al. (1991) J. Biol. Chem. 266, 15790-15796). In this study, we have demonstrated that wheat germ agglutinin caused an increase in the autophosphorylation activity of p72syk which preceded an increase of protein-tyrosine phosphorylation observed at the same time in porcine splenocyte. The increase of p72syk activity was dose dependent and was inhibited by the coexistence of N-acetyl-D-glucosamine. Upon stimulation by the combination of 4 beta-phorbol 12-myristate 13-acetate and ionophore A23187, we could not detect the increase of activity of p72syk suggesting that the activation of p72syk was independent of protein kinase C and calcium ions.

Acetylglucosamine↗

PubMed1991

Molecular cloning of a porcine gene syk that encodes a 72-kDa protein-tyrosine kinase showing high susceptibility to proteolysis.

By using antibodies raised against a portion of N terminus of 40-kDa kinase (Kobayashi, T., Nakamura, S., Taniguchi, T., and Yamamura, H. (1990) Eur. J. Biochem. 188, 535-540), not only 40-kDa protein but also 72-kDa protein were detected on immunoblot analysis of porcine spleen homogenate. In splenocytes preparation, the antibodies could immunoprecipitate protein-tyrosine kinase activity of the 72-kDa protein but not detect the 40-kDa protein even on immunoblot. After incubation of crude spleen homogenate at 37 degrees C with or without various protease inhibitors, immunoblot analysis revealed proteolytic breakdown of the 72-kDa protein to 40-kDa fragment. Next, using oligonucleotides designed according to partially sequenced information of the 40-kDa kinase as a probe, we have isolated a clone containing entire coding sequence for the 40-kDa kinase from a porcine spleen cDNA library. This clone had a 1884-base-pair-long open reading frame encoding 628-amino-acid polypeptide with a calculated molecular weight of 71,618. The deduced amino acid sequence did not contain a ligand binding or membrane spanning region but did a well-conserved protein-tyrosine kinase domain and two src homology region 2 domains. The sequences of these domains showed 30-40% identity to those of other protein-tyrosine kinases, but those of remaining parts were quite unique. From these results, we concluded that the 40-kDa kinase was generated by proteolysis from the 72-kDa holoprotein which was a new member of nonreceptor protein-tyrosine kinase so far reported. We therefore designated this gene as syk after spleen tyrosine kinase.

Amino Acid Sequence↗

PubMed1991

The lectin wheat germ agglutinin induces rapid protein-tyrosine phosphorylation in human platelets.

In response to wheat germ agglutinin (WGA), platelet aggregation and stimulation of protein-tyrosine phosphorylation were observed in a dose dependent manner. These reactions were completely inhibited by coexistence of N-acetyl-D-glucosamine with WGA. Upon stimulation by this agonist, protein-tyrosine phosphorylation of seven bands with molecular masses of 140-, 130-, 80-, 76-, 53-, 38- and 35-kDa proteins was observed by immunoblot. These protein-tyrosine phosphorylations were divided into three groups by kinetics. Considering the previous report from our laboratory that thrombin and collagen induced tyrosine phosphorylation in 135-, 124- and 76-kDa proteins (Nakamura, S. and Yamamura, H. (1989) J. Biol. Chem. 264, 7089-7091.), there may be another signal transduction pathway in tyrosine phosphorylation of human platelets.

Blood Platelets↗

PubMed1991

Ionophore A23187-induced protein-tyrosine phosphorylation of human platelets: possible synergism between Ca2+ mobilization and protein kinase C activation.

