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IWASAKI TetsushiBiosignal Research CenterAssistant Professor
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■ Award■ Paper
- Springer Science and Business Media LLC, Feb. 2025, Scientific Reports, 15(1) (1), English[Refereed]Scientific journal
- Abstract Cellular senescence is defined as irreversible growth arrest induced by various stress, such as DNA damage and oxidative stress. Senescent cells exhibit various characteristic morphological changes including enlarged morphology. In our recent study, we identified Nectin-4 to be upregulated in cellular senescence by comparative transcriptomic analysis. However, there are few reports on the relationship between Nectin-4 and senescence. Therefore, we analyzed the function of Nectin-4 in senescence and its biological significance. When overexpressed with Nectin-4, the cells exhibited the enlarged cell morphology closely resembling senescent cells. In addition, the cell size enlargement during DNA damage-induced senescence was suppressed by knockdown of Nectin-4, while there were no significant changes in senescence induction. These results suggest that Nectin-4 is not involved in the regulation of senescence itself but contributes to the senescence-associated cell size increase. Furthermore, the Nectin-4-dependent cell size increase was found to be mediated by Src family kinase (SFK)/PI3 kinase (PI3K)/Rac1 pathway. To explore the functional consequences of cell size enlargement, we analyzed cell survival in Nectin-4-depleted senescent cells. Single-cell tracking experiments revealed that Nectin-4 knockdown induced apoptosis in senescent cells, and there is a strong positive correlation between cell size and survival rate. These results collectively indicate that Nectin-4 plays a causative role in the senescence-associated cell size enlargement via SFK/PI3K/Rac1, which can contribute to survival of senescent cells.Springer Science and Business Media LLC, Dec. 2023, Scientific Reports, 13(1) (1), English, International magazine[Refereed]Scientific journal
- Wiley, Sep. 2022, FEBS Letters, 596(21) (21), 2768 - 2780, English[Refereed]Scientific journal
- Amyloid fibrils have been an important subject as they are involved in the development of many amyloidoses and neurodegenerative diseases. The formation of amyloid fibrils is typically initiated by nucleation, whereas its exact mechanisms are largely unknown. With this situation, we have previously identified prefibrillar aggregates in the formation of insulin B chain amyloid fibrils, which have provided an insight into the mechanisms of protein assembly involved in nucleation. Here, we have investigated the formation of insulin B chain amyloid fibrils under different pH conditions to better understand amyloid nucleation mediated by prefibrillar aggregates. The B chain showed strong propensity to form amyloid fibrils over a wide pH range, and prefibrillar aggregates were formed under all examined conditions. In particular, different structures of amyloid fibrils were found at pH 5.2 and pH 8.7, making it possible to compare different pathways. Detailed investigations at pH 5.2 in comparison with those at pH 8.7 have suggested that the evolution of protofibril-like aggregates is a common mechanism. In addition, different processes of evolution of the prefibrillar aggregates have also been identified, suggesting that the nucleation processes diversify depending on the polymorphism of amyloid fibrils.MDPI AG, Jun. 2022, Molecules, 27(13) (13), 3964 - 3964[Refereed]Scientific journal
- The pro-apoptotic tumor suppressor BIN1 inhibits the activities of the neoplastic transcription factor MYC, poly (ADP-ribose) polymerase-1 (PARP1), and ATM Ser/Thr kinase (ATM) by separate mechanisms. Although BIN1 deficits increase cancer-cell resistance to DNA-damaging chemotherapeutics, such as cisplatin, it is not fully understood when BIN1 deficiency occurs and how it provokes cisplatin resistance. Here, we report that the coordinated actions of MYC, PARP1, and ATM assist cancer cells in acquiring cisplatin resistance by BIN1 deficits. Forced BIN1 depletion compromised cisplatin sensitivity irrespective of Ser15-phosphorylated, pro-apoptotic TP53 tumor suppressor. The BIN1 deficit facilitated ATM to phosphorylate the DNA-damage-response (DDR) effectors, including MDC1. Consequently, another DDR protein, RNF8, bound to ATM-phosphorylated MDC1 and protected MDC1 from caspase-3-dependent proteolytic cleavage to hinder cisplatin sensitivity. Of note, long-term and repeated exposure to cisplatin naturally recapitulated the BIN1 loss and accompanying RNF8-dependent cisplatin resistance. Simultaneously, endogenous MYC was remarkably activated by PARP1, thereby repressing the BIN1 promoter, whereas PARP inhibition abolished the hyperactivated MYC-dependent BIN1 suppression and restored cisplatin sensitivity. Since the BIN1 gene rarely mutates in human cancers, our results suggest that simultaneous inhibition of PARP1 and ATM provokes a new BRCAness-independent synthetic lethal effect and ultimately re-establishes cisplatin sensitivity even in platinum-refractory cancer cells.Dec. 2021, International journal of molecular sciences, 22(24) (24), English, International magazine[Refereed]Scientific journal
- Cellular senescence is a state of permanent proliferative arrest induced by a variety of stresses, such as DNA damage. The transcriptional activity of p53 has been known to be essential for senescence induction. It remains unknown, however, whether among the downstream genes of p53, there is a gene that has anti-senescence function. Our recent studies have indicated that the expression of SLC52A1 (also known as GPR172B/RFVT1), a riboflavin transporter, is upregulated specifically in senescent cells depending on p53, but the relationship between senescence and SLC52A1 or riboflavin has not been described. Here, we examined the role of SLC52A1 in senescence. We found that knockdown of SLC52A1 promoted senescence phenotypes induced by DNA damage in tumor and normal cells. The senescence suppressive-action of SLC52A1 was dependent on its riboflavin transport activity. Furthermore, elevation of intracellular riboflavin led to activation of mitochondrial membrane potential (MMP) mediated by the mitochondrial electron transport chain complex II. Finally, the SLC52A1-dependent activation of MMP inhibited the AMPK-p53 pathway, a central mediator of mitochondria dysfunction-related senescence. These results suggest that SLC52A1 contributes to suppress senescence through the uptake of riboflavin and acts downstream of p53 as a negative feedback mechanism to limit aberrant senescence induction.American Society for Cell Biology (ASCB), Sep. 2021, Molecular Biology of the Cell, 32(21) (21), mbc.E21 - 05, English, International magazine[Refereed]Scientific journal
- American Society for Biochemistry and Molecular Biology Inc., Jan. 2021, Journal of Biological Chemistry, 296, EnglishScientific journal
- Amyloid fibrils are aberrant protein aggregates associated with various amyloidoses and neurodegenerative diseases. It is recently indicated that structural diversity of amyloid fibrils often results in different pathological phenotypes, including cytotoxicity and infectivity. The diverse structures are predicted to propagate by seed-dependent growth, which is one of the characteristic properties of amyloid fibrils. However, much remains unknown regarding how exactly the amyloid structures are inherited to subsequent generations by seeding reaction. Here, we investigated the behaviors of self- and cross-seeding of amyloid fibrils of human and bovine insulin in terms of thioflavin T fluorescence, morphology, secondary structure, and iodine staining. Insulin amyloid fibrils exhibited different structures, depending on species, each of which replicated in self-seeding. In contrast, gradual structural changes were observed in cross-seeding, and a new type of amyloid structure with distinct morphology and cytotoxicity was formed when human insulin was seeded with bovine insulin seeds. Remarkably, iodine staining tracked changes in amyloid structure sensitively, and singular value decomposition analysis of the ultraviolet-visible absorption spectra of the fibril-bound iodine has revealed the presence of one or more intermediate metastable states during the structural changes. From these findings, we propose a propagation scheme with multistep structural changes in cross-seeding between two heterologous proteins, which is accounted for as a consequence of the rugged energy landscape of amyloid formation.Elsevier BV, Jan. 2021, Biophysical Journal, 120(2) (2), 284 - 295, English, International magazine[Refereed]Scientific journal
- Although senescent cells display various morphological changes including vacuole formation, it is still unclear how these processes are regulated. We have recently identified the gene, lymphocyte antigen 6 complex, locus D (LY6D), to be upregulated specifically in senescent cells. LY6D is a glycosylphosphatidylinositol-anchored cell-surface protein whose function remains unknown. Here, we analyzed the functional relationship between LY6D and the senescence processes. We found that overexpression of LY6D induced vacuole formation and knockdown of LY6D suppressed the senescence-associated vacuole formation. The LY6D-induced vacuoles were derived from macropinocytosis, a distinct form of endocytosis. Furthermore, Src family kinases and Ras were found to be recruited to membrane lipid rafts in an LY6D-dependent manner, and inhibition of their activity impaired the LY6D-induced macropinocytosis. Finally, reduction of senescent-cell survival induced by glutamine deprivation was recovered by albumin supplementation to the culture media in an LY6D-dependent manner. Because macropinocytosis acts as an amino acid supply route, these results suggest that LY6D-mediated macropinocytosis contributes to senescent-cell survival through the incorporation of extracellular nutrients.Elsevier BV, 2021, Journal of Biological Chemistry, 296, 100049 - 100049, English, International magazine[Refereed]Scientific journal
- Catharanthus roseus is a medicinal plant well known for producing bioactive compounds such as vinblastine and vincristine, which are classified as terpenoid indole alkaloids (TIAs). Although the leaves of this plant are the main source of these antitumour drugs, much remains unknown on how TIAs are biosynthesised from a central precursor, strictosidine, to various TIAs in planta. Here, we have succeeded in showing, for the first time in leaf tissue of C. roseus, cell-specific TIAs localisation and accumulation with 10 μm spatial resolution Imaging mass spectrometry (Imaging MS) and live single-cell mass spectrometry (single-cell MS). These metabolomic studies revealed that most TIA precursors (iridoids) are localised in the epidermal cells, but major TIAs including serpentine and vindoline are localised instead in idioblast cells. Interestingly, the central TIA intermediate strictosidine also accumulates in both epidermal and idioblast cells of C. roseus. Moreover, we also found that vindoline accumulation increases in laticifer cells as the leaf expands. These discoveries highlight the complexity of intercellular localisation in plant specialised metabolism.Oct. 2019, The New phytologist, 224(2) (2), 848 - 859, English, International magazine[Refereed]Scientific journal
- Mitogen-activated protein kinases (MAPKs) are involved in the regulation of various cellular processes, including cell survival and apoptosis. Here, we report that Xenopus p42 MAPK becomes phosphorylated in apoptotic eggs, however this modification does not activate the enzyme. Using phosphorylation residue-specific antibodies, we demonstrate that this modification occurs on the Tyr residue in the MAPK activation segment, pinpointing the autophosphorylation mechanism. Notably, MAPK phosphorylation in apoptotic Xenopus eggs coincides with prominent intracellular acidification accompanying apoptosis in these cells. Furthermore, autophosphorylation of recombinant Xenopus MAPK is stimulated and phosphorylation of a protein substrate is inhibited under low pH conditions. Thus, acidic intracellular conditions inactivate MAPK and effectively disable the MAPK-mediated survival pathway in the apoptotic eggs. Given that cell acidification is a rather common feature of apoptosis, we hypothesize that stimulation of MAPK autophosphorylation and shutdown of the MAPK pathway may represent universal traits of apoptotic cell death.Sep. 2019, Biochemical and biophysical research communications, 517(1) (1), 140 - 145, English, International magazine[Refereed]Scientific journal
- The tumor suppressor bridging integrator 1 (BIN1) is a corepressor of the transcription factor E2F1 and inhibits cell-cycle progression. BIN1 also curbs cellular poly(ADP-ribosyl)ation (PARylation) and increases sensitivity of cancer cells to DNA-damaging therapeutic agents such as cisplatin. However, how BIN1 deficiency, a hallmark of advanced cancer cells, increases cisplatin resistance remains elusive. Here, we report that BIN1 inactivates ataxia telangiectasia-mutated (ATM) serine/threonine kinase, particularly when BIN1 binds E2F1. BIN1 + 12A (a cancer-associated BIN1 splicing variant) also inhibited cellular PARylation, but only BIN1 increased cisplatin sensitivity. BIN1 prevented E2F1 from transcriptionally activating the human ATM promoter, whereas BIN1 + 12A did not physically interact with E2F1. Conversely, BIN1 loss significantly increased E2F1-dependent formation of MRE11A/RAD50/NBS1 DNA end-binding protein complex and efficiently promoted ATM autophosphorylation. Even in the absence of dsDNA breaks (DSBs), BIN1 loss promoted ATM-dependent phosphorylation of histone H2A family member X (forming γH2AX, a DSB biomarker) and mediator of DNA damage checkpoint 1 (MDC1, a γH2AX-binding adaptor protein for DSB repair). Of note, even in the presence of transcriptionally active (i.e. proapoptotic) TP53 tumor suppressor, BIN1 loss generally increased cisplatin resistance, which was conversely alleviated by ATM inactivation or E2F1 reduction. However, E2F2 or E2F3 depletion did not recapitulate the cisplatin sensitivity elicited by E2F1 elimination. Our study unveils an E2F1-specific signaling circuit that constitutively activates ATM and provokes cisplatin resistance in BIN1-deficient cancer cells and further reveals that γH2AX emergence may not always reflect DSBs if BIN1 is absent.Lead, Apr. 2019, The Journal of biological chemistry, 294(14) (14), 5700 - 5719, English, International magazine[Refereed]Scientific journal
- d-amino acid oxidase (DAO) is a flavin adenine dinucleotide (FAD)-dependent oxidase metabolizing neutral and polar d-amino acids. Unlike l-amino acids, the amounts of d-amino acids in mammalian tissues are extremely low, and therefore, little has been investigated regarding the physiological role of DAO. We have recently identified DAO to be up-regulated in cellular senescence, a permanent cell cycle arrest induced by various stresses, such as persistent DNA damage and oxidative stress. Because DAO produces reactive oxygen species (ROS) as byproducts of substrate oxidation and the accumulation of ROS mediates the senescence induction, we explored the relationship between DAO and senescence. We found that inhibition of DAO impaired senescence induced by DNA damage, and ectopic expression of wild-type DAO, but not enzymatically inactive mutant, enhanced it in an ROS-dependent manner. Furthermore, addition of d-amino acids and riboflavin, a metabolic precursor of FAD, to the medium potentiated the senescence-promoting effect of DAO. These results indicate that DAO promotes senescence through the enzymatic ROS generation, and its activity is regulated by the availability of its substrate and coenzyme.Feb. 2019, Life science alliance, 2(1) (1), e201800045, English, International magazine[Refereed]Scientific journal
- The MEKK1 kinase is a key regulator of stress signaling in Arabidopsis; however, little is known about the regulation of its kinase activity. Here, we found that recombinant MEKK1, expressed in both mammalian HEK293 cells and Escherichia coli, shows a mobility shift in SDS-PAGE, and immunoblotting detected phosphorylation of serine, threonine, and tyrosine residues. N-terminal deletions, site-directed mutagenesis, and protein phosphatase treatment revealed that the mobility shift results from autophosphorylation of the kinase domain. We identified the tyrosine autophosphorylation sites in the N-terminal region of MEKK1. Tyrosine to phenylalanine mutations decrease phosphorylation of the substrate MKK1, suggesting the important role of this residue in the regulation of MEKK1 kinase activity. The present study is the first to show that plant MAPKKKs are regulated by tyrosine phosphorylation.Oct. 2018, FEBS letters, 592(19) (19), 3327 - 3334, English, International magazine[Refereed]Scientific journal
- Apr. 2017, Journal of cell science, 130(8) (8), 1413 - 1420, English, International magazine[Refereed]Scientific journal
- Mar. 2017, RNA biology, 14(3) (3), 339 - 346, English, International magazine[Refereed]Scientific journal
- Feb. 2016, Genes to cells : devoted to molecular & cellular mechanisms, 21(2) (2), 185 - 99, English, International magazine[Refereed]Scientific journal
- 2016, The International journal of developmental biology, 60(7-8-9) (7-8-9), 289 - 296, English, International magazine[Refereed]Scientific journal
- Feb. 2015, CELL DEATH AND DIFFERENTIATION, 22(2) (2), 311 - 322, English[Refereed]Scientific journal
- Jan. 2014, The FEBS journal, 281(1) (1), 104 - 14, English, International magazine[Refereed]Scientific journal
- BACKGROUND: In several species with external fertilization, including frogs, laid unfertilized eggs were found to die by apoptosis outside of the animal body. However, there is no apparent reason for the externally laid eggs to degrade by this process, considering that apoptosis developed as a mechanism to reduce the damaging effect of individual cell death to the whole organism. RESULTS: Here, we demonstrate that a number of eggs are retained in the genital tract of the African clawed frog Xenopus laevis after gonadotropin-induced ovulation. The majority of these eggs exit meiotic arrest within 24 hours of hormone administration. Subsequently, post-meiotic eggs die in the frog genital tract by a well-defined apoptotic process. The hallmarks of egg degradation include prominent morphological changes, cytochrome c release, caspase activation, increase in ADP/ATP ratio, progressive intracellular acidification, egg swelling and all-out proteolysis of egg proteins. The sustained presence of post-apoptotic eggs in the genital tract of ageing frogs evidenced age-associated worsening of apoptotic clearance. CONCLUSIONS: The direct observation of egg degradation in the Xenopus genital tract provides a clue to the physiological relevance of frog egg apoptosis. It works to eliminate the mature unlaid eggs retained in the animal body after ovulation. Our findings establish egg apoptosis as a major physiological process accompanying ovulation in frogs.Mar. 2013, BMC cell biology, 14(1) (1), 11 - 11, English, International magazine[Refereed]Scientific journal
- Oct. 2012, Biology open, 1(10) (10), 1024 - 34, English, International magazine[Refereed]Scientific journal
- Jul. 2012, The Journal of investigative dermatology, 132(7) (7), 1877 - 85, English, International magazine[Refereed]Scientific journal
- BACKGROUND: A characteristic feature of frog reproduction is external fertilization accomplished outside the female's body. Mature fertilization-competent frog eggs are arrested at the meiotic metaphase II with high activity of the key meiotic regulators, maturation promoting factor (MPF) and cytostatic factor (CSF), awaiting fertilization. If the eggs are not fertilized within several hours of ovulation, they deteriorate and ultimately die by as yet unknown mechanism. RESULTS: Here, we report that the vast majority of naturally laid unfertilized eggs of the African clawed frog Xenopus laevis spontaneously exit metaphase arrest under various environmental conditions and degrade by a well-defined apoptotic process within 48 hours after ovulation. The main features of this process include cytochrome c release, caspase activation, ATP depletion, increase of ADP/ATP ratio, apoptotic nuclear morphology, progressive intracellular acidification, and egg swelling. Meiotic exit seems to be a prerequisite for execution of the apoptotic program, since (i) it precedes apoptosis, (ii) apoptotic events cannot be observed in the eggs maintaining high activity of MPF and CSF, and (iii) apoptosis in unfertilized frog eggs is accelerated upon early meiotic exit. The apoptotic features cannot be observed in the immature prophase-arrested oocytes, however, the maturation-inducing hormone progesterone renders oocytes susceptible to apoptosis. CONCLUSIONS: The study reveals that naturally laid intact frog eggs die by apoptosis if they are not fertilized. A maternal apoptotic program is evoked in frog oocytes upon maturation and executed after meiotic exit in unfertilized eggs. The meiotic exit is required for execution of the apoptotic program in eggs. The emerging anti-apoptotic role of meiotic metaphase arrest needs further investigation.Dec. 2011, BMC cell biology, 12, 56 - 56, English, International magazine[Refereed]Scientific journal
- 2011, FUNCTIONAL PLANT BIOLOGY, 38(4) (4), 282 - 292, English, International magazine[Refereed]Scientific journal
- Expression, phosphorylation, and mRNA-binding of heterogeneous nuclear ribonucleoprotein K in Xenopus oocytes, eggs, and early embryos.Here we show that heterogeneous nuclear ribonucleoprotein K (hnRNP K), a member of the K homology domain-containing proteins, is expressed in Xenopus immature oocytes, unfertilized eggs, and early embryos. Fertilization or egg activation treatment involving upregulation of the egg tyrosine kinase Src promotes a rapid and transient tyrosine phosphorylation of hnRNP K. HnRNP K is also phosphorylated on serine/threonine residues in unfertilized eggs, dephosphorylated after fertilization, and re-phosphorylated during the premitotic phase of early embryogenesis. In vitro, Src and mitogen-activated protein kinase (MAPK) were capable of phosphorylating hnRNP K on tyrosine and serine/threonine residues, respectively. In support of this, pretreatment of oocytes, eggs, or embryos with inhibitors for Src (PP2) and MAPK (U0126) blocked effectively the phosphorylation of hnRNP K. We also identify some maternal mRNAs that coimmunoprecipitate with hnRNP K in unfertilized eggs. Specific binding of these mRNAs to hnRNP K was verified by reverse transcriptase-polymerase chain reaction (RT-PCR). In addition, real-time PCR analyses revealed a subset of the mRNAs whose binding to hnRNP K might be up or downregulated in activated eggs. In vitro binding assay with the use of poly U monopolymeric RNA-coupled beads demonstrated that the RNA-binding property of hnRNP K is negatively regulated by tyrosine phosphorylation and positively or neutrally regulated by serine/threonine phosphorylation. Taken together, it is attractive to suggest that hnRNP K is in association with certain pools of maternal mRNAs whose translational activation are modulated by the Src/MAPK phosphorylation of hnRNP K during oocyte-egg-embryo transition.Jan. 2008, Development, growth & differentiation, 50(1) (1), 23 - 40, English, Domestic magazine[Refereed]Scientific journal
- Characterization of Xenopus egg membrane microdomains containing uroplakin Ib/III complex: roles of their molecular interactions for subcellular localization and signal transduction.A single-transmembrane protein uroplakin III (UPIII) and its tetraspanin binding-partner uroplakin Ib (UPIb) are members of the UP proteins that were originally identified in mammalian urothelium. In Xenopus laevis eggs, these proteins: xUPIII and xUPIb, are components of the cholesterol-enriched membrane microdomains or "rafts" and involved in the sperm-egg membrane interaction and subsequent egg activation signaling via Src tyrosine kinase at fertilization. Here, we investigate whether the xUPIII-xUPIb complex is in close proximity to CD9, a tetraspanin that has been implicated in the sperm-egg fusion in the mouse and GM1, a ganglioside typically enriched in egg rafts. Preparation of the egg membrane microdomains using different non-ionic detergents (Brij 98 and Triton X-100), chemical cross-linking, co-immunoprecipitation, in vitro kinase assay and in vitro fertilization experiments demonstrated that GM1, but not CD9, is in association with the xUPIII-xUPIb complex and contributes to the sperm-dependent egg activation. Transfection experiments using HEK293 cells demonstrated that xUPIII and xUPIb localized efficiently to the cholesterol-dependent membrane microdomains when they were co-expressed, whereas co-expression of xUPIII and CD9, instead of xUPIb, did not show this effect. Furthermore, xUPIII and xUPIb were shown to suppress kinase activity of the wild type, but not a constitutively active form of, Xenopus Src protein co-expressed in HEK293 cells. These results provide novel insight into the molecular architecture of the egg membrane microdomains containing xUPIII, xUPIb and Src, which may contribute to the understanding of sperm-egg interaction and signaling during Xenopus fertilization.Feb. 2007, Genes to cells : devoted to molecular & cellular mechanisms, 12(2) (2), 251 - 67, English, International magazine[Refereed]Scientific journal
- Activation of Arabidopsis MAPK kinase kinase (AtMEKK1) and induction of AtMEKK1-AtMEK1 pathway by wounding.We have constructed a series of deletion mutants of Arabidopsis MAPK kinase kinase (AtMEKK1) and obtained a constitutively active mutant, AtMEKK1Delta166, which lacks in self-inhibitory sequence of N-terminal 166 amino acids but still has substrate specificity. AtMEKK1Delta166 predominantly phosphorylates AtMEK1, an Arabidopsis MAPKK, but not its double mutant (AtMEK1T218A/S224E), suggesting that Thr-218 and Ser-224 are the phosphorylation sites. In wounded seedlings, AtMEKK1 was activated and phosphorylated its downstream AtMEK1. Furthermore, analysis using anti-AtMEKK1 and anti-AtMEK1 antibodies revealed that the interaction between the two proteins was signal dependent. These results suggest the presence of AtMEKK1-AtMEK1 pathway induced by wounding.Mar. 2006, Planta, 223(4) (4), 708 - 13, English, International magazine[Refereed]Scientific journal
- Mar. 2006, Journal of biochemistry, 139(3) (3), 347 - 54, English, International magazine[Refereed]Scientific journal
- Studying fertilization in cell-free extracts: focusing on membrane/lipid raft functions and proteomics.Xenopus oocytes, eggs, and embryos serve as an ideal model system to study several aspects of animal development (e.g., gametogenesis, fertilization, embryogenesis, and organogenesis). In particular, the Xenopus system has been extensively employed not only as a "living cell" system but also as a "cell-free" or "reconstitutional" system. In this chapter, we describe a protocol for studying the molecular mechanism of egg fertilization with the use of cell-free extracts and membrane/lipid rafts prepared from unfertilized, metaphase II-arrested Xenopus eggs. By using this experimental system, we have reconstituted a series of signal transduction events associated with egg fertilization, such as sperm-egg membrane interaction, activation of Src tyrosine kinase and phospholipase Cgamma, production of inositol trisphosphate, transient calcium release, and cell cycle transition. This type of reconstitutional system may allow us to perform focused proteomics (e.g., rafts) as well as global protein analysis (i.e., whole egg proteome) of fertilization in a cell-free manner. As one of these proteomics approaches, we provide a protocol for molecular identification of Xenopus egg raft proteins using mass spectrometry and database mining.2006, Methods in molecular biology (Clifton, N.J.), 322, 395 - 411, English, International magazine[Refereed]Scientific journal
- Uroplakin III, a novel Src substrate in Xenopus egg rafts, is a target for sperm protease essential for fertilization.In a previous study, we identified Xenopus egg uroplakin III (xUPIII), a single-transmembrane protein that localized to lipid/membrane rafts and was tyrosine-phosphorylated upon fertilization. An antibody against the xUPIII extracellular domain abolishes fertilization, suggesting that xUPIII acts not only as tyrosine kinase substrate but also as a receptor for sperm. Previously, it has been shown that the protease cathepsin B can promote a transient Ca2+ release and egg activation as seen in fertilized eggs (Mizote, A., Okamoto, S., Iwao, Y., 1999. Activation of Xenopus eggs by proteases: possible involvement of a sperm protease in fertilization. Dev. Biol. 208, 79-92). Here, we show that activation of Xenopus eggs by cathepsin B is accompanied by tyrosine phosphorylation of egg-raft-associated Src, phospholipase Cgamma, and xUPIII. Cathepsin B also promotes a partial digestion of xUPIII both in vitro and in vivo. A synthetic xUPIII-GRR peptide, which contains a potential proteolytic site, inhibits the cathepsin-B-mediated proteolysis and tyrosine phosphorylation of xUPIII and egg activation. Importantly, this peptide also inhibits sperm-induced tyrosine phosphorylation of xUPIII and egg activation. Protease activity that digests xUPIII in an xUPIII-GRR peptide-sensitive manner is present in Xenopus sperm. Several protease inhibitors, which have been identified to be inhibitory toward Xenopus fertilization, are shown to inhibit sperm-induced tyrosine phosphorylation of xUPIII. Uroplakin Ib, a tetraspanin UP member, is found to be associated with xUPIII in egg rafts. Our results highlight novel mechanisms of fertilization signaling by which xUPIII serves as a potential target for sperm protease essential for fertilization.Oct. 2005, Developmental biology, 286(2) (2), 483 - 92, English, International magazine[Refereed]Scientific journal
- Generation of an antibody specific to Xenopus fertilized eggs by subtractive immunization.Here we report the generation and characterization of a monoclonal antibody, mAb 5H7-G1, which recognizes egg antigens in the animal cortex of fertilized, but not unfertilized, Xenopus eggs. The mAb 5H7-G1 was generated by subtractive immunization of mice: primary immunization with unfertilized egg extract followed by immunosuppression treatment with cyclophosphamide and repeated immunization with fertilized egg extract. In immunoblotting analysis, mAb 5H7-G1 recognizes multiple protein bands of fertilized (but not unfertilized or the ionophore-activated) Xenopus eggs. N-linked polysaccharide is most likely the target of mAb 5H7-G1 because immunoreactivity of mAb 5H7-G1 is effectively diminished when protein samples are treated with N-glycosidase F. Moreover, mAb 5H7-G1 recognizes some, but not all, tyrosine-phosphorylated proteins in eggs treated with H2O2, an artificial activator of the egg tyrosine kinase Src, suggesting that these proteins also contain N-linked sugars. When microinjected into fertilized Xenopus embryos, mAb 5H7-G1 causes a retardation or complete inhibition of first cell cleavage, suggesting that the mAb 5H7-G1-reactive antigens play an important role in this event. These results demonstrate that mAb 5H7-G1 is useful to analyze differential proteome display during fertilization and early development. More generally, subtractive immunization may work as a strategy to uncover cellular events that operate during different cellular conditions of interest.Apr. 2005, Genes to cells : devoted to molecular & cellular mechanisms, 10(4) (4), 345 - 56, English, International magazine[Refereed]Scientific journal
- Regulation of Src kinase activity during Xenopus oocyte maturation.Expression of constitutively active Src protein tyrosine kinase in Xenopus oocytes has been shown to accelerate oocyte maturation suggesting that Src may be involved in meiotic progression. However, meiotic regulation of endogenous Src kinase in oocytes has not been investigated in detail. To address this problem, we measured the activity, expression level, and phosphorylation state of the endogenous Xenopus Src (xSrc) and overexpressed xSrc mutants in the process of progesterone-induced oocyte maturation. We found that the enzyme is first transiently activated in the plasma membrane-containing fraction of oocytes within 3 min of progesterone administration. This event represents one of the earliest responses of oocytes to the hormone and should be related to triggering some early signaling pathways of maturation. Thereafter, xSrc activity increases again at the time of germinal vesicle breakdown (GVBD) and remains elevated till the completion of maturation. This elevation of xSrc activity is associated with a 2-fold increase of xSrc protein content in the absence of change in its specific activity and xSrc mRNA content. No significant changes in the phosphorylation state of C-terminal regulatory phosphotyrosine can be registered either in endogenous xSrc or in overexpressed kinase-negative and wild-type xSrc proteins during maturation. Altogether, these results indicate that upregulation of xSrc in the meiotic metaphase occurs at the translation level. We also demonstrate here that the expression of constitutively active xSrc in Xenopus oocytes is accompanied by the activation of mitogen-activated protein kinase (MAPK). Our data suggest that the Src kinase acts through the MAPK pathway to accelerate oocyte maturation.Feb. 2005, Developmental biology, 278(2) (2), 289 - 300, English, International magazine[Refereed]Scientific journal
- Molecular dissection of egg fertilization signaling with the aid of tyrosine kinase-specific inhibitor and activator strategies.Fertilization is triggered by sperm-egg interaction and fusion that initiate a transient rise(s) in the free intracellular calcium ([Ca(2+)](i)) that is responsible for a series of biochemical and cell biological events, so-called "egg activation". Calcium-dependent egg activation leads to the initiation of developmental program that culminates in the birth of individuals. A growing body of knowledge has uncovered the molecular mechanisms underlying sperm-induced transient [Ca(2+)](i) increase(s) to some extent; namely, in most animals so far studied, a second messenger inositol 1,4,5-trisphosphate (IP(3)) seems to play a pivotal role in inducing [Ca(2+)](i) transient(s) at fertilization. However, signaling mechanisms used by sperm to initiate IP(3)-[Ca(2+)](i) transient pathway have not been elucidated. To approach this problem, we have employed African clawed frog, Xenopus laevis, as a model animal and conducted experiments designed specifically to determine the role of the Src family protein-tyrosine kinases (SFKs or Src family PTKs) in the sperm-induced egg activation. This review compiles information about the use of PTK-specific inhibitors and activators for analyzing signal transduction events in egg fertilization. Specifically, we focus on molecular identification of Xenopus Src and the signaling mechanism of the Src-dependent egg activation that has been established recently. We also summarize recent advances in understanding the role of the Src family kinases in egg fertilization of other model organisms, and discuss future directions of the field.Mar. 2004, Biochimica et biophysica acta, 1697(1-2) (1-2), 103 - 21, English, International magazine[Refereed]Scientific journal
- Mar. 2004, BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS, 1697(1-2) (1-2), 103 - 121, English[Refereed]Scientific journal
- Src-dependent phosphorylation of the EGF receptor Tyr-845 mediates Stat-p21waf1 pathway in A431 cells.BACKGROUND: Cell surface receptor for the epidermal growth factor (EGFR) and cytoplasmic tyrosine kinase c-Src co-operate in several cellular functions such as proliferation and apoptosis. Our previous studies have shown that ectopic expression of the adaptor protein p52shc or p66shc, but not p46shc, and EGF stimulation lead to the activation of c-Src that is accompanied by phosphorylation of signal transducers and activators of transcription (Stat) in A431 cells. RESULTS: Here, we show that by using A431 cells as a model system, expression of p52shc, or cell stimulation with EGF or H2O2 leads to phosphorylation of EGFR on Tyr 845 that is located to the activation segment of the catalytic domain. The phosphorylation of Tyr 845 can be inhibited by PP2, but not by AG1478, and is associated with Src activation and Stat 3/5 phosphorylation, but not with MAP (mitogen-activated protein) kinase phosphorylation. Phosphorylation of Stat 3/5 in response to p52shc expression, EGF or H2O2 could also be inhibited by introduction into cells of phospho-Tyr 845-specific antibody or by expression of dominant-negative version of c-Src. Co-incubation of purified c-Src and EGFR results in phosphorylation of Tyr 845 in vitro, indicating that c-Src can directly phosphorylate EGFR on Tyr 845. CONCLUSION: These results indicate that multiple signals for c-Src activation can promote Stat 3/5 phosphorylations through Src-dependent phosphorylation of EGFR on Tyr 845.Dec. 2003, Genes to cells : devoted to molecular & cellular mechanisms, 8(12) (12), 995 - 1003, English, International magazine[Refereed]Scientific journal
- Reconstitution of Src-dependent phospholipase Cgamma phosphorylation and transient calcium release by using membrane rafts and cell-free extracts from Xenopus eggs.We reported previously that egg membrane rafts serve as a subcellular microdomain for sperm-dependent tyrosine kinase signaling in Xenopus fertilization. Moreover, we demonstrated that raft-associated Src tyrosine kinase was activated by sperm in vitro. Here we show that egg rafts incubated with sperm or hydrogen peroxide (H2O2) can promote Src-dependent phosphorylation of phospholipase Cgamma (PLCgamma) and transient calcium release in the extracts of unfertilized Xenopus eggs. In vivo egg activation by sperm or H2O2 also promotes tyrosine phosphorylation and raft-translocalization of PLCgamma. Immunodepletion of PLCgamma from the egg extracts inhibits the raft-dependent calcium release. Rafts prepared from H2O2-activated eggs also promote Src-dependent dephosphorylation of p42 mitogen-activated protein kinase and cell cycle transition from metaphase II to interphase in egg extracts. PLCgamma phosphorylation and calcium release in egg extracts can be promoted by rafts prepared from COS-7 cells expressing the Xenopus Src gene. These results demonstrate that the signaling events elicited by fertilization in Xenopus eggs can be reconstituted in vitro. The development of such experimental platforms will allow us to dissect the molecular mechanism of sperm-dependent activation of raft-associated Src and subsequent up-regulation of PLCgamma and egg activation machinery in Xenopus eggs.Oct. 2003, The Journal of biological chemistry, 278(40) (40), 38413 - 20, English, International magazine[Refereed]Scientific journal
- 2003, CELLULAR & MOLECULAR BIOLOGY LETTERS, 8(2A) (2A), 541 - 542, EnglishMolecular dissection of fertilisation signalling with the aid of tyrosine-kinase-specific inhibitors[Refereed]Scientific journal
- Towards the molecular dissection of fertilization signaling: Our functional genomic/proteomic strategies.Recent advances in DNA sequencing techniques and automated informatics has led to clarification of all genome sequence of some model organisms in a very short period. The demonstration of the first draft sequence of the human genome has prompted us to elaborate new approaches in biology, pharmacology and medicine. Such new research will focus on high throughput methods to function on collections of genes, and hopefully, on a genome-wide, quantitative modeling of the cell system as a whole. In this review article, we discuss the present status of "post genome sequencing" approaches in line with our strategies for understanding the molecular mechanism of fertilization and activation of development using the African clawed frog, Xenopus laevis, as a model system.Sep. 2002, Proteomics, 2(9) (9), 1079 - 89, English, International magazine[Refereed]Scientific journal
- Adaptor protein Shc is an isoform-specific direct activator of the tyrosine kinase c-Src.The activity of c-Src protein-tyrosine kinase is up-regulated under a number of receptor signaling pathways. However, the activation mechanism of c-Src under physiological conditions has remained unclear. We show here that the Shc adaptor protein is a novel direct activator of c-Src in epidermal growth factor receptor signaling in A431 human epidermoid carcinoma cells. Among the three Shc isoforms, P66 and P52, but not P46, were found to interact with and activate c-Src in vitro and in vivo. Activation of c-Src accompanied autophosphorylation of c-Src in the activation segment, but the carboxyl-terminal dephosphorylation was not observed. We have identified the interaction sites between Shc and c-Src and constructed a point mutant of Shc that abolishes the c-Src activation. Using this mutant, we have confirmed that the Shc-mediated c-Src activation triggers Stat-p21/WAF1/Cip1 pathway that has been implicated in the cell cycle arrest and apoptosis of epidermal growth factor-stimulated A431 cells.Aug. 2002, The Journal of biological chemistry, 277(33) (33), 29568 - 76, English, International magazine[Refereed]Scientific journal
- Feb. 2002, PHARMACOLOGY & THERAPEUTICS, 93(2-3) (2-3), 263 - 270, EnglishInhibition and activation of c-Src: the head and tail of a coin[Refereed]Scientific journal
- Low density detergent-insoluble membrane of Xenopus eggs: subcellular microdomain for tyrosine kinase signaling in fertilization.Protein-tyrosine phosphorylation plays an important role in egg activation signaling at fertilization. We show that in Xenopus, fertilization stimulates a rapid and transient tyrosine phosphorylation of egg proteins, as revealed by immunoblotting with anti-phosphotyrosine antibody. Immunofluorescent microscopic analysis demonstrated that the phosphorylation occurs in cortical area of the egg animal hemisphere. To further characterize subcellular compartment for fertilization-dependent tyrosine kinase signaling, we isolated low density detergent-insoluble membrane (LD-DIM) fraction from Xenopus eggs. The egg LD-DIM was enriched in cholesterol and GM1 ganglioside. It also contained signaling molecules such as Xyk (Xenopus egg Src), Gq alpha, Ras, integrin beta 1 and CD9. Fertilization stimulated tyrosine phosphorylation of Xyk and some other LD-DIM proteins. Remarkably, sperm stimulated tyrosine phosphorylation of the LD-DIM proteins in vitro. The sperm-dependent phosphorylation was sensitive to the tyrosine kinase inhibitors PP2 and genistein. We found that pretreatment of eggs with methyl-beta-cyclodextrin, a cholesterol-binding substance, led to a decrease in cholesterol, Xyk and sperm-induced tyrosine phosphorylation in LD-DIM. In methyl-beta-cyclodextrin-treated eggs, sperm-induced Ca(2+) transient and first cell division were also inhibited. These findings suggest that the egg LD-DIM might serve as subcellular microdomain for tyrosine kinase signaling in Xenopus egg fertilization.Feb. 2002, Development (Cambridge, England), 129(4) (4), 885 - 96, English, International magazine[Refereed]Scientific journal
- May 1999, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 258(2) (2), 265 - 270, EnglishA 58-kDa Shc protein is present in Xenopus eggs and is phosphorylated on tyrosine residues upon egg activation[Refereed]Scientific journal
- May 1999, DEVELOPMENTAL BIOLOGY, 209(2) (2), 308 - 320, EnglishEvidence for the involvement of a Src-related tyrosine kinase in Xenopus egg activation[Refereed]Scientific journal
- Mar. 1998, FEBS Letters, 424(1-2) (1-2), 113 - 118, English[Refereed]Scientific journal
- Nov. 2016, MOLECULAR CANCER RESEARCH, 14, English[Refereed]Summary international conference
- May 2015, JOURNAL OF INVESTIGATIVE DERMATOLOGY, 135, S107 - S107, EnglishHigh expression of Mcl-1 mediated by MEK-ERK1/2-STAT3 signaling pathway protects melanocytes and melanoma cells against ultraviolet B-induced apoptosisSummary international conference
- 15 Oct. 2014, International journal of molecular sciences, 15(10) (10), 18659 - 76, English, International magazine[Refereed]
- May 2012, JOURNAL OF INVESTIGATIVE DERMATOLOGY, 132, S24 - S24, EnglishSer727 phosphorylation in STAT3 plays a crucial role in cell survival and nuclear translocation of STAT3 in human melanocytes and melanoma cells[Refereed]Summary international conference
- Aug. 2011, PIGMENT CELL & MELANOMA RESEARCH, 24(4) (4), 791 - 792, EnglishSer727 phosphorylation in STAT3 plays a crucial role in nuclear translocation of STAT3 and growth in human melanoma cells and melanocytesSummary international conference
- Sep. 2010, JOURNAL OF INVESTIGATIVE DERMATOLOGY, 130, S96 - S96, EnglishTPA inhibits melanoma growth by dephosphorylation of Tyr705 on STAT3 through PKC-activated tyrosine phosphatase(s)Summary international conference
- Intracellular signaling during egg activation/fertilization has been extensively studied using intact eggs, which can be manipulated by microinjection of different mRNAs, proteins, or chemical drugs. Furthermore, egg extracts, which retain high CSF activity (CSF-arrested extracts), were developed for studying fertilization/activation signal transduction, which have significant advantages as a model system. The addition of calcium to CSF-arrested extracts initiates a plethora of signaling events that take place during egg activation. Hence, the signaling downstream of calcium mobilization has been successfully studied in the egg extracts. Moreover, despite disruption of membrane-associated signaling compartments and ordered compartmentalization during extract preparation, CSF-arrested extracts can be successfully used to study early signaling events, which occur upstream of calcium release during egg activation/fertilization. In combination with the CSF-arrested extracts, activated egg rafts can reproduce some events of egg activation, including PLCgamma activation, IP3 production, transient calcium release, MAPK inactivation, and meiotic exit. This becomes possible due to complementation of the sperm-induced egg activation signaling machinery present in the rafts with the components of signal transduction system localized in the extracts. Herein, we describe protocols for studying molecular mechanisms of egg fertilization/activation using cell-free extracts and membrane rafts prepared from metaphase-arrested Xenopus eggs.May 2010, Methods (San Diego, Calif.), 51(1) (1), 177 - 82, English, International magazine[Refereed]
- BACKGROUND: Studies have examined the function of PI 3-kinase in the early developmental processes that operate in oocytes or early embryos of various species. However, the roles of egg-associated PI 3-kinase and Akt, especially in signal transduction at fertilization, are not well understood. RESULTS: Here we show that in Xenopus eggs, a potent inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase), LY294002 inhibits sperm-induced activation of the tyrosine kinase Src and a transient increase in the intracellular concentration of Ca2+ at fertilization. LY294002 also inhibits sperm-induced dephosphorylation of mitogen-activated protein kinase, breakdown of cyclin B2 and Mos, and first embryonic cleavage, all of which are events of Ca2+-dependent egg activation. In fertilized eggs, an 85-kDa subunit of PI 3-kinase (p85) undergoes a transient translocation to the low-density, detergent-insoluble membranes (membrane microdomains) where Src tyrosine kinase signaling is operating. However, the tyrosine phosphorylation of p85 in fertilized eggs is not as evident as that in H2O2-activated eggs, arguing against the possibility that PI 3-kinase is activated by Src phosphorylation. Nevertheless, sperm-induced activation of PI 3-kinase has been demonstrated by the finding that Akt, a serine/threonine-specific protein kinase, is phosphorylated at threonine-308. The threonine-phosphorylated Akt also localizes to the membrane microdomains of fertilized eggs. Application of bp(V), an inhibitor of PTEN that dephosphorylates PIP3, the enzymatic product of PI 3-kinase, promotes parthenogenetic activation of Xenopus eggs. In vitro kinase assays demonstrate that PIP3 activates Src in a dose-dependent manner. CONCLUSIONS: These results suggest that PI 3-kinase is involved in sperm-induced egg activation via production of PIP3 that would act as a positive regulator of the Src signaling pathway in Xenopus fertilization.17 Dec. 2009, BMC developmental biology, 9, 68 - 68, English, International magazine[Refereed]
- The growth of most melanoma cells in vitro is inhibited by the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). In this study, the involvement of the signal transducer and activator of transcription 3 (STAT3) in the TPA-induced growth inhibition of melanoma cells was examined. The in vitro growth and DNA synthesis of five melanoma cell lines, whose STAT3 was activated (phosphorylated), was inhibited by TPA, whereas that of WM35 and WM39 cells, whose STAT3 activity was at negligible levels, was considerably slow and not affected by TPA. Blockade of STAT3 activity by small interfering RNAs suppressed the growth of WM1205Lu cells containing constitutively activated STAT3. Treatment of WM1205Lu cells with TPA decreased both the phosphorylated STAT3 and the DNA-binding activity of STAT3. Pretreatment of WM1205Lu cells with either a protein-tyrosine phosphatase inhibitor or a protein kinase C (PKC) inhibitor prevented the inhibitory effects of TPA on the level of phosphorylated STAT3. The five melanoma cell lines containing phosphorylated STAT3 commonly expressed PKCalpha, PKCdelta, and PKCepsilon. Introduction of the dominant negative mutant of one of these PKC isoforms into WM1205Lu cells inhibited the TPA-induced dephosphorylation of STAT3. A Src inhibitor attenuated the STAT3 phosphorylation in WM1205Lu cells. These results indicate that constitutively activated STAT3 is positively regulated by c-Src and negatively regulated by a PKC-activated tyrosine phosphatase(s) in melanoma cells. Because TPA did not affect c-Src activity, we conclude that the growth inhibitory effect of TPA on melanoma cells is mediated through inactivation of STAT3 by a PKC-activated tyrosine phosphatase(s).30 Oct. 2009, The Journal of biological chemistry, 284(44) (44), 30416 - 23, English, International magazine[Refereed]
- Jun. 2008, FEBS JOURNAL, 275, 132 - 132, EnglishStimulation of T7 RNA polymerase in Xenopus oocyte and egg extractsSummary international conference
- Jun. 2006, FEBS JOURNAL, 273, 102 - 102, EnglishFunctional relationships among egg raft-associated molecules UPIB, UPIII and Src in Xenopus fertilizationSummary international conference
- Jun. 2005, CELL STRUCTURE AND FUNCTION, 30, 29 - 29, EnglishCharacterization of hnRNP K and Sam 68 in Xenopus eggsSummary international conference
- Here we describe mass spectrometric identification, molecular cloning, and biochemical characterization of a lipid/membrane raft-associated protein that is tyrosine-phosphorylated upon Xenopus egg fertilization. This protein is homologous to mammalian uroplakin III, a member of the uroplakin family proteins (UPs) that constitute asymmetric unit membranes in the mammalian urothelial tissues, thus termed Xenopus uroplakin III (xUPIII). xUPIII contains N-linked sugars and is highly expressed in Xenopus eggs, ovary, urinary tract, and kidney. In unfertilized eggs, xUPIII is predominantly localized to the lipid/membrane rafts and exposed on the cell surface, as judged by surface biotinylation experiments and indirect immunofluorescent studies. After fertilization or hydrogen peroxide-induced egg activation, xUPIII becomes rapidly phosphorylated on tyrosine residue-249, which locates in the carboxyl-terminal cytoplasmic tail of the molecule. Raft localization and tyrosine phosphorylation of xUPIII can be reconstituted in HEK293 cells by coexpression of xUPIII, and Xenopus c-Src, a tyrosine kinase whose fertilization-induced activation in egg rafts is required for initiation of development. In mammals, UPIII is forming a complex with a tetraspanin molecule uroplakin Ib. As another tetraspanin, CD9, is known to be a critical component for sperm-egg fusion in the mouse, we have assumed that xUPIII is involved in sperm-egg interaction. An antibody against the extracellular domain of xUPIII blocks sperm-egg interaction, as judged by the occurrence of egg activation and first cell cleