48 results on '"Morita, T"'
Search Results
2. Coagulation factor X activating enzyme from Russell's viper venom (RVV-X). A novel metalloproteinase with disintegrin (platelet aggregation inhibitor)-like and C-type lectin-like domains.
- Author
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Takeya, H, primary, Nishida, S, additional, Miyata, T, additional, Kawada, S, additional, Saisaka, Y, additional, Morita, T, additional, and Iwanaga, S, additional
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- 1992
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3. The primary structure of coagulation factor IX/factor X-binding protein isolated from the venom of Trimeresurus flavoviridis. Homology with asialoglycoprotein receptors, proteoglycan core protein, tetranectin, and lymphocyte Fc epsilon receptor for immunoglobulin E
- Author
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Atoda, H., primary, Hyuga, M., additional, and Morita, T., additional
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- 1991
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4. Molecular cloning and tissue-specific expression of a novel murine laminin gamma3 chain.
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Iivanainen, A, Morita, T, and Tryggvason, K
- Abstract
A novel laminin gamma3 chain was identified from the expressed sequence tag data base at the National Center for Biotechnology Information. A complete cDNAderived peptide sequence reveals a 1592-amino acid-long primary translation product, including a tentative 33-amino acid-long signal peptide. Comparison with the laminin gamma1 chain predicts that the two polypeptides have equal spatial dimensions. In addition, the well conserved domains VI and III(LE4) predict that gamma3 containing laminins are able to integrate to the laminin network and also via nidogen connect to other protein networks in the basement membranes. Combination of Northern analysis and in situ hybridization experiments indicate that expression of the gamma3 chain is highly tissue- and cell-specific, being significantly strong in capillaries and arterioles of kidney as well as in interstitial Leydig cells of testis.
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- 1999
5. Murine matrix metalloproteinase 9 gene. 5'-upstream region contains cis-acting elements for expression in osteoclasts and migrating keratinocytes in transgenic mice.
- Author
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Munaut, C, Salonurmi, T, Kontusaari, S, Reponen, P, Morita, T, Foidart, J M, and Tryggvason, K
- Abstract
Knowledge about the regulation of cell lineage-specific expression of extracellular matrix metalloproteinases is limited. In the present work, the murine matrix metalloproteinase 9 (MMP-9) gene was shown to contain 13 exons, and the 2.8-kilobase pair upstream region was found to contain several common promoter elements including a TATA box-like motif, three GC boxes, four AP-1-like binding sites, an AP-2 site, and three PEA3 consensus sequences that may be important for basic activity of the gene. In order to identify cell-specific regulatory elements, constructs containing varying lengths of the upstream region in front of a LacZ reporter gene were made and studied for expression in transgenic mice generated by microinjection into fertilized oocytes. Analyses of the mice revealed that the presence of sequences between -2722 and -7745 allowed for expression in osteoclasts and migrating keratinocytes, i. e. cells that have been shown to normally express the enzyme in vivo. The results represent the first in vivo demonstration of the location of cell-specific control elements in a matrix metalloproteinase gene and show that element(s) regulating most cell-specific activities of 92-kDa type collagenase are located in the -2722 to -7745 base pair region.
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- 1999
6. Purification, molecular cloning, and catalytic activity of Schizosaccharomyces pombe pyridoxal reductase. A possible additional family in the aldo-keto reductase superfamily.
- Author
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Nakano, M, Morita, T, Yamamoto, T, Sano, H, Ashiuchi, M, Masui, R, Kuramitsu, S, and Yagi, T
- Abstract
Pyridoxal reductase (PL reductase), which catalyzes reduction of PL by NADPH to form pyridoxine and NADP(+), was purified from Schizosaccharomyces pombe. The purified enzyme was very unstable but was stabilized by low concentrations of various detergents such as Tween 40. The enzyme was a monomeric protein with the native molecular weight of 41,000 +/- 1,600. The enzyme showed a single absorption peak at 280 nm (E(1%) = 10.0). PL and 2-nitrobenzaldehyde were excellent substrates, and no measurable activity was observed with short chain aliphatic aldehydes; substrate specificity of PL reductase was obviously different from those of yeast aldo-keto reductases (AKRs) so far purified. The peptide sequences of PL reductase were identical with those in a hypothetical 333-amino acid protein from S. pombe (the DDBJ/EMBL/GenBank(TM) accession number D89205). The gene corresponding to this protein was expressed in Escherichia coli, and the purified protein was found to have PL reductase activity. The recombinant PL reductase showed the same properties as those of native PL reductase. PL reductase showed only low sequence identities with members of AKR superfamily established to date; it shows the highest identity (18.5%) with human Shaker-related voltage-gated K(+) channel beta2 subunit. The elements of secondary structure of PL reductase, however, distributed similarly to those demonstrated in the three-dimensional structure of human aldose reductase except that loop A region is lost, and loop B region is extended. Amino acid residues involved in substrate binding or catalysis are also conserved. Conservation of these features, together with the major modifications, establish PL reductase as the first member of a new AKR family, AKR8.
- Published
- 1999
7. Carbon monoxide and nitric oxide suppress the hypoxic induction of vascular endothelial growth factor gene via the 5' enhancer.
- Author
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Liu, Y, Christou, H, Morita, T, Laughner, E, Semenza, G L, and Kourembanas, S
- Abstract
Vascular endothelial growth factor (VEGF) plays an important role in angiogenesis and blood vessel remodeling. Its expression is up-regulated in vascular smooth muscle cells by a number of conditions, including hypoxia. Hypoxia increases the transcriptional rate of VEGF via a 28-base pair enhancer located in the 5'-upstream region of the gene. The gas molecules nitric oxide (NO) and carbon monoxide (CO) are important vasodilating agents. We report here that these biological molecules can suppress the hypoxia-induced production of VEGF mRNA and protein in smooth muscle cells. In transient expression studies, both NO and CO inhibited the ability of the hypoxic enhancer we have previously identified to activate gene transcription. Furthermore, electrophoretic mobility shift assays indicated decreased binding of hypoxia-inducible factor 1 (HIF-1) to this enhancer by nuclear proteins isolated from CO-treated cells, although HIF-1 protein levels were unaffected by CO. Given that both CO and NO activate guanylyl cyclase to produce cGMP and that a cGMP analog (8-Br-cGMP) showed a similar suppressive effect on the hypoxic induction of the VEGF enhancer, we speculate that the suppression of VEGF by these two gas molecules occurs via a cyclic GMP-mediated pathway.
- Published
- 1998
8. Magnesium(II) is a crucial constituent of the blood coagulation cascade. Potentiation of coagulant activities of factor IX by Mg2+ ions.
- Author
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Sekiya, F, Yoshida, M, Yamashita, T, and Morita, T
- Abstract
We recently showed that not only Ca2+ ions but also Mg2+ ions play a crucial role in stabilizing the native conformation of coagulation factor IX. We here report that Mg2+ ions at physiological concentrations greatly augment the biological activities of factor IX. In clotting assays with dialyzed plasma, addition of Mg2+ ions enhanced the apparent coagulant activity of factor IXa, while that of factor Xa was scarcely affected. Activation of factor X by factor IXa in the presence of factor VIIIa, phospholipids, and Ca2+ ions was accelerated by Mg2+ ions. It appeared that the cation increased the affinity between factor IXa and factor VIIIa, thereby increasing the apparent catalytic efficacy of the enzyme. We also evaluated the effect of Mg2+ ions in the coagulation pathway initiated by tissue factor and found that activation of factor IX by factor VIIa*tissue factor was accelerated by the cation. Consequently, clotting of normal plasma induced by factor VIIa*tissue factor was shortened by the cation, while no such effect was observed in plasma deficient in factor IX or VIII. These results indicate that the previously unrecognized plasma component, Mg2+ ions, plays crucial roles in blood coagulation and, moreover, that contributions of factors IX and VIII in the coagulation cascade have been seriously underestimated in previous investigations.
- Published
- 1996
9. Carbon monoxide controls the proliferation of hypoxic vascular smooth muscle cells.
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Morita, T, Mitsialis, S A, Koike, H, Liu, Y, and Kourembanas, S
- Abstract
Excess vascular smooth muscle cell (VSMC) proliferation and contractility are key events in the pathophysiology of vascular disorders induced by hypoxia. We have recently reported that carbon monoxide (CO), produced by VSMC under conditions of hypoxia, can be a modulator of cGMP levels in both endothelial and smooth muscle cells. In this respect, some of the physiologic effects of CO in the vasculature parallel those of nitric oxide (NO), a well characterized regulator of vascular tone. We report here that under hypoxia, VSMC-derived CO is an important regulator of VSMC proliferation. Inhibiting CO formation or scavenging CO with hemoglobin increased VSMC proliferation in response to serum or to mitogens such as endothelin, whereas increasing CO production or exposing cells to exogenous CO lead to a markedly attenuated growth response. The effects of CO on VSMC proliferation correlated with changes in E2F-1 expression, the prototype member of a family of transcription factors that participate in the control of cell cycle progression. CO significantly suppressed E2F-1 expression, whereas, removal of CO from the cultures with hemoglobin lead to increased E2F-1 gene transcription, mRNA, and protein production as well as mRNA levels of c-myc, a target gene of E2F-1. Moreover, the actions of CO were mediated by the second messenger molecule, cGMP. Limiting VSMC growth by increasing the release of CO may represent a key event in the body's compensatory responses to hypoxia.