Addition of ionophore A23187 to washed human platelets caused a time- and dose-dependent increase in the phosphotyrosyl content of 135, 124 and 76 kDa proteins. Platelets loaded with intracellular Ca2+ chelator 5,5'-dimethyl-bis-(o-aminophenoxy)-ethane-N, N, N', N'-tetraacetic acid before addition of A23187 exhibited no protein-tyrosine phosphorylation. Replenishment of such platelets with extracellular CaCl2 restored A23187-induced protein-tyrosine phosphorylation. Upon stimulation with A23187, both aspirin and ADP scavengers-treated platelets exhibited protein-tyrosine phosphorylation without phosphoinositide hydrolysis and protein kinase C activation. These data show (a) that A23187 stimulates protein-tyrosine phosphorylation by the elevation of intracellular Ca2+, and (b) that A23187-induced protein-tyrosine phosphorylation is independent of formation of endoperoxides/thromboxane A2, released ADP, phosphoinositide hydrolysis and protein kinase C activation. Furthermore, a synergistic effect of A23187 and protein kinase C activators in stimulating protein-tyrosine phosphorylation is suggested.

Blood Platelets↗

PubMed1991

The prevalence at birth of cogenital malformations at a maternity hospital in Osaka City, 1948-1990.

The frequencies of congenital malformations at St. Barnabas' Hospital, Osaka were studied during the period 1948-1990. There were 1,418 malformed infants diagnosed during the first week of life among a total number of 131,996 births during the period. Of the 1.07% of malformed children, 0.90% had single and 0.17% had multiple malformations. The birth prevalence was 1.07% in singletons and 1.20% in twins. There was significant difference in prevalence between males (1.15%) and females (0.97%). The prevalence was 0.88% for the period 1948-1958 and increased with each year cohort up to the recent period when the value was 1.38%. Varus deformities of feet was the most common defect (23.4 per 10,000 births), followed by polydactyly (finger) (7.8), anencephaly (7.1), cleft lip and palate (6.5), syndactyly (toe) (6.5), cleft lip (6.3), and Down's syndrome (6.1). The prevalence of conjoined twins was 1 in 65,551 deliveries. Significant differences occurred between males and females for cleft palate, cleft lip and palate, syndactyly (finger), oligodactyly (finger), and hypoplastic auricle. The prevalences for valgus deformities of feet, syndactyly (toe) and atresia ani increased significantly with the year cohort, whereas the prevalence of cleft palate decreased significantly with the year cohort. Overall prevalence was significantly higher in 1970-1990 (1.28% than in 1948-1969 (0.88%). Similar results were examined for cleft lip and palate, syndactyly (finger), syndactyly (toe), hypospadias, hydrocele testis, and Down's syndrome.

Congenital Abnormalities↗

PubMed1991

Possible endogenous substrate proteins for cytosolic protein-tyrosine kinase from porcine spleen.

1. Endogenous phosphate acceptor proteins by cytosolic protein-tyrosine kinase from porcine spleen (CPTK-40) were studied using subcellular fractions of porcine spleen and supernatant fraction of rat various tissues. 2. At least 13 phosphate acceptor proteins ranging from 94 to 26 kDa were observed in all but mitochondrial subcellular fractions. 3. Among the supernatant fraction of rat tissues, brain, testis and spleen contained many phosphate acceptor proteins. 4. The most heavily phosphorylated band of around 55 kDa which was commonly recognized among various tissues was confirmed as tubulin by the immunoreactivity with anti-tubulin antibody. 5. The results obtained in this paper indicate that CPTK-40 has the potential to catalyze the phosphorylation of numerous endogenous proteins including tubulin.

Animals↗

PubMed1991

Studies on protein kinase C tightly-bound to rat liver plasma membrane and its protease-activated form.

