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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

The mechanisms of cell death induced by hypoxia or ischemia are not yet fully understood. We have previously demonstrated that cell death induced by hypoxia occurs independently of caspases, and is mediated by phospholipase A(2) (PLA(2)).
Here, we show that p38 mitogen-activated protein kinase is activated under hypoxia. A selective inhibitor of p38 or decrease in the p38alpha protein level prevents hypoxia-induced cell death. The p38 inhibitor abolishes PLA(2) activation by hypoxia, indicating that p38 acts upstream of PLA(2). The antioxidant N-acetyl-cysteine inhibits activation of p38 and cell death induced by hypoxia, indicating that reactive oxygen species (ROS) are responsible for p38 activation. These results demonstrate that the ROS/p38/PLA(2) signaling axis has a crucial role in caspase-independent cell death induced by hypoxia. Crown Copyright (C) 2009 Published by Elsevier B. V. on behalf of Federation of European Biochemical society. All rights reserved.

DOI： 10.1016/j.febslet.2009.04.028

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Language：English   Publisher：SPRINGER TOKYO  

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Language：English   Publisher：SPRINGER TOKYO  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：IOS PRESS  

The extract from Panax ginseng has been reported to improve the microcirculation in various organs. However, the mechanisms underlying this phenomenon are still poorly understood. In the present study, using the rheological properties of erythrocytes as an index, we have screened the components of Panax ginseng extract and identified Rg(2) and Rh-1 as the active ingredients. These two ginsenosides prevented the oxidative stress-induced elevation of erythrocyte suspension viscosity and the impairment of erythrocyte elongation in response to shear stress. Rg2 and Rh1 ginsenosides did not have antioxidant activity in an aqueous phase and did not inhibit the peroxidation of membrane lipids, either. However, they inhibited the oxidation-induced decrease of SH-groups in band 3 (anion exchanger-1), one of the important structural proteins of the erythrocyte membrane, but not in other structural proteins: bands 1 and 2 (spectrins), band 4.2 or band 5 (actin). These results suggest that ginsenosides Rg2 and Rh1 protect the rheological functions of erythrocytes against oxidative stress by preventing the oxidation of SH-groups in band 3 protein.

DOI： 10.3233/BIR-2008-0516

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：IOS PRESS  

Erythrocytes are very susceptible to oxidative stress, having a high content of intracellular oxygen and hemoglobin. In the present study, exposure to oxidative stress resulted in a significant impairment of erythrocyte membrane functions, such as deformability and anion exchange. Band 3 protein, also known as anion exchanger-1, plays an important role in these two functions. We show that oxidative stress activated caspase-3 inside the erythrocytes, which resulted in band 3 protein cleavage. Interestingly, inhibition of the caspase-3 with its specific inhibitor not only suppressed the digestion of band 3 protein, but also blunted the functional damage to erythrocytes, such as deformability and anion exchange, without changing the level of peroxidation of membrane lipids. These results provide experimental evidence that activation of caspase-3 plays an important role in the oxidative stress-induced impairment of membrane functions of erythrocytes.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Nicastrin interacts with gamma-secretase complex components predominantly via the N-terminal third of the transmembrane domain. The authentic transmembrane domain is critically required for the interaction with gamma-secretase complex components and for formation of an active gamma-secretase complex. In this study, we have identified a novel alternatively spliced transcript of nicastrun in human brain tissue. This transcript (NCSTN-Delta E16) lacks exon 16 of nicastrin mRNA, which leads to deletion of 71 amino acids just upstream of its transmembrane domain. Its expression pattern was analyzed in the hippocampus of patients with pathologically diagnosed Alzheimer disease (cases) and non-Alzheimer dementia (controls). In patients with the APOE-epsilon 4 allele, the frequency of Alzheimer disease appeared to be increased in the NCSTN-Delta E16-positive group, but the association was not statistically significant. In conclusion, the expression of NCSTN-Delta E16 transcript may confer some additional risk for developing Alzheimer disease beyond the risk due to ApoE-epsilon 4 allele. Further investigation in larger scale population would be necessary to address its potential implication in Alzheimer disease. (c) 2005 Elsevier Inc All rights reserved.