- Published
- 1997
10. Primary structure, developmental expression, and immunolocalization of the murine laminin alpha4 chain.
- Author
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Iivanainen, A, Kortesmaa, J, Sahlberg, C, Morita, T, Bergmann, U, Thesleff, I, and Tryggvason, K
- Abstract
The complete primary structure of the mouse laminin alpha4 chain was derived from cDNA clones. The translation product contains a 24-residue signal peptide preceding the mature alpha4 chain of 1,792 residues. Northern analysis on whole mouse embryos revealed that the expression was weak at day 7, but it later increased and peaked at day 15. In adult tissues the strongest expression was observed in lung and cardiac and skeletal muscles. Weak expression was also seen in other adult tissues such as brain, spleen, liver, kidney, and testis. By in situ hybridization of fetal and newborn tissues, expression of the laminin alpha4 chain was mainly localized to mesenchymal cells. Strong expression was seen in the villi and submucosa of the developing intestine, the mesenchymal stroma surrounding the branching lung epithelia, and the external root sheath of vibrissae follicles, as well as in cardiac and skeletal muscle fibers. In the developing kidney, intense but transient expression was associated with the differentiation of epithelial kidney tubules from the nephrogenic mesenchyme. Immunohistologic staining with affinity-purified IgG localized the laminin alpha4 chain primarily to lung septa, heart, and skeletal muscle, capillaries, and perineurium.
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- 1997
11. Transport of carbamyl phosphate synthetase I and ornithine transcarbamylase into mitochondria. Inhibition by rhodamine 123 and accumulation of enzyme precursors in isolated hepatocytes.
- Author
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Morita, T, Mori, M, Ikeda, F, and Tatibana, M
- Abstract
Carbamoyl-phosphate synthetase (ammonia) (EC 6.3.4.16) and ornithine carbamoyltransferase (EC 2.1.3.3) are matrix enzymes synthesized outside the mitochondria in the form of larger precursors and are transported rapidly into mitochondria, in association with post-translational proteolytic processing to the mature enzymes. Treatment of isolated rat hepatocytes with 40 micrograms/ml of rhodamine 123, a laser dye which specifically stains mitochondria, resulted both in a strong inhibition of the processing of the enzyme precursors and in accumulation. Rhodamine 123 did not specifically inhibit the synthesis of the synthetase and the transcarbamylase precursors in a reticulocyte lysate cell-free system programmed with rat liver free polysomes. The dye strongly inhibited the uptake and processing of the ornithine transcarbamylase precursor by isolated rat liver mitochondria. When the mitochondria and the medium were separated by centrifugation, the unprocessed precursor was recovered almost exclusively in the medium. These results indicate that rhodamine 123 inhibits either the binding of the enzyme precursors to the mitochondria or their transport into the organelle.
- Published
- 1982
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12. Primary structure of limulus anticoagulant anti-lipopolysaccharide factor.
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Aketagawa, J, Miyata, T, Ohtsubo, S, Nakamura, T, Morita, T, Hayashida, H, Miyata, T, Iwanaga, S, Takao, T, and Shimonishi, Y
- Abstract
A potent anticoagulant, anti-lipopolysaccharide (LPS) factor, found in limulus hemocytes inhibits the LPS-mediated activation of limulus coagulation cascade and shows an antibacterial action against R-types of Gram-negative bacteria (Morita, T., Ohtsubo, S., Nakamura, T., Tanaka, S., Iwanaga, S., Ohashi, K., and Niwa, M. (1985) J. Biochem. (Tokyo) 97, 1611-1620). The complete amino acid sequence of this substance was determined by sequencing the peptides obtained by selective proteolytic cleavage. The NH2-terminal end of anti-LPS factor was pyroglutamic acid. Anti-LPS factor had two variant residues at position 36 and the COOH-terminal end, respectively. The following sequence was assigned to anti-LPS factor, and it was also confirmed by fast atom bombardment mass spectrometry. less than EGGIWTQLALALVKNLATLWQSGDFQFLGHE (formula; see text) Limulus anti-LPS factor consisted of a single chain of 102 residues with 2 half-cystines in disulfide linkage. Its NH2-terminal region up to 20 residues was highly hydrophobic, and positively charged residues were clustered mainly within the disulfide loop. By searching the homologous sequence in known protein sequences with that of anti-LPS factor, we found a structural homology between anti-LPS factor and alpha-lactalbumin/lysozyme family.
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- 1986
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13. The amino acid sequence of the procoagulant- and prothrombin-binding domain isolated from staphylocoagulase.
- Author
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Kawabata, S, Miyata, T, Morita, T, Miyata, T, Iwanaga, S, and Igarashi, H
- Abstract
The primary structure of the procoagulant- and prothrombin-binding domains, the 43- and 30-kDa fragments previously isolated from staphylocoagulase, has been determined by sequencing peptides derived from various chemical (CNBr and 2-(2-nitrophenylsulfenyl)-3-methyl-3-bromoindolenine) and enzymatic (trypsin and alpha-chymotrypsin) cleavages. Carboxypeptidase Y was also used to deduce the COOH-terminal sequence. The 43-kDa fragment contained 324 amino acids and had a calculated molecular weight of 38,098. It included the entire structure of the 30-kDa fragment located in the COOH-terminal portion (positions 126-324). The 43-kDa fragment had an unusual amino acid composition based on the sequence, in which the sum of Asp (28 residues), Asn (22), Glu (35), Gln (9), and Lys (52) residues accounted for more than 45% of the total. In addition, the frequent occurrence of repetitions of the various kinds of dipeptides was found along the whole sequence. Structural comparison of the NH2-terminal portion of the 43-kDa fragment of staphylocoagulase with that of streptokinase did not reveal any obvious sequence homologies. There was also no sequence homology with that of trypsin, alpha-chymotrypsin, and elastase.
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- 1986
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14. Isolation and characterization of staphylocoagulase chymotryptic fragment. Localization of the procoagulant- and prothrombin-binding domain of this protein.
- Author
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Kawabata, S, Morita, T, Miyata, T, Iwanaga, S, and Igarashi, H
- Abstract
Several strains of Staphylococcus aureus secrete a protein, staphylocoagulase, that binds stoichiometrically to human prothrombin, resulting in a coagulant complex designated staphylothrombin. In the present study, staphylocoagulase was digested with alpha-chymotrypsin and the resulting fragments were isolated by gel filtration. One fragment (Mr 43,000) exhibited a high affinity for human prothrombin (Kd = 1.7 X 10(-9) M), which is comparable to the affinity observed using intact staphylocoagulase (Kd = 4.6 X 10(-10) M). A complex of the Mr 43,000 fragment and prothrombin possessed both clotting and amidase activity essentially identical to that observed in a complex of intact staphylocoagulase and prothrombin. A second fragment (Mr 30,000) exhibited weaker affinity for prothrombin (Kd = 1.2 X 10(-7) M). While clotting activity was not observed with a complex of this fragment and prothrombin, it nonetheless possessed a weak amidase activity. A third fragment (Mr 20,000) was found to bind to prothrombin, but the resultant complex did not exhibit clotting or amidase activity. Amino-terminal sequence analyses of these staphylocoagulase fragments revealed that the Mr 43,000 fragment constitutes the amino-terminal portion of staphylocoagulase and also contains the Mr 30,000 and 20,000 fragments. Moreover, the amino-terminal sequence of the Mr 20,000 fragment was identical to that observed for the Mr 30,000 fragment. From these results, we conclude that the functional region of staphylocoagulase for binding and activation of human prothrombin is localized in the amino-terminal region of the intact bacterial protein.
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- 1986
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15. Isolation and characterization of carinactivase, a novel prothrombin activator in Echis carinatus venom with a unique catalytic mechanism.
- Author
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Yamada, D, Sekiya, F, and Morita, T
- Abstract
The venom of the viper Echis carinatus contains a metalloprotease, ecarin, that is a potent prothrombin activator. We here show that the venom is also rich in another prothrombin activator, which does not belong to any known category of prothrombin activators. The novel enzyme, designated carinactivase-1 (CA-1), consists of two subunits held together non-covalently but very tightly. One subunit is a 62-kDa polypeptide that has metalloprotease activity and is homologous to the single-chain enzyme ecarin; the other subunit of 25 kDa consists of two disulfide-linked polypeptides of 17 and 14 kDa, and this subunit resembles the anticoagulant in the habu snake venom, IX/X-bp, that specifically binds the Gla domains of coagulation factors IX and X in a Ca2+-dependent fashion. The activation of prothrombin by CA-1 requires Ca2+ ions at millimolar concentrations and in the absence of Ca2+ ions this enzyme is virtually inactive. By contrast, activation by ecarin is completely independent of Ca2+ ions. CA-1, unlike ecarin, does not activate prothrombin derivatives, in which binding of Ca2+ ions has been perturbed, namely prethrombin-1 and acarboxyprothrombin. Furthermore, the isolated catalytic subunit, although its activity is greatly reduced as compared to that of the holoenzyme, no longer requires Ca2+ ions for the activation of prothrombin. Reconstitution with the non-catalytic 25-kDa subunit restores high level activity and the dependence on Ca2+ ions. Finally, prothrombin activation by CA-1 is inhibited by prothrombin fragment 1, and the isolated non-catalytic subunit is capable of binding fragment 1 in the presence of Ca2+ ions. From these observations, we postulate the following unique mechanism for the activation of prothrombin by CA-1. The enzyme primarily recognizes the Ca2+-bound conformation of the Gla domain in prothrombin via the 25-kDa regulatory subunit, and the subsequent conversion of prothrombin to active thrombin is catalyzed by the 62-kDa catalytic subunit.