1. Rat liver plasma membrane contained two types of protein kinase C which could be extracted by Ca2(+)-chelator and detergent, respectively. The activities of these two enzymes were nearly equivalent. 2. The detergent-extracted protein kinase C, tightly-bound to membrane, was separated into two subtypes by hydroxyapatite column chromatography. Based on the elution profile and the Ca2+/phospholipid requirement, the major and the minor components were identified as type III and type II protein kinase C, respectively. 3. The detergent-extracted protein kinase C was converted to an active fragment with Mr 45,000 by limited proteolysis with trypsin. Incubation under physiological level of ionic strength increased the stability of this active enzyme and protected it from further inactivation by trypsin. 4. Phosphorylation of H1 histone by the protease-activated kinase was stimulated 1.5-2-fold by phosphatidylserine. However, this enzyme phosphorylated multiple proteins in rat liver subcellular fractions in Ca2(+)- and phospholipid-independent manner. 5. These results suggest that the protein kinase C (mainly type III enzyme) tightly-bound to rat liver plasma membrane may have important role through protein phosphorylation by the native or the protease-activated kinase.

Animals↗

PubMed1991

Effect of SN-408 (salmeterol hydroxynaphthoate) on passive cutaneous anaphylaxis and anaphylactic chemical mediator release in rats and guinea pigs.

The influence of SN-408 (salmeterol hydroxynaphthoate), a new selective beta 2-stimulant, on homologous passive cutaneous anaphylaxis (PCA), histamine- or serotonin-induced skin reaction and anaphylactic chemical mediator release was investigated in rats and guinea pigs, and its efficacy was compared with that of salbutamol, isoproterenol or disodium cromoglycate (DSCG). Intravenous administration of SN-408 (0.1-10 micrograms/kg) 1 min before, the antigen challenge dose-dependently inhibited rat homologous PCA to a similar extent to salbutamol. The inhibitory effect of these beta-agonists was quickly attenuated along with time between the administration of the drug and the antigen challenge. SN-408, however, still showed significant inhibition at the time of administration 5 min before the antigen challenge at which other agonists did not significantly affect any more. Ten micrograms/kg of SN-408 exhibited approximately 50% inhibition of skin reaction induced by either histamine or serotonin in rats. In addition, the compound represented concentration-dependent inhibition of the anaphylactic histamine release from the isolated rat peritoneal exudate cell, indicating that the inhibitory effect of SN-408 on rat homologous PCA is due to the suppression of mediator release in addition to the antagonism to mediator(s) released. The anaphylactic release of either histamine, immunoreactive (i-) leukotriene (LT)B4 or i-LTC4 from guinea pig lung fragments was concentration-dependently inhibited by 10(-10)-10(-7) g/ml SN-408, and the inhibitory potency appeared to be enhanced by prolongation of the pretreatment interval with the drug.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic beta-Agonists↗

PubMed1991

Successful 0 degree C liquid preservation of red blood cells.

In an effort to determine the lowest acceptable temperature for liquid preservation of red blood cells, the latter were stored at 5, 0, -2 and -5 degrees C. When polyvinylchloride (PVC) bags plasticized with di(2-ethylhexyl)phthalate (DEHP) were used as containers, hemolysis of red cells stored for 42 days at 0 degree C was lower than at the other three temperatures. Adenosine triphosphate (ATP) levels, morphology scores, and deformability indices were also best maintained at 0 degree C, and erythrocyte osmotic fragility did not increase at that temperature. On the other hand, when blood was stored in glass bottles at 0 degree C, hemolysis and erythrocyte osmotic fragility increased. The addition of DEHP to red cells stored in glass bottles at 0 degree C decreased hemolysis and erythrocyte osmotic fragility to levels equal to those in PVC bags. Thus, storage of red cells can be done at temperatures as low as 0 degree C, if PVC bags are used as the container. Previous negative assessments of storage temperatures below 4 degrees C, may be due to the use of glass bottles as containers for blood preservation.

Adenosine Triphosphate↗

PubMed1991

Effect of long-term feeding of nivalenol on aflatoxin B1-initiated hepatocarcinogenesis in mice.