DOI： 10.1016/j.lfs.2005.10.007

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MACMILLAN MAGAZINES LTD  

Apoptosis is defined by several unique morphological nuclear changes, such as chromatin condensation and nuclear fragmentation(1). These changes are triggered by the activation of a family of cysteine proteases called caspases(2,3), and caspase-activated DNase (CAD/DPP40)(4,5) and Iamin protease (caspase-6)(6,7) have been implicated in some of these changes. CAD/DFF40 induces chromatin condensation in purified nuclei, but distinct caspase-activated factor(s) may be responsible for chromatin condensations. Here we use an in vitro system to identify a new nuclear factor, designated Acinus, which induces apoptotic chromatin condensation after cleavage by caspase-3 without inducing DNA fragmentation. Immunodepletion experiments showed that Acinus is essential for apoptotic chromatin condensation in vitro, and an antisense study revealed that Acinus is also important in the induction of apoptotic chromatin condensation in cells.

DOI： 10.1038/43678

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC  

A 58-kDa protein was detected in Xenopus egg lysate by SDS-PAGE and immunoblotting with an antibody raised against adaptor protein Shc, a well known tyrosine kinase substrate in numerous biological events. Tyrosine phosphorylation of the Xenopus Shc protein (p58 xShc) was found to increase 2.3 +/- 0.4-fold (n = 3) upon fertilization. Pretreatment of eggs with the tyrosine kinase inhibitor genistein effectively blocked the fertilization-dependent phosphorylation. Tyrosine phosphorylation of p58 xShc was also observed when eggs were activated parthenogenetically by an integrin-interacting RODS-peptide which is known to cause egg activation accompanied by intracellular calcium release. On the other hand, other egg-activating treatments such as electrical shock and calcium ionophore, which directly induce the elevation of intracellular calcium, did not show such an effect. It is also suggested that the phosphorylated p58 xShc may play a role unique to the egg activation process because we found that there was no increase of Shc-Grb2 complex after fertilization. These results demonstrate that p58 xShc is a substrate of egg tyrosine kinases which may be activated by sperm-egg interaction and suggest that the phosphorylated p58 xShc may act upstream of the calcium-dependent pathway of egg activation. (C) 1999 Academic Press.

DOI： 10.1006/bbrc.1999.0624

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

We have analyzed tyrosine-phosphorylated proteins in Xenopus laevis eggs before and after fertilization by immunoblotting with anti-phosphotyrosine antibody. A number of egg proteins with different subcellular distribution became tyrosine-phosphorylated or dephosphorylated within 30 min after insemination. Tyrosine kinase-specific inhibitors genistein and herbimycin A were found to inhibit sperm-induced egg activation judged by the egg cortical contraction. Surprisingly, sodium orthovanadate, a tyrosine phosphatase inhibitor, also inhibited the egg activation. Moreover, we found that fertilization-dependent tyrosine dephosphorylation of 42-kDa mitogen-activated protein kinase was inhibited in genistein-treated eggs. These results suggest that both protein-tyrosine phosphorylation and dephosphorylation pathways play an important role in the sperm-induced Xenopus egg activation. (C) 1998 Federation of European Biochemical.