- Published
- 1996
16. Regulation of the mouse histone H2A.X gene promoter by the transcription factor E2F and CCAAT binding protein.
- Author
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Yagi, H, Kato, T, Nagata, T, Habu, T, Nozaki, M, Matsushiro, A, Nishimune, Y, and Morita, T
- Abstract
We have molecularly cloned the genomic gene encoding the mouse histone variant H2A.X and characterized the promoter. The promoter region of the H2A.X gene was characterized by chloramphenicol acetyltransferase analysis using Balb/c 3T3 cells. Maximal promoter activity was found in the construct containing up to -282 base pairs H2A.X upstream region. Within this region, we found two sequences regulating the promoter activation; one was an E2F site and another was a CCAAT box. These sequences were also required for the DNA/protein binding activities. Thus, these activities corresponded to the promoter activities, implying that the promoter activity H2A.X gene was controlled by both the transcription factor E2F and H1TF2 through the E2F and CCAAT element. The CCAAT box binding activity was constitutive when cell cycle was progressed by release from G1 arrest, but transiently transfected chloramphenicol acetyltransferase activity slightly increased when cells entered S phase. Similarly, the level of the smallest form of E2F (free E2F) became higher when cells reentered the cell cycle, indicating that the free E2F was one capable of inducing the promoter activation. Thus, the free E2F and CCAAT DNA binding activity correlated with regulation of the promoter activity.
- Published
- 1995
17. Regulation of the tertiary structure and function of coagulation factor IX by magnesium (II) ions.
- Author
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Sekiya, F, Yamashita, T, Atoda, H, Komiyama, Y, and Morita, T
- Abstract
The indispensable role of Ca2+ ions in the maintenance of the functional tertiary structures of vitamin K-dependent coagulation factors has been definitively established but the participation of Mg2+ ions, another alkaline-earth metal that is present abundantly in blood plasma, in such a process is not yet understood. We show here that the Ca(2+)-stabilized conformation of coagulation factor IX undergoes a further conformational change upon binding of Mg2+ ions using three independent structural probes. The probes we used were (i) IX/X-bp, a snake venom anticoagulant that recognizes the Gla domains in coagulation factors IX and X, (ii) conformation-specific polyclonal antibodies against bovine factor IX, and (iii) monoclonal antibodies against the Gla domain of human factor IX. The binding of all these probes had an absolute requirement for Ca2+ ions, and Mg2+ ions alone were ineffective. However, when added together with Ca2+ ions, Mg2+ ions at physiological concentrations greatly augmented the binding of these probes to factor IX; the required concentration of Ca2+ ions was much reduced, and the affinity of each probe for factor IX was increased even in the presence of an excess of Ca2+ ions. These results suggest the presence of a Mg(2+)-specific binding site that does not interact with Ca2+ ions in factor IX. Furthermore, Mg2+ ions potentiated the susceptibility of factor IX to activation by factor XIa, concomitant with their effect on the conformation. Similarly, the required Ca2+ concentration was reduced by Mg2+ ions, and the rate of conversion to factor IXa was increased by Mg2+ ions in the presence of an excess of Ca2+ ions. At a saturating concentration of Ca2+ ions (5 mM), addition of 1 mM Mg2+ reduced the apparent Km value for factor IX from 0.31 to 0.18 microM, and in the presence of a physiological concentration of Ca2+ ions (1 mM), the reduction in Km by Mg2+ ions was far more striking (from 0.91 to 0.24 microM). The apparent Vmax values were hardly affected by Mg2+ ions. Our present data reveal a hitherto novel physiological role of the Mg2+ ions in plasma. Not only Ca2+ ions but also Mg2+ ions are important regulators of the stabilization of the native conformation of factor IX as well as of its efficient activation.
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- 1995
18. Derivatives of blood coagulation factor IX contain a high affinity Ca2+-binding site that lacks gamma-carboxyglutamic acid.
- Author
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Morita, T, Isaacs, B S, Esmon, C T, and Johnson, A E
- Abstract
We have examined the calcium-binding properties and metal ion-dependent conformational changes of proteolytically modified derivatives of factor IX that lack gamma-carboxyglutamic acid (Gla) residues. Equilibrium dialysis experiments demonstrated that a Gla-domainless factor IX species retained a single high affinity calcium ion-binding site (Kd = 85 +/- 5 microM). Ca2+ binding to this site was accompanied by a decrease in intrinsic fluorescence emission intensity (Kd = 63 +/- 15 microM). These spectral changes were reversed upon the addition of EDTA. Titration with Sr2+ resulted in little change in fluorescence intensity below 1 mM, while titration with Tb3+ caused fluorescence changes similar to those observed with Ca2+. Tb3+ and Ca2+ appear to bind to the same site because tryptophan-dependent terbium emission was reduced by the addition of Ca2+. Similar results were obtained with a Gla-domainless factor IX species lacking the activation peptide. Gla domain-containing factor IX species exhibited fluorescence changes similar to those of the Gla-domainless proteins at low Ca2+, but an additional structural transition was found at higher Ca2+ concentrations (apparent Kd greater than 0.8 mM). Thus, the conformations of factor IX proteins are nucleated and/or stabilized by calcium binding to a high affinity site which does not contain Gla residues. The binding of Ca2+ to lower affinity Gla domain-dependent metal ion-binding sites elicits an additional conformational change. The strong similarities between these results and those obtained with protein C (Johnson, A. E., Esmon, N. L., Laue, T. M. & Esmon, C. T. (1983) J. Biol. Chem. 258, 5554-5560), coupled with the remarkable sequence homologies of the vitamin K-dependent proteins, suggest that the high affinity Gla-independent Ca2+-binding site may be a common feature of vitamin K-dependent proteins.
- Published
- 1984
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19. Preparation and properties of derivatives of bovine factor X and factor Xa from which the gamma-carboxyglutamic acid containing domain has been removed.
- Author
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Morita, T and Jackson, C M
- Abstract
Limited proteolysis of bovine blood coagulation Factor X by chymotrypsin produces a derivative in which the light chain is cleaved between Tyr 44 and Lys 45. Two peptide products, residues 1-44 of the Factor X light chain and a modified zymogen, Factor X(-GD) have been isolated and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, elution behavior on anion-exchange chromatography, amino acid composition, and by partial amino acid sequence determination. Factor X(-GD) no longer contains the 12 gamma-carboxyglutamic acid residues of the native zymogen and thus serves as a model for investigation of the properties conferred on Factor X by the presence of gamma-carboxyglutamic acid. Cleavage of Factor X at Tyr 44 by chymotrypsin is inhibited by Ca2+ and Mg2+ ions. Factor X(-GD) is activated by the coagulation factor activator of Vipera russellii venom, but at less than 1% of the rate of activation of native Factor X. The susceptibility of Tyr 44 to chymotryptic cleavage implies that this residue is on the surface of the light chain of Factor X. Factor Xa(-GD) is indistinguishable from native Factor Xa in its activity on Benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide, on prothrombin alone, and on prothrombin plus Factor Va. In the presence of phospholipid the rate of prothrombin activation catalyzed by Factor Xa(-GD) is the same as in the absence of phospholipid.
- Published
- 1986
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20. Localization of the structural difference between bovine blood coagulation factors X1 and X2 to tyrosine 18 in the activation peptide.
- Author
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Morita, T and Jackson, C M
- Abstract
Bovine Factor X is isolated in two chromatographically separable forms, Factor X1 and Factor X2. Whereas only a single form of Factor Xa, the active protease, exists, the activation peptides also exist as two chromatographically distinct species. These peptides have been shown to differ at a tyrosyl residue by ultraviolet spectrophotometry, and in their composition after alkaline hydrolysis. On the basis of the spectral properties, and elution position of the modified tyrosine on Dowex 1 columns and on an amino acid analyzer, it has been concluded that Factor X2 contains a tyrosyl-O-SO4 residue at position 18 in the activation peptide whereas Factor X1 contains only tyrosine. Alternative explanations such as differences in carbohydrate composition, differences in phosphate content, or differences in the number of gamma-carboxyglutamic acid residues were demonstrated to be unrelated to the difference in chromatographic behavior between bovine Factors X1 and X2.
- Published
- 1986
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21. Terminal deoxynucleotidyltransferase of 60,000 daltons from mouse, rat, and calf thymus. Purification by immunoadsorbent chromatography and comparison of peptide structures.
- Author
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Nakamura, H., primary, Tanabe, K., additional, Yoshida, S., additional, and Morita, T., additional
- Published
- 1981
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22. Uptake and processing of the precursor for rat liver ornithine transcarbamylase by isolated mitochondria. Inhibition by uncouplers.
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Mori, M., primary, Morita, T., additional, Miura, S., additional, and Tatibana, M., additional
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- 1981
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23. Paraoxonase 2 serves a proapopotic function in mouse and human cells in response to the Pseudomonas aeruginosa quorum-sensing molecule N-(3-Oxododecanoyl)-homoserine lactone.
- Author
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Schwarzer C, Fu Z, Morita T, Whitt AG, Neely AM, Li C, and Machen TE
- Subjects
- 4-Butyrolactone metabolism, Animals, Cells, Cultured, HEK293 Cells, Homoserine metabolism, Host-Pathogen Interactions, Humans, Mice, Pseudomonas Infections microbiology, Pseudomonas Infections pathology, 4-Butyrolactone analogs & derivatives, Apoptosis, Aryldialkylphosphatase metabolism, Homoserine analogs & derivatives, Pseudomonas Infections metabolism, Pseudomonas aeruginosa physiology, Quorum Sensing
- Abstract
Pseudomonas aeruginosa use quorum-sensing molecules, including N-(3-oxododecanoyl)-homoserine lactone (C12), for intercellular communication. C12 activated apoptosis in mouse embryo fibroblasts (MEF) from both wild type (WT) and Bax/Bak double knock-out mice (WT MEF and DKO MEF that were responsive to C12, DKOR MEF): nuclei fragmented; mitochondrial membrane potential (Δψmito) depolarized; Ca(2+) was released from the endoplasmic reticulum (ER), increasing cytosolic [Ca(2+)] (Cacyto); and caspase 3/7 was activated. DKOR MEF had been isolated from a nonclonal pool of DKO MEF that were non-responsive to C12 (DKONR MEF). RNAseq analysis, quantitative PCR, and Western blots showed that WT and DKOR MEF both expressed genes associated with cancer, including paraoxonase 2 (PON2), whereas DKONR MEF expressed little PON2. Adenovirus-mediated expression of human PON2 in DKONR MEF rendered them responsive to C12: Δψmito depolarized, Cacyto increased, and caspase 3/7 activated. Human embryonic kidney 293T (HEK293T) cells expressed low levels of endogenous PON2, and these cells were also less responsive to C12. Overexpression of PON2, but not PON2-H114Q (no lactonase activity) in HEK293T cells caused them to become sensitive to C12. Because [C12] may reach high levels in biofilms in lungs of cystic fibrosis (CF) patients, PON2 lactonase activity may control Δψmito, Ca(2+) release from the ER, and apoptosis in CF airway epithelia. Coupled with previous data, these results also indicate that PON2 uses its lactonase activity to prevent Bax- and Bak-dependent apoptosis in response to common proapoptotic drugs like doxorubicin and staurosporine, but activates Bax- and Bak-independent apoptosis in response to C12., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)
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- 2015
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24. Differences in the nuclear export mechanism between myocardin and myocardin-related transcription factor A.
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Hayashi K and Morita T
- Subjects
- Actins metabolism, Animals, COS Cells, Cell Nucleus metabolism, Chlorocebus aethiops, Cytoplasm metabolism, Karyopherins metabolism, Models, Biological, Plasmids metabolism, Protein Interaction Mapping, RNA, Small Interfering metabolism, Rats, Receptors, Cytoplasmic and Nuclear metabolism, Serum Response Factor metabolism, Exportin 1 Protein, Active Transport, Cell Nucleus, Nuclear Proteins metabolism, Trans-Activators metabolism, Transcription Factors metabolism
- Abstract
Myocardin (Mycd), a key factor in smooth muscle cell differentiation, is constitutively located in the nucleus, whereas myocardin-related transcription factors A and B (MRTF-A/B) reside mostly in the cytoplasm and translocate to the nucleus in a Rho-dependent manner. Here, we investigated the nuclear export of Mycd family members. They possess two leucine-rich sequences: L1 in the N terminus and L2 in the Gln-rich domain. Although L2 (but not L1) served as a CRM1-binding site for Mycd, CRM1-mediated nuclear export did not affect its subcellular localization. Serum response factor (SRF) competitively inhibited Mycd/CRM1 interaction. Furthermore, such interaction was autonomously inhibited. The N terminus of Mycd bound intramolecularly to Mycd, resulting in masking L2. In contrast, the CRM1-binding affinity of MRTF-A was much higher than that of Mycd because both L1 and L2 of MRTF-A served as functional CRM1-binding sites, and the autoinhibition observed in the Mycd/CRM1 interaction was absent in the MRTF-A/CRM1 interaction. Additionally, because the SRF-binding affinity of MRTF-A was lower than that of Mycd, the inhibitory effect of SRF on the MRTF-A/CRM1 interaction was weak. Thus, MRTF-A is much more likely to be exported from the nucleus. These differences could be the reason for the distinct subcellular localization of Mycd and MRTF-A.
- Published
- 2013
- Full Text
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25. Photo-induced regulation of the chromatic adaptive gene expression by Anabaena sensory rhodopsin.
- Author
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Irieda H, Morita T, Maki K, Homma M, Aiba H, and Sudo Y
- Subjects
- Anabaena, Bacterial Proteins genetics, Escherichia coli genetics, Escherichia coli metabolism, Gene Expression Regulation, Bacterial physiology, Mutation, Phycocyanin biosynthesis, Phycocyanin genetics, Protein Structure, Tertiary, Rhodopsins, Microbial genetics, Bacterial Proteins biosynthesis, Gene Expression Regulation, Bacterial radiation effects, Light, Promoter Regions, Genetic physiology, Rhodopsins, Microbial biosynthesis
- Abstract
Rhodopsin molecules are photochemically reactive membrane-embedded proteins, with seven transmembrane α-helices, which bind the chromophore retinal (vitamin A aldehyde). They are roughly divided into two groups according to their basic functions: (i) ion transporters such as proton pumps, chloride pumps, and cation channels; and (ii) photo-sensors such as sensory rhodopsin from microbes and visual pigments from animals. Anabaena sensory rhodopsin (ASR), found in 2003 in the cyanobacterium Anabaena PCC7120, is categorized as a microbial sensory rhodopsin. To investigate the function of ASR in vivo, ASR and the promoter sequence of the pigment protein phycocyanin were co-introduced into Escherichia coli cells with the reporter gene crp. The result clearly showed that ASR functions as a repressor of the CRP protein expression and that this is fully inhibited by the light activation of ASR, suggesting that ASR would directly regulate the transcription of crp. The repression is also clearly inhibited by the truncation of the C-terminal region of ASR, or mutations on the C-terminal Arg residues, indicating the functional importance of the C-terminal region. Thus, our results demonstrate a novel function of rhodopsin molecules and raise the possibility that the membrane-spanning protein ASR could work as a transcriptional factor. In the future, the ASR activity could be utilized as a tool for arbitrary protein expression in vivo regulated by visible light.
- Published
- 2012
- Full Text
- View/download PDF
26. Caldesmon regulates axon extension through interaction with myosin II.
- Author
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Morita T, Mayanagi T, and Sobue K
- Subjects
- Animals, Calmodulin-Binding Proteins genetics, Cells, Cultured, Cytoskeleton genetics, Hippocampus cytology, Humans, Myosin Type II genetics, Neural Stem Cells cytology, Neural Stem Cells metabolism, Rats, Axons metabolism, Calmodulin-Binding Proteins metabolism, Cytoskeleton metabolism, Hippocampus metabolism, Myosin Type II metabolism
- Abstract
To begin the process of forming neural circuits, new neurons first establish their polarity and extend their axon. Axon extension is guided and regulated by highly coordinated cytoskeletal dynamics. Here we demonstrate that in hippocampal neurons, the actin-binding protein caldesmon accumulates in distal axons, and its N-terminal interaction with myosin II enhances axon extension. In cortical neural progenitor cells, caldesmon knockdown suppresses axon extension and neuronal polarity. These results indicate that caldesmon is an important regulator of axon development.
- Published
- 2012
- Full Text
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27. Calpain protects the heart from hemodynamic stress.
- Author
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Taneike M, Mizote I, Morita T, Watanabe T, Hikoso S, Yamaguchi O, Takeda T, Oka T, Tamai T, Oyabu J, Murakawa T, Nakayama H, Nishida K, Takeda J, Mochizuki N, Komuro I, and Otsu K
- Subjects
- Animals, Calcium metabolism, Calpain genetics, Cell Membrane enzymology, Cell Membrane genetics, Cell Membrane pathology, Heart Failure genetics, Heart Failure physiopathology, Mice, Mice, Knockout, Muscle Proteins genetics, Myocardium pathology, Stress, Physiological, Blood Pressure, Calpain metabolism, Heart Failure enzymology, Muscle Proteins metabolism, Myocardial Contraction, Myocardium enzymology
- Abstract
Calpains make up a family of Ca(2+)-dependent intracellular cysteine proteases that include ubiquitously expressed μ- and m-calpains. Both are heterodimers consisting of a distinct large catalytic subunit (calpain 1 for μ-calpain and calpain 2 for m-calpain) and a common regulatory subunit (calpain 4). The physiological roles of calpain remain unclear in the organs, including the heart, but it has been suggested that calpain is activated by Ca(2+) overload in diseased hearts, resulting in cardiac dysfunction. In this study, cardiac-specific calpain 4-deficient mice were generated to elucidate the role of calpain in the heart in response to hemodynamic stress. Cardiac-specific deletion of calpain 4 resulted in decreased protein levels of calpains 1 and 2 and showed no cardiac phenotypes under base-line conditions but caused left ventricle dilatation, contractile dysfunction, and heart failure with interstitial fibrosis 1 week after pressure overload. Pressure-overloaded calpain 4-deficient hearts took up a membrane-impermeant dye, Evans blue, indicating plasma membrane disruption. Membrane repair assays using a two-photon laser-scanning microscope revealed that calpain 4-deficient cardiomyocytes failed to reseal a plasma membrane that had been disrupted by laser irradiation. Thus, the data indicate that calpain protects the heart from hemodynamic stresses, such as pressure overload.
- Published
- 2011
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28. Specification of neuronal polarity regulated by local translation of CRMP2 and Tau via the mTOR-p70S6K pathway.
- Author
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Morita T and Sobue K
- Subjects
- Amino Acid Sequence, Animals, Axons metabolism, Base Sequence, Enzyme Activation, Gene Expression Regulation, Humans, Intercellular Signaling Peptides and Proteins, Mice, Molecular Sequence Data, Nerve Tissue Proteins genetics, Nerve Tissue Proteins metabolism, RNA, Messenger genetics, RNA, Messenger metabolism, Rats, Signal Transduction, Sirolimus pharmacology, TOR Serine-Threonine Kinases, tau Proteins genetics, tau Proteins metabolism, Cell Polarity drug effects, Nerve Tissue Proteins biosynthesis, Neurons cytology, Neurons metabolism, Protein Biosynthesis, Protein Kinases metabolism, Ribosomal Protein S6 Kinases, 70-kDa metabolism, tau Proteins biosynthesis
- Abstract
Mammalian target of rapamycin (mTOR) is an important regulator of neuronal development and functions. Although it was reported recently that mTOR signaling is critical for neuronal polarity, the underlying mechanism remains unclear. Here, we describe the molecular pathway of mTOR-dependent axon specification, in which the collapsing response mediator protein 2 (CRMP2) and Tau are major downstream targets. The activity of mTOR effector 70-kDa ribosomal protein S6 kinase (p70S6K) specifically increases in the axon during neuronal polarity formation. The mTOR inhibitor rapamycin suppresses the translation of some neuronal polarity proteins, including CRMP2 and Tau, thereby inhibiting axon formation. In contrast, constitutively active p70S6K up-regulates the translation of these molecules, thus inducing multiple axons. Exogenous CRMP2 and Tau facilitate axon formation, even in the presence of rapamycin. In the 5'-untranslated region of Tau and CRMP2 mRNAs, we identified a 5'-terminal oligopyrimidine tract, which mediates mTOR-governed protein synthesis. The 5'-terminal oligopyrimidine tract sequences of CRMP2 and Tau mRNAs strongly contribute to the up-regulation of their translation in the axon in response to the axonal activation of the mTOR-p70S6K pathway. Taken together, we conclude that the local translation of CRMP2 and Tau, regulated by mTOR-p70S6K, is critical for the specification of neuronal polarity.
- Published
- 2009
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29. Snake venom Vascular Endothelial Growth Factors (VEGF-Fs) exclusively vary their structures and functions among species.
- Author
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Yamazaki Y, Matsunaga Y, Tokunaga Y, Obayashi S, Saito M, and Morita T
- Subjects
- Animals, Cloning, Molecular, Exons, Humans, Introns, Kinetics, Models, Genetic, Molecular Sequence Data, Phylogeny, Protein Binding, Protein Structure, Tertiary, Species Specificity, Time Factors, Snake Venoms metabolism, Vascular Endothelial Growth Factor A metabolism
- Abstract
Vascular endothelial growth factor (VEGF-A) and its family proteins are crucial regulators of blood vessel formation and vascular permeability. Snake venom has recently been shown to be an exogenous source of unique VEGF (known as VEGF-F), and now, two types of VEGF-F with distinct biochemical properties have been reported. Here, we show that VEGF-Fs (venom-type VEGFs) are highly variable in structure and function among species, in contrast to endogenous tissue-type VEGFs (VEGF-As) of snakes. Although the structures of tissue-type VEGFs are highly conserved among venomous snake species and even among all vertebrates, including humans, those of venom-type VEGFs are extensively variegated, especially in the regions around receptor-binding loops and C-terminal putative coreceptor-binding regions, indicating that highly frequent variations are located around functionally key regions of the proteins. Genetic analyses suggest that venom-type VEGF gene may have developed from a tissue-type gene and that the unique sequence of its C-terminal region was generated by an alteration in the translation frame in the corresponding exons. We further verified that a novel venom-type VEGF from Bitis arietans displays unique properties distinct from already known VEGFs. Our results may provide evidence of a novel mechanism causing the generation of multiple snake toxins and also of a new model of molecular evolution.
- Published
- 2009
- Full Text
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30. Use of Fluorescence Resonance Energy Transfer-based Biosensors for the Quantitative Analysis of Inositol 1,4,5-Trisphosphate Dynamics in Calcium Oscillations.
- Author
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Tanimura A, Morita T, Nezu A, Shitara A, Hashimoto N, and Tojyo Y
- Subjects
- Animals, COS Cells, Calcium analysis, Chlorocebus aethiops, Humans, Inositol 1,4,5-Trisphosphate analysis, Biological Clocks, Biosensing Techniques methods, Calcium metabolism, Calcium Signaling, Fluorescence Resonance Energy Transfer methods, Inositol 1,4,5-Trisphosphate metabolism
- Abstract
Inositol 1,4,5-trisphosphate (IP(3)) is an intracellular messenger that elicits a wide range of spatial and temporal Ca(2+) signals, and this signaling versatility is exploited to regulate diverse cellular responses. In this study, we have developed a series of IP(3) biosensors that exhibit strong pH stability and varying affinities for IP(3), as well as a method for the quantitative measurement of cytosolic concentrations of IP(3) ([IP(3)](i)) in single living cells. We applied this method to elucidate IP(3) dynamics during agonist-induced Ca(2+) oscillations, and we demonstrated cell type-dependent differences in IP(3) dynamics, a nonfluctuating rise in [IP(3)](i) and repetitive IP(3) spikes during Ca(2+) oscillations in COS-7 cells and HSY-EA1 cells, respectively. The size of the IP(3) spikes in HSY-EA1 cells varied from 10 to 100 nm, and the [IP(3)](i) spike peak was preceded by a Ca(2+) spike peak. These results suggest that repetitive IP(3) spikes in HSY-EA1 cells are passive reflections of Ca(2+) oscillations, and are unlikely to be essential for driving Ca(2+) oscillations. In addition, the interspike periods of Ca(2+) oscillations that occurred during the slow rise in [IP(3)](i) were not shortened by the rise in [IP(3)](i), indicating that IP(3)-dependent and -independent mechanisms may regulate the frequency of Ca(2+) oscillations. The novel method described herein as well as the quantitative information obtained by using this method should provide a valuable and sound basis for future studies on the spatial and temporal regulations of IP(3) and Ca(2+).
- Published
- 2009
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31. Glucocorticoid receptor-mediated expression of caldesmon regulates cell migration via the reorganization of the actin cytoskeleton.
- Author
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Mayanagi T, Morita T, Hayashi K, Fukumoto K, and Sobue K
- Subjects
- Actins genetics, Calmodulin-Binding Proteins genetics, Cell Line, Tumor, Cell Movement drug effects, Cytoskeleton genetics, Glucocorticoids metabolism, Glucocorticoids pharmacology, Humans, Receptors, Glucocorticoid genetics, Response Elements genetics, Transcriptional Activation drug effects, Transcriptional Activation physiology, Up-Regulation drug effects, Actins metabolism, Calmodulin-Binding Proteins biosynthesis, Cell Movement physiology, Cytoskeleton metabolism, Receptors, Glucocorticoid metabolism, Response Elements physiology, Up-Regulation physiology
- Abstract
Glucocorticoids (GCs) play important roles in numerous cellular processes, including growth, development, homeostasis, inhibition of inflammation, and immunosuppression. Here we found that GC-treated human lung carcinoma A549 cells exhibited the enhanced formation of the thick stress fibers and focal adhesions, resulting in suppression of cell migration. In a screen for GC-responsive genes encoding actin-interacting proteins, we identified caldesmon (CaD), which is specifically up-regulated in response to GCs. CaD is a regulatory protein involved in actomyosin-based contraction and the stability of actin filaments. We further demonstrated that the up-regulation of CaD expression was controlled by glucocorticoid receptor (GR). An activated form of GR directly bound to the two glucocorticoid-response element-like sequences in the human CALD1 promoter and transactivated the CALD1 gene, thereby up-regulating the CaD protein. Forced expression of CaD, without GC treatment, also enhanced the formation of thick stress fibers and focal adhesions and suppressed cell migration. Conversely, depletion of CaD abrogated the GC-induced phenotypes. The results of this study suggest that the GR-dependent up-regulation of CaD plays a pivotal role in regulating cell migration via the reorganization of the actin cytoskeleton.
- Published
- 2008
- Full Text
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32. Distant N- and C-terminal domains are required for intrinsic kinase activity of SMG-1, a critical component of nonsense-mediated mRNA decay.
- Author
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Morita T, Yamashita A, Kashima I, Ogata K, Ishiura S, and Ohno S
- Subjects
- Cell Line, Gene Deletion, HeLa Cells, Humans, Models, Genetic, Mutation, Phosphorylation, Plasmids metabolism, Protein Binding, Protein Biosynthesis, Protein Serine-Threonine Kinases, Protein Structure, Tertiary, Transfection, Phosphatidylinositol 3-Kinases metabolism, RNA, Messenger metabolism
- Abstract
Phosphatidylinositol 3-kinase-related kinases (PIKKs) consisting of SMG-1, ATM, ATR, DNA-PKcs, and mTOR are a family of proteins involved in the surveillance of gene expression in eukaryotic cells. They are involved in mechanisms responsible for genome stability, mRNA quality, and translation. They share a large N-terminal domain and a C-terminal FATC domain in addition to the unique serine/threonine protein kinase (PIKK) domain that is different from classical protein kinases. However, structure-function relationships of PIKKs remain unclear. Here we have focused on one of the PIKK members, SMG-1, which is involved in RNA surveillance, termed nonsense-mediated mRNA decay (NMD), to analyze the roles of conserved and SMG-1-specific sequences on the intrinsic kinase activity. Analyses of sets of point and deletion mutants of SMG-1 in a purified system and intact cells revealed that the long N-terminal region and the conserved leucine in the FATC domain were essential for SMG-1 kinase activity. However, the conserved tryptophan in the TOR SMG-1 (TS) homology domain and the FATC domain was not. In addition, the long insertion region between PIKK and FATC domains was not essential for SMG-1 kinase activity. These results indicated an unexpected feature of SMG-1, i.e. that distantly located N- and C-terminal sequences were essential for the intrinsic kinase activity.
- Published
- 2007
- Full Text
- View/download PDF
33. Changes in the balance between caldesmon regulated by p21-activated kinases and the Arp2/3 complex govern podosome formation.
- Author
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Morita T, Mayanagi T, Yoshio T, and Sobue K
- Subjects
- Actins chemistry, Animals, Calmodulin-Binding Proteins metabolism, Fibroblasts metabolism, Genetic Vectors, Models, Biological, Phosphorylation, Protein Binding, Rats, Rous sarcoma virus metabolism, Transfection, Tropomyosin chemistry, Wiskott-Aldrich Syndrome Protein, Neuronal chemistry, p21-Activated Kinases, Actin-Related Protein 2 metabolism, Actin-Related Protein 3 metabolism, Calmodulin-Binding Proteins chemistry, Protein Serine-Threonine Kinases metabolism
- Abstract
Podosomes are dynamic cell adhesion structures that degrade the extracellular matrix, permitting extracellular matrix remodeling. Accumulating evidence suggests that actin and its associated proteins play a crucial role in podosome dynamics. Caldesmon is localized to the podosomes, and its expression is down-regulated in transformed and cancer cells. Here we studied the regulatory mode of caldesmon in podosome formation in Rous sarcoma virus-transformed fibroblasts. Exogenous expression analyses revealed that caldesmon represses podosome formation triggered by the N-WASP-Arp2/3 pathway. Conversely, depletion of caldesmon by RNA interference induces numerous small-sized podosomes with high dynamics. Caldesmon competes with the Arp2/3 complex for actin binding and thereby inhibits podosome formation. p21-activated kinases (PAK)1 and 2 are also repressors of podosome formation via phosphorylation of caldesmon. Consequently, phosphorylation of caldesmon by PAK1/2 enhances this regulatory mode of caldesmon. Taken together, we conclude that in Rous sarcoma virus-transformed cells, changes in the balance between PAK1/2-regulated caldesmon and the Arp2/3 complex govern the formation of podosomes.
- Published
- 2007
- Full Text
- View/download PDF
34. Characterization of a monoclonal antibody B1 that recognizes phosphorylated Ser-158 in the activation peptide region of human coagulation factor IX.
- Author
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Atoda H, Yokota E, and Morita T
- Subjects
- Amino Acid Sequence, Binding Sites, Epitopes, Glycosylation, Humans, Hybridomas, Molecular Sequence Data, Peptides, Protein Binding, Protein Processing, Post-Translational, Serine, Snake Venoms, Antibodies, Monoclonal immunology, Factor IX metabolism, Phosphoserine chemistry
- Abstract
Blood coagulation factor IX (FIX) undergoes various post-translational modifications such as gamma-carboxylation and glycosylation. Non-phosphorylated recombinant FIX has been reported to rapidly disappear from plasma, indicating that phosphorylation of FIX plays an important role in the physiological activity of this coagulation factor. In this study, we characterized the human FIX activation peptide (AP) using a monoclonal antibody that recognizes phosphorylated Ser-158 in the AP region. Murine monoclonal antibody B1 against human FIX recognized FIX with an apparent K(d) value of 5 nm in the presence of Ca(2+) (EC(50) = 0.58 mm). B1 bound to the isolated AP of FIX and retained the Ca(2+) dependence of binding to the isolated AP. The deglycosylation of AP did not affect the binding of B1 to AP, while B1 failed to bind to recombinant AP expressed in Escherichia coli. MALDI-TOF mass spectrometry showed that the m/z of plasma-derived deglycosylated AP is 82.54 Da greater than that of recombinant AP. The binding ability of B1 to AP was lost by the dephosphorylation of plasma-derived AP. B1 bound to synthetic peptide AP-(5-19), including phosphoserine-13, but not to the non-phosphorylated AP-(5-19) in the presence of Ca(2+). These data provide direct evidence that Ser-13 of the plasma-derived FIX AP region (Ser-158 of FIX) is phosphorylated and that B1 recognizes the epitope, which includes Ca(2+)-bound phosphoserine-158. B1 should be useful in the quality control of biologically active recombinant FIX containing phosphoserine-158.
- Published
- 2006
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35. Identification of vascular endothelial growth factor receptor-binding protein in the venom of eastern cottonmouth. A new role of snake venom myotoxic Lys49-phospholipase A2.
- Author
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Yamazaki Y, Matsunaga Y, Nakano Y, and Morita T
- Subjects
- Agkistrodon, Amino Acid Sequence, Animals, Aspartic Acid chemistry, Calcium chemistry, Dose-Response Relationship, Drug, Electrophoresis, Polyacrylamide Gel, Group II Phospholipases A2, Immunoglobulin G chemistry, Kinetics, Ligands, Lysine chemistry, Molecular Sequence Data, Muscle, Skeletal metabolism, Neurotoxins chemistry, Phospholipases A chemistry, Phospholipases A2, Phosphorylation, Protein Binding, Protein Structure, Tertiary, Proteins chemistry, Reptilian Proteins, Sequence Analysis, DNA, Time Factors, Vascular Endothelial Growth Factor A chemistry, Vascular Endothelial Growth Factor Receptor-2 chemistry, Viper Venoms chemistry, Phospholipases A physiology
- Abstract
Vascular endothelial growth factor (VEGF165) and its receptor KDR (kinase insert domain-containing receptor) are central regulators of blood vessel formation. We herein report a KDR-binding protein we have isolated in the venom of eastern cottonmouth (Agkistrodon piscivorus piscivorus). Sequence analysis revealed the isolated KDR-binding protein (designated KDR-bp) is identical to Lys49-phosholipase A2 (Lys49PLA2), an inactive PLA2 homologue with strong myotoxicity, in which Lys49 substitutes Asp49, a key residue for binding the essential cofactor Ca2+. KDR-bp binds to the extracellular domain of KDR with subnanomolar affinity. KDR-bp also binds to a lesser extent with Flt-1 and IgG but not to other receptors with similar immunoglobulin-like domain structures such as platelet-derived growth factor receptor alpha. The interaction between KDR-bp and KDR was blocked by VEGF165, and KDR-bp specifically inhibited VEGF165-stimulated endothelial cell proliferation, indicating KDR-bp is an antagonistic ligand for KDR. Lys49PLA2s from another snake venom were found to exhibit similar receptor binding properties to KDR-bp. This is the first report to demonstrate that an exogenous factor antagonizes VEGF and its receptor system. Our observation offers further insight into the as yet unknown molecular mechanism of myotoxic activity of snake venom Lys49PLA2s. Furthermore, KDR-bp would make a valuable tool for studying the structure and function of KDR, such as that expressed on skeletal muscle cells.
- Published
- 2005
- Full Text
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36. Crystal structures of novel vascular endothelial growth factors (VEGF) from snake venoms: insight into selective VEGF binding to kinase insert domain-containing receptor but not to fms-like tyrosine kinase-1.
- Author
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Suto K, Yamazaki Y, Morita T, and Mizuno H
- Subjects
- Amino Acid Sequence, Animals, Crystallography, X-Ray, Molecular Sequence Data, Protein Binding, Protein Conformation, Sequence Homology, Amino Acid, Vascular Endothelial Growth Factor A metabolism, Receptor, Macrophage Colony-Stimulating Factor metabolism, Snake Venoms chemistry, Vascular Endothelial Growth Factor A chemistry
- Abstract
Vascular endothelial growth factor-A (VEGF-A(165)) exerts multiple effects upon binding to the fms-like tyrosine kinase-1 (Flt-1) and the kinase insert domain-containing receptor (KDR). We recently identified two novel snake venom VEGFs (vammin and VR-1) having unique properties. These VEGFs, designated VEGF-Fs, are highly specific ligands for the kinase insert domain-containing receptor and exhibit potent biological activity both in vitro and in vivo when compared with VEGF-A(165). Here, we solved the crystal structures of vammin and VR-1 at 1.9 and 2.0 A resolutions, respectively. Both structures are very similar to each other, and these structures exhibit similar but significantly different features from the known structures of other VEGFs. These differences include a conformational difference in receptor-binding loop 3 caused by an amino acid residue insertion and a difference in surface potential on the possible binding surface for domain 3 of the receptor. These structural differences may be related to the highly selective ligand properties of VEGF-F.
- Published
- 2005
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37. Insulin receptor substrate-1/SHP-2 interaction, a phenotype-dependent switching machinery of insulin-like growth factor-I signaling in vascular smooth muscle cells.
- Author
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Hayashi K, Shibata K, Morita T, Iwasaki K, Watanabe M, and Sobue K
- Subjects
- Animals, Cell Division, Cell Movement, Dose-Response Relationship, Drug, Enzyme Activation, Enzyme Inhibitors pharmacology, Flavonoids pharmacology, Insulin Receptor Substrate Proteins, Insulin-Like Growth Factor I metabolism, Intracellular Signaling Peptides and Proteins, Mitogen-Activated Protein Kinases metabolism, Models, Biological, Phenotype, Phosphorylation, Protein Binding, Protein Tyrosine Phosphatase, Non-Receptor Type 11, Rats, Rats, Sprague-Dawley, Signal Transduction, Time Factors, Tyrosine metabolism, p38 Mitogen-Activated Protein Kinases, ras Proteins metabolism, Muscle, Smooth, Vascular cytology, Myocytes, Smooth Muscle cytology, Phosphoproteins metabolism, Protein Tyrosine Phosphatases metabolism
- Abstract
Insulin-like growth factor-I (IGF-I) plays a role in mutually exclusive processes such as proliferation and differentiation in a variety of cell types. IGF-I is a potent mitogen and motogen for dedifferentiated vascular smooth muscle cells (VSMCs) in vivo and in vitro. However, in differentiated VSMCs, IGF-I is only required for maintaining the differentiated phenotype. Here we investigated the VSMC phenotype-dependent signaling and biological processes triggered by IGF-I. In differentiated VSMCs, IGF-I activated a protein-tyrosine phosphatase, SHP-2, recruited by insulin receptor substrate-1 (IRS-1). The activated SHP-2 then dephosphorylated IRS-1 Tyr(P)-895, resulting in blockade of the pathways from IRS-1/Grb2/Sos to the ERK and p38 MAPK. Conversely, such negative regulation was silent in dedifferentiated VSMCs, where IGF-I activated both MAPKs via IRS-1/Grb2/Sos interaction-linked Ras activation, leading to proliferation and migration. Thus, our present results demonstrate that the IRS-1/SHP-2 interaction acts as a switch controlling VSMC phenotype-dependent IGF-I-induced signaling pathways and biological processes, and this mechanism is likely to be applicable to other cells., (Copyright 2004 American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2004
- Full Text
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38. Fluorescent biosensor for quantitative real-time measurements of inositol 1,4,5-trisphosphate in single living cells.
- Author
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Tanimura A, Nezu A, Morita T, Turner RJ, and Tojyo Y
- Subjects
- Adenosine Triphosphate chemistry, Amino Acid Sequence, Animals, Bacterial Proteins metabolism, Chelating Agents pharmacology, Green Fluorescent Proteins, Hydrogen-Ion Concentration, Ionomycin pharmacology, Luminescent Proteins chemistry, Luminescent Proteins metabolism, Molecular Sequence Data, Plasmids metabolism, Protein Binding, Protein Structure, Tertiary, Rats, Receptors, Cholinergic metabolism, Receptors, Muscarinic metabolism, Recombinant Fusion Proteins metabolism, Signal Transduction, Spectrometry, Fluorescence, Time Factors, Transfection, Biosensing Techniques methods, Calcium metabolism, Fluorescence Resonance Energy Transfer methods, Inositol 1,4,5-Trisphosphate chemistry
- Abstract
The second messenger inositol 1,4,5-trisphosphate (IP(3)) plays a central role in the generation of a variety of spatiotemporally complex intracellular Ca(2+) signals involved in the regulation of many essential physiological processes. Here we describe the development of "LIBRA", a novel ratiometric fluorescent IP(3) biosensor that allows for the quantitative monitoring of intracellular IP(3) concentrations in single living cells in real time. LIBRA consists of the IP(3)-binding domain of the rat type 3 IP(3) receptor fused between the fluorescence resonance energy transfer pair cyan fluorescent protein and yellow fluorescent protein and preceded by a membrane-targeting signal. We show that the LIBRA fluorescent signal is highly selective for IP(3) and unaffected by concentrations of Ca(2+) and ATP in the physiological range. In addition, LIBRA can be calibrated in situ. We demonstrate the utility of LIBRA by monitoring the temporal relationship between the responses intracellular IP(3) and Ca(2+) concentrations in SH-SY5Y cells following acetylcholine stimulation.
- Published
- 2004
- Full Text
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39. Snake venom vascular endothelial growth factors (VEGFs) exhibit potent activity through their specific recognition of KDR (VEGF receptor 2).
- Author
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Yamazaki Y, Takani K, Atoda H, and Morita T
- Subjects
- Amino Acid Sequence, Animals, Dose-Response Relationship, Drug, Kinetics, Ligands, Molecular Sequence Data, Protein Binding, Sequence Homology, Amino Acid, Snakes, Time Factors, Snake Venoms metabolism, Vascular Endothelial Growth Factor A metabolism, Vascular Endothelial Growth Factor Receptor-2 metabolism, Vascular Endothelial Growth Factors metabolism, Viper Venoms metabolism
- Abstract
Vascular endothelial growth factor (VEGF165) exhibits multiple effects via the activation of two distinct endothelial receptor tyrosine kinases: Flt-1 (fms-like tyrosine kinase-1) and KDR (kinase insert domain-containing receptor). KDR shows strong ligand-dependent tyrosine phosphorylation in comparison with Flt-1 and mainly mediates the mitogenic, angiogenic, and permeability-enhancing effects of VEGF165. Here we show the isolation of two VEGFs from viper venoms and the characterization of their unique biological properties. Snake venom VEGFs strongly stimulated proliferation of vascular endothelial cells in vitro. Interestingly, the maximum activities were almost twice that of VEGF165. They also induced strong hypotension on rat arterial blood pressure compared with VEGF165 in vivo. A receptor binding assay revealed that snake venom VEGFs bound to KDR-IgG with high affinity (Kd = approximately 0.1 nm) as well as to VEGF165 but did not interact with Flt-1, Flt-4, or neuropilin-1 at all. Our data clearly indicate that snake venom VEGFs act through the specific activation of KDR and show potent effects. Snake venom VEGFs are a highly specific ligand to KDR and form a new group of the VEGF family.
- Published
- 2003
- Full Text
- View/download PDF
40. Crystal structure of von Willebrand factor A1 domain complexed with snake venom, bitiscetin: insight into glycoprotein Ibalpha binding mechanism induced by snake venom proteins.
- Author
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Maita N, Nishio K, Nishimoto E, Matsui T, Shikamoto Y, Morita T, Sadler JE, and Mizuno H
- Subjects
- Crystallization, Models, Molecular, Peptides pharmacology, Protein Conformation, Protein Subunits, Snake Venoms, Peptides chemistry, Platelet Glycoprotein GPIb-IX Complex metabolism, von Willebrand Factor chemistry
- Abstract
Bitiscetin, a platelet adhesion inducer isolated from venom of the snake Bitis arietans, activates the binding of the von Willebrand factor (VWF) A1 domain to glycoprotein Ib (GPIb) in vitro. This activation requires the formation of a bitiscetin-VWF A1 complex, suggesting an allosteric mechanism of action. Here, we report the crystal structure of bitiscetin-VWF A1 domain complex solved at 2.85 A. In the complex structure, helix alpha5 of VWF A1 domain lies on a concave depression on bitiscetin, and binding sites are located at both ends of the depression. The binding sites correspond well with those proposed previously based on alanine-scanning mutagenesis (Matsui, T., Hamako, J., Matsushita, T., Nakayama, T., Fujimura, Y., and Titani, K. (2002) Biochemistry 41, 7939-7946). Against our expectations, the structure of the VWF A1 domain bound to bitiscetin does not differ significantly from the structure of the free A1 domain. These results are similar to the case of botrocetin, another snake-derived inducer of platelet aggregation, although the binding modes of botrocetin and bitiscetin are different. The modeled structure of the ternary bitiscetin-VWF A1-GPIb complex suggests that an electropositive surface of bitiscetin may interact with a favorably positioned anionic region of GPIb. These results suggest that snake venom proteins induce VWF A1-GPIbalpha binding by interacting with both proteins, and not by causing conformational changes in VWF A1.
- Published
- 2003
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41. Structure-based analysis of high pressure adaptation of alpha-actin.
- Author
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Morita T
- Subjects
- Actins metabolism, Adenosine Triphosphate metabolism, Animals, Calcium metabolism, Cloning, Molecular, DNA, Complementary metabolism, Deoxyribonuclease I chemistry, Deoxyribonuclease I metabolism, Fishes, Lysine chemistry, Models, Molecular, Molecular Sequence Data, Phylogeny, Pressure, Reverse Transcriptase Polymerase Chain Reaction, Species Specificity, Tryptophan chemistry, Actins chemistry
- Abstract
Deep-sea fishes occur to depths of several thousand meters, and at these abyssal depths encounter pressures that shallower living fishes cannot tolerate. Tolerance of abyssal pressures by deep-sea fish is likely to depend in part on adaptive modifications of proteins. However, the types of structural modifications to proteins that allow function at high pressure have not been discovered. To elucidate the mechanisms of protein adaptation to high pressure, we cloned the alpha-skeletal actin cDNAs from two abyssal Coryphaenoides species, C. armatus and C. yaquinae, and identified three amino acid substitutions, V54A or L67P, Q137K, and A155S, that distinguish these abyssal actins from orthologs of alpha-actin from non-abyssal Coryphaenoides. These substitutions, Q137K and A155S, prevent the dissociation reactions of ATP and Ca2+ from being influenced by high pressure. In particular, the lysine residue at position 137 results in a much smaller apparent volume change in the Ca2+ dissociation reaction. The V54A or L67P substitution reduces the volume change associated with actin polymerization and has a role in maintaining the DNase I activity of actin at high pressure. Together, these results indicate that a few amino acid substitutions in key functional positions can adaptively alter the pressure sensitivity of a protein.
- Published
- 2003
- Full Text
- View/download PDF
42. Crystal structure of Mg2+- and Ca2+-bound Gla domain of factor IX complexed with binding protein.
- Author
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Shikamoto Y, Morita T, Fujimoto Z, and Mizuno H
- Subjects
- Animals, Blood Coagulation, Cattle, Crystallization, Molecular Structure, Protein Binding, Protein Conformation, X-Ray Diffraction, 1-Carboxyglutamic Acid chemistry, Calcium chemistry, Factor IX chemistry, Magnesium chemistry
- Abstract
Factor IX is an indispensable protein required in the blood coagulation cascade. It binds to the surface of phospholipid membrane by means of a gamma-carboxyglutamic acid (Gla) domain situated at the N terminus. Recently, we showed that physiological concentrations of Mg2+ ions affect the native conformation of the Gla domain and in doing so augment the biological activity of factor IXa and binding affinity with its binding protein even in the presence of Ca2+ ions. Here we report on the crystal structures of the Mg2+/Ca2+-bound and Ca2+-bound (Mg2+-free) factor IX Gla domain (IXGD1-46) in complex with its binding protein (IX-bp) at 1.55 and 1.80 A resolutions, respectively. Three Mg2+ and five Ca2+ ions were bound in the Mg2+/Ca2+-bound IXGD1-46, and the Mg2+ ions were replaced by Ca2+ ions in Mg2+-free IXGD1-46. Comparison of Mg2+/Ca2+-bound with Ca2+-bound structures of the complexes showed that Mg2+ ion, which formed a bridge between IXGD1-46 and IX-bp, forced IXGD1-46 to rotate 4 degrees relative to IX-bp and hence might be the cause of a more tight interaction between the molecules than in the case of the Mg2+-free structure. The results clearly suggest that Mg2+ ions are required to maintain native conformation and in vivo function of factor IX Gla domain during blood coagulation.
- Published
- 2003
- Full Text
- View/download PDF
43. Accumulation of glucose 6-phosphate or fructose 6-phosphate is responsible for destabilization of glucose transporter mRNA in Escherichia coli.
- Author
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Morita T, El-Kazzaz W, Tanaka Y, Inada T, and Aiba H
- Subjects
- Glucose metabolism, Glucosephosphate Dehydrogenase physiology, Glycolysis, Escherichia coli metabolism, Fructosephosphates metabolism, Glucose-6-Phosphate metabolism, Phosphoenolpyruvate Sugar Phosphotransferase System genetics, RNA, Messenger metabolism
- Abstract
Previously we found that a mutation in either pgi or pfkA, encoding phosphoglucose isomerase or phosphofructokinase A, respectively, facilitates degradation of the ptsG mRNA in an RNase E-dependent manner in Escherichia coli (1). In this study, we examined the effects of a series of glycolytic genes on the degradation of ptsG mRNA and how the mutations destabilize the ptsG mRNA. The conditional lethal mutation ts8 in fda, encoding fructose-1,6-P(2) aldolase just downstream of pfkA in the glycolytic pathway, caused the destabilization of ptsG mRNA at the nonpermissive temperature. Mutations in any other gene did not destabilize the ptsG mRNA; rather, they reduced the ptsG transcription mainly by affecting the cAMP level. The rapid degradation of ptsG mRNA in mutant strains was completely dependent upon the presence of glucose or any one of its compounds, which enter the Embden-Meyerhof glycolytic pathway before the block points. A significant increase in the intracellular glucose-6-P level was observed in the presence of glucose in the pgi strain. An overexpression of glucose-6-phosphate dehydrogenase eliminated both the accumulation and the degradation of ptsG mRNA in the pgi strain. In addition, accumulation of fructose-6-P led to the rapid degradation of ptsG mRNA in a pgi pfkA mutant strain lacking glucose-6-P. We conclude that the RNase E-dependent destabilization of ptsG mRNA occurs in response to accumulation of glucose-6-P or fructose-6-P.
- Published
- 2003
- Full Text
- View/download PDF
44. Mutations in autolytic loop-2 and at Asp554 of human prothrombin that enhance protein C activation by meizothrombin.
- Author
-
Koike H, Okuda D, and Morita T
- Subjects
- Autolysis, Binding Sites genetics, Blood Coagulation Tests, Enzyme Activation, Enzyme Precursors genetics, Humans, Mutagenesis, Site-Directed, Protein Structure, Tertiary, Prothrombin genetics, Sodium metabolism, Enzyme Precursors metabolism, Mutation, Protein C metabolism, Prothrombin metabolism, Thrombin metabolism
- Abstract
Thrombin acts on many protein substrates during the hemostatic process. Its specificity for these substrates is modulated through interactions at regions remote from the active site of the thrombin molecule, designated exosites. Exosite interactions can be with the substrate, cofactors such as thrombomodulin, or fragments from prothrombin. The relative activity of alpha-thrombin for fibrinogen is 10 times greater than that for protein C. However, the relative activity of meizothrombin for protein C is 14 times greater than that for fibrinogen. Modulation of thrombin specificity is linked to its Na(+)-binding site and residues in autolytic loop-2 that interact with the Na(+)-binding site. Recombinant prothrombins that yield recombinant meizothrombin (rMT) and rMT des-fragment 1 (rMT(desF1)) enable comparisons of the effects of mutations at the Na(+)-binding residue (Asp(554)) and deletion of loop-2 (Glu(466)-Thr(469)) on the relative activity of meizothrombin for several substrates. Hydrolysis of t-butoxycarbonyl-VPR-p-nitroanilide by alpha-thrombin, recombinant alpha-thrombin, or rMT(desF1) was almost identical, but that by rMT was only 40% of that by alpha-thrombin. Clotting of fibrinogen by rMT and rMT(desF1) was 12-16% of that by alpha-thrombin, as already known. Strikingly, however, although meizothrombins modified by substitution of Asp(554) with either Ala or Leu or by deletion of loop-2 had 6-8 and <1%, respectively, of the clotting activity of alpha-thrombin, the activity of these meizothrombins for protein C was increased to >10 times that of alpha-thrombin. It is proposed that interactions within thrombin that involve autolytic loop-2 and the Na(+)-binding site primarily enhance thrombin action on fibrinogen, but impair thrombin action on protein C.
- Published
- 2003
- Full Text
- View/download PDF
45. Interplay between calcium, diacylglycerol, and phosphorylation in the spatial and temporal regulation of PKCalpha-GFP.
- Author
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Tanimura A, Nezu A, Morita T, Hashimoto N, and Tojyo Y
- Subjects
- Adenosine Triphosphate metabolism, Carcinogens, Cell Line, Cell Membrane metabolism, Cytosol metabolism, Dose-Response Relationship, Drug, Enzyme Inhibitors pharmacology, Green Fluorescent Proteins, Humans, Ionomycin pharmacology, Ionophores pharmacology, Magnesium metabolism, Phosphorylation, Precipitin Tests, Protein Binding, Protein Kinase C-alpha, Recombinant Fusion Proteins metabolism, Saponins pharmacology, Staurosporine pharmacology, Tetradecanoylphorbol Acetate metabolism, Time Factors, Transfection, Calcium metabolism, Diglycerides metabolism, Isoenzymes metabolism, Luminescent Proteins metabolism, Protein Kinase C metabolism, Staurosporine metabolism
- Abstract
The function of protein kinase C (PKC) is closely regulated by its subcellular localization. We expressed PKCalpha fused to green fluorescent protein (PKCalpha-GFP) and examined its translocation in living and permeabilized cells of the human parotid cell line, HSY-EB. ATP induced an oscillatory translocation of PKCalpha-GFP to and from the plasma membrane that paralleled the appearance of repetitive Ca2+ spikes. Staurosporine attenuated the relocation of PKCalpha-GFP to the cytosol and caused a stepwise accumulation of PKCalpha-GFP at the plasma membrane during ATP stimulation. Diacylglycerol enhanced the amplitude and duration of the ATP-induced oscillatory translocation of PKCalpha-GFP. Ionomycin induced a transient translocation of PKCalpha-GFP to the plasma membrane despite the continuous elevation of cytosolic Ca2+. The ionomycin-induced transient translocation of PKCalpha-GFP was prolonged by staurosporine, diacylglycerol, and phorbol myristate acetate. Experiments using permeabilized cells showed that staurosporine or the elimination of ATP and Mg2+ decreases the rate of dissociation of PKCalpha-GFP from the membrane. Diacylglycerol slowed the dissociation of PKCalpha-GFP from the membrane regardless of the Ca2+ concentration. The effect of diacylglycerol was attenuated by ATP plus Mg2+ at low concentrations of Ca2+ (<500 nm) but not at high concentrations of Ca2+ (>1000 nm). These data suggest a complex interplay between Ca2+, diacylglycerol, and phosphorylation in the regulation of the membrane binding of PKCalpha.
- Published
- 2002
- Full Text
- View/download PDF
46. Rhodocytin induces platelet aggregation by interacting with glycoprotein Ia/IIa (GPIa/IIa, Integrin alpha 2beta 1). Involvement of GPIa/IIa-associated src and protein tyrosine phosphorylation.
- Author
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Suzuki-Inoue K, Ozaki Y, Kainoh M, Shin Y, Wu Y, Yatomi Y, Ohmori T, Tanaka T, Satoh K, and Morita T
- Subjects
- Animals, Antibodies, Monoclonal pharmacology, Blood Platelets drug effects, Blood Proteins metabolism, Dinoprostone pharmacology, Humans, In Vitro Techniques, Integrins drug effects, Kinetics, Mice, Mice, Inbred C57BL, Phosphorylation, Phosphotyrosine metabolism, Platelet Adhesiveness physiology, Platelet Aggregation drug effects, Receptors, Collagen, Receptors, IgG deficiency, Receptors, IgG genetics, Receptors, IgG physiology, Tyrosine blood, Viper Venoms, Viperidae, Blood Platelets physiology, Integrins physiology, Lectins pharmacology, Lectins, C-Type, Platelet Aggregation physiology
- Abstract
Although glycoprotein Ia/IIa (GPIa/IIa, integrin alpha(2)beta(1)) has established its role as a collagen receptor, it remains unclear whether GPIa/IIa mediates activation signals. In this study, we show that rhodocytin, purified from the Calloselasma rhodostoma venom, induces platelet aggregation, which can be blocked by anti-GPIa monoclonal antibodies. Studies with rhodocytin-coupled beads and liposomes loaded with recombinant GPIa/IIa demonstrated that rhodocytin directly binds to GPIa/IIa independently of divalent cations. In vitro kinase assays and Western blotting of GPIa immunoprecipitates revealed that Src and Lyn constitutively associate with GPIa/IIa and that Src activity increases transiently after rhodocytin stimulation. Src specifically associates with p130 Crk-associated substrate (Cas) in a manner dependent upon Cas phosphorylation, suggesting that Src is responsible for Cas tyrosine phosphorylation. While all these phenomena occur early after rhodocytin stimulation in a cAMP-resistant manner, tyrosine phosphorylation of Syk and phospholipase Cgamma2, intracellular Ca(2+) mobilization, and platelet aggregation occur later in a cAMP-sensitive manner. Cytochalasin D, which interferes with actin polymerization and blocks receptor clustering, inhibits all the rhodocytin-mediated signals we examined in this study. We suggest that rhodocytin, by clustering GPIa/IIa, activates GPIa/IIa-associated Src, which then mediates downstream activation signals.
- Published
- 2001
- Full Text
- View/download PDF
47. Glucosamine and the action of insulin.
- Author
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WICK AN, DRURY DR, NAKADA HI, BARNET HN, and MORITA TN
- Subjects
- Humans, Glucosamine metabolism, Insulin pharmacology
- Published
- 1955
48. Studies on the relationships between insulin, glucosamine, and glucose in rat diaphragms.
- Author
-
NAKADA HI, MORITA TN, and WICK AN
- Subjects
- Animals, Rats, Abdominal Muscles, Carbohydrate Metabolism, Diaphragm metabolism, Glucosamine metabolism, Glucose metabolism, Insulin metabolism
- Published
- 1955
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