Nivalenol, a trichothecene, occurs widely in cereals and foods; our current two-year feeding trial has revealed no tumorigenic activity in female mice. To investigate whether dietary nivalenol modulates the development of aflatoxin B1 (AFB1)-initiated hepatocarcinogenesis, one-week old C57Bl/6 x C3H F1 mice were injected intraperitoneally with 6 mg/kg bw AFB1 and six weeks later fed diets containing 0, 6 or 12 ppm nivalenol for one year. Male mice in all three groups developed hepatocellular carcinomas and adenomas, while the incidences in females were 31% in those given AFB1 alone and 20% and 0 in those given AFB1 with 6 and 12 ppm nivalenol, respectively. These findings indicate that dietary nivalenol suppresses AFB1-initiated hepatocarcinogenesis in female mice, presumably by acting on the promotion step.

Aflatoxin B1↗

PubMed1991

[A case report of pseudoxanthoma elasticum with upper gastrointestinal hemorrhage].

A gastric hemorrhage in a patient with pseudoxanthoma elasticum (PXE) is reported. A 44-year-old woman was admitted to our hospital with hematemesis. Bleeding point was located at upper body of the stomach. As neither endoscopic alcohol injection nor operative hemostasis by over sewing under gastrotomy were unsuccessful, total gastrectomy was performed. Histologic examination of the stomach revealed fragmentation of elastic fiber in the internal lamina of arteriole and degeneration of muscle layer. This change was observed fundamentally in all specimens obtained from different region of the stomach. In addition to describing clinical and pathological feature of this case, the management of gastric hemorrhage in patients with PXE is discussed.

Adult↗

PubMed1991

Protease-activated protein kinase C in rat liver.

In regenerating rat liver, an elevated protein kinase activity was detected which phosphorylated ribosomal protein S6 and histones. The properties of this enzyme were closely similar with those of protease-activated protein kinase C with Mr 45,000. During the study of the mechanism of proteolytic activation, type III protein kinase C (encoding alpha-sequence) was shown to be subjected to limited proteolysis by trypsin-like protease and converted to protein kinase M in ionic strength- and pH-dependent manner. This reaction was stimulated in the presence of Ca2+ and phospholipid under slightly higher ionic strength condition than physiological level (greater than 140 mM NaCl) and alkaline pH (7.5-8.0). These results suggest that activation of Na+/H+ exchanger in plasma membrane may trigger this type of proteolytic activation of protein kinase C. In addition to protein kinase M, another type of protease-activated kinase with Mr 80,000 was detected when limited proteolysis of protein kinase C was performed on inactive form of this enzyme (in the absence of either Ca2+ or phospholipid or both activators) under lower ionic strength condition. The molecular mass of this active enzyme was slightly smaller (approximately 200) than that of native protein kinase C. However, it is not clear at this time whether this small fragment was released from amino-terminal or carboxy-terminal domain to make protein kinase C partially active in the absence of Ca2+ and phospholipid. Although it has been proposed that proteolytic degradation of protein kinase C is involved in down regulation of this enzyme, the physiological significance of these two types of protease-activated forms of protein kinases in liver has remained obscure.

Animals↗

PubMed1990

Protein-tyrosine phosphorylation and aggregation of intact human platelets by vanadate with H2O2.

The ability of vanadate with H2O2 to stimulate protein-tyrosine phosphorylation and aggregation in intact human platelets was observed. Upon stimulation by these agents, there was a change in the amount of protein-tyrosine phosphorylation of four bands with molecular masses of 80-, 76-, 53- and 38-kDa proteins. The tyrosine phosphorylation in these four bands increased gradually and reached a maximum level at 2 min and then decreased. Aggregation of platelets was also induced by these agents in a dose dependent manner. The observation that the aggregation was preceded by the increase in tyrosine phosphorylation of these proteins suggests that tyrosine phosphorylation may be involved in an early stage of platelet aggregation.

Blood Platelets↗

PubMed1990

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

A cytosolic protein-tyrosine kinase has been highly purified from porcine spleen using [Val5]angiotensin II as a substrate. The purification procedure involves sequential column chromatographies on phosphocellulose, Sephacryl S-200, casein-Sepharose 4B, heparin-Sepharose CL-6B and anti-(4-aminobenzyl phosphonic acid)--Sepharose 4B. Analysis of the most highly purified preparation by SDS/PAGE revealed a major silver-stained band of molecular mass 40 kDa. The 40-kDa cytosolic protein-tyrosine kinase was purified approximately 10,000-fold with an overall yield of about 7%. It had autophosphorylation activity which was carried out by intramolecular catalysis. The stoichiometry of phosphate incorporation was about 1 mol phosphate/mol enzyme. In the autophosphorylation reaction, the apparent Km value for ATP was relatively low, 0.35 microM; Mn2+ was slightly preferred to Mg2+ as divalent cation. [Val5]Angiotensin II phosphorylation activity of the 40-kDa kinase increased with the amount of phosphate incorporated into the enzyme. A phosphate exchange reaction was observed during the autophosphorylation. These results suggest that the 40-kDa kinase described here is a different type of protein-tyrosine kinase than the enzymes so far reported.

Adenosine Triphosphate↗

PubMed1990

Diverse effects of poly-basic amino acids, heparin and ionic strength on the phosphorylation of various substrates by cytosolic protein-tyrosine kinase from porcine spleen.

1. Effects of poly-basic amino acids, heparin and ionic strength on the activity of cytosolic protein-tyrosine kinase from porcine spleen (CPTK-40) have been studied. 2. Both polylysine and polyarginine stimulated the phosphorylation of [Val5]angiotensin II and E11 G1 (synthetic peptide of EDAEYAARRRG), but could neither stimulate nor inhibit the phosphorylation of random copolymers; poly(EY)4:1 and poly(EAY)6:3:1. 3. Heparin stimulated the phosphorylation of poly(EY)4:1 by 2.5-fold, however, it inhibited those of E11G1, poly(EAY)6:3:1, casein and H2B histone. 4. Elevation of ionic strength of either NaCl, KCl or (NH4)2SO4 stimulated the phosphorylation of poly(EY)4:1 by greater than 5-fold, but inhibited those of casein, tubulin, H2B histone, E11G1 and poly(EAY)6:3:1. 5. These effectors did not change the Km for substrates but increased the Vmax. 6. These results suggest that the effects of poly-basic amino acids, heparin and ionic strength on the activity of CPTK-40 are mainly on the substrates employed rather than on the enzyme itself.

Amino Acids↗

PubMed1990

Comparison of substrate recognition by protein kinase C (type III) between rat liver cytosolic and particulate fractions.

1. Phosphorylation of rat liver endogenous substrates by protein kinase C (type III) was compared between cytosolic and particulate (mitochondria, microsomes and plasma membrane) fractions. 2. The rate and the maximum level of protein phosphorylation were several-fold higher in particulate fractions than in cytosolic fraction. 3. Protein phosphorylation in cytosolic fraction was dependent on both Ca2+ and phospholipid, but only Ca2+ was necessary in phosphorylation of particulate fractions. 4. These results suggest that protein kinase C (type III) has much more target proteins in particulate fractions rather than in cytosolic fraction and Ca2+ was important regulator in particulate protein phosphorylation.

Animals↗

PubMed1990

Mechanism of proteolytic activation of rat liver protein kinase C generating Ca2(+)-phospholipid-independent form with apparent molecular mass of 80,000.

1. New Ca2(+)-phospholipid-independent form of protein kinase C was produced by limited proteolysis with trypsin. 2. The molecular mass of this active enzyme was slightly smaller than that of original protein kinase C. 3. The active enzyme cross-reacted with antibody against the pseudosubstrate region on amino-terminal end of protein kinase C. 4. The active enzyme was inhibited by the peptide inhibitor derived from the pseudosubstrate region. 5. These results suggest that the limited proteolysis at or near the pseudosubstrate region made protein kinase C active without Ca2+ and phospholipid.

Animals↗