DOI： 10.1016/S0014-5793(98)00123-9

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS  

In the previous study (Sate K.-I. et al. (1997) FEES Lett. 410, 136-140), we showed that the phosphorylation of Shc protein by c-Src is dependent on the binding of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) to the PTB domain of Shc. In this study, we demonstrate that, in contrast to c-Src, v-Src and epidermal growth factor (EGF) receptor can phosphorylate Shc in a PtdIns(4,5)P2-independent manner and at different phosphorylation sites. To determine the phosphorylation sites in Shc, we used mutant Shc proteins in which tyrosine residues (Y) 317 and/or 239 and 240 were replaced by phenylalanine residues (F), We found that Y317F Shc but not Y239/240P or Y239/240/317F Shc was phosphorylated by c-Src. The reaction was PtdIns(4,5)P2-dependent and inhibited by the addition of PTB domain of Shc. On the other hand, v-Src and EGF receptor were able to phosphorylate both Y317F and Y239/240F but not Y239/240/317F Shc in a PtdIns (4,5)P2-independent manner. These results highlight the difference between c-Src and v-Src or EGF receptor and suggest that c-Src can phosphorylate predominantly on Tyr239/240 of Shc only when Shc PTB domain is bound to PtdIns(4,5)P2. (C) 1997 Academic Press.

DOI： 10.1006/bbrc.1997.7667

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

The adaptor protein She was prepared as glutathione S-transferase fusion proteins (GST-Shc) and used as in vitro substrate for c-Src. Since phosphotyrosine-binding domain of She has been shown to bind phosphatidyl-inositol 4,5-bisphosphate (PtdIns(4,5)P2) [Zhou et al. (1995) Nature 378, 584-592], effect of PtdIns(4,5)P2 on the phosphorylation of GST-Shc by c-Src was examined, PtdIns(4,5)P2 stimulated the phosphorylation of GST-Shc without any effect on the c-Src activity as judged by both its autophosphorylation and phosphorylation of exogenous substrate, Cdc2 peptide. On the other hand, phosphatidylserine, phosphatidic acid, phosphatidylinositol, and phosphatidylinositol 4-phosphate but not phosphatidylcholine stimulated the c-Src activity itself. K-m for GST-Shc in the presence of 1 mu M PtdIns(4,5)P2 was calculated to be 90 nM. The PtdIns(4,5)P2-dependent phosphorylation of GST-Shc was inhibited by a GST-fusion protein containing the phosphotyrosine-binding domain of She. These results suggest that PtdIns(4,5)P2 can act as a regulator of phosphorylation of She by c-Src through its binding to She. (C) 1997 Federation of European Biochemical Societies.

DOI： 10.1016/S0014-5793(97)00539-5

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

In the previous study (Fukami, Y., Sate, K.-I., Ikeda, H., Kamisango, K., Koizumi, K., and Matsuno, T. (1993) J. Biol. Chem. 268, 1132-1140), we found that an antibody termed anti-pepY antibody causes a severalfold activation of bovine brain c-Src. The anti-pepY antibody was raised against a synthetic peptide corresponding to residues 410-428 of chicken c-Src, one of the most conserved regions among the Src family protein-tyrosine kinases. In this study, we have used this antibody as an in vitro activator and purified a c-Src-related protein-tyrosine kinase from the particulate fraction of Xenopus laevis oocytes. A synthetic peptide corresponding to residues 7-26 of fission yeast Cdc2 was used as substrate. Immunoreactivity toward the antibody was also monitored during the purification. The purified kinase displayed a single polypeptide of 57 kDa on SDS-gel electrophoresis and showed a specific activity of 2.37 and 20.1 nmol/min/mg protein in the absence and the presence of the anti-pepY antibody, respectively. The purified enzyme underwent autophosphorylation and phosphorylated actin and the Cdc2 peptide exclusively on tyrosine residues. Specific antibodies against c-Src, Fyn, c-Yes, c-Fgr, Lck, Lyn, Hck, and Blk proteins did not recognize the p57 Xenopus tyrosine kinase. The kinase activity of the Xenopus enzyme was not affected by oocyte maturation but was found to be elevated severalfold upon fertilization. Fertilization also caused a translocation of the activated enzyme from the particulate fraction to the cytosolic fraction. The activation and translocation was observed within 1 min after fertilization. These results suggest a possible involvement of the p57 Xenopus tyrosine kinase in the signal transduction of fertilization.

DOI： 10.1074/jbc.271.22.13250

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

To explore the structural basis required for the holoenzyme formation of cAMP-dependent protein kinase, we have prepared rabbit anti-peptide antibodies that can block the holoenzyme formation without affecting the catalytic activity of the enzyme. The antibodies were raised against a specific site in the catalytic (C)-subunit, termed IDA (Inter-DFG-APE) region, which lies between the kinase subdomains VII and VIII. Although the C-subunit immunoprecipitated with anti-IDA antibodies could not form a stable complex with regulatory (R)-subunit, it was still susceptible to inhibition by the R-subunit or by PKI, a specific inhibitor peptide containing a pseudosubstrate site. These results indicate that there exists an IDA region-mediated interaction between the R- and C-subunits, which is distinct from that mediated through the substrate site and substrate binding site, In accordance with this idea, association of synthetic IDA peptides with the R-subunit was directly demonstrated by resonance mirror analysis, The calculated association constants of IDA peptides were high enough to suggest a possible involvement of the IDA region in the initial step of holoenzyme formation.

DOI： 10.1016/0014-5793(96)00302-X

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS  

In the previous study [Sato et al. (1995) Biochem. Biophys. Res. Commun. 210, 844-851], we found that c-Src was associated with epidermal growth factor (EGF) receptor and activated upon EGF treatment in A431 cells. In the present study, we investigated the phosphorylation of EGF receptor by c-Src in the c-Src-EGF receptor complex. We have focused our attention to tyrosine residue 845 (Y845) of EGF receptor as a candidate for the phosphorylation site. A synthetic peptide containing Y845, named Y845 peptide, which corresponds to residue 837 to 856 of EGF receptor, was found to be phosphorylated by c-Src and used to provide the standard phosphopeptide. In addition to the autophosphorylated peptide of 25 kDa, a phosphopeptide of 7 kDa was detected in the cyanogen bromide-digested fragments of the c-Src-associated EGF receptor phosphorylated in vitro in an EGF-dependent manner. In phosphopeptide mapping, tryptic digest of the 7-kDa phosphopeptide was shown to co-migrate with that of the phosphorylated Y845 peptide. The 7-kDa phosphopeptide was found to be phosphorylated exclusively on tyrosine. These results suggest that c-Src can phosphorylate EGF receptor on Y845 in an EGF-dependent manner. Furthermore, we confirmed that the same site of the c-Src-associatcd EGF receptor was phosphorylated in EGF-treated A431 cells. (C) 1995 Academic Press, Inc.

DOI： 10.1006/bbrc.1995.2574

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

We have examined the interaction between c-Src and epidermal growth factor (EGF) receptor in A431 cells. c-Src was found exclusively in the Triton X-100-solubilized particulate fraction and activated up to 3-fold within 1 min after EGF treatment of the cells. Association between c-Src and EGF receptor was detected by immunoprecipitation of c-Src followed by immunoblotting with anti-EGF receptor antibody. The c-Src-EGF receptor complex was found in both EGF-treated and untreated cells, but an augmented complex formation was observed in EGF-treated cells. We have isolated the complex by DEAE-cellulose column chromatography and found that a site-specific anti-c-Src antibody, which was raised against a synthetic peptide corresponding to residues 413 to 431 of human c-Src, did not recognize the c-Src protein in the complex, while other c-Src-specific antibodies tested did. Incubation of the complex with this synthetic peptide resulted in a partial dissociation of the complex. These results suggest that the specific region of c-Src is involved in the association with EGF receptor. (C) 1995 Academic Press, Inc.

DOI： 10.1006/bbrc.1995.1735

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

DOI： 10.1016/S0006-291X(05)80118-4

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Authorship：Last author,　Corresponding author   Language：English  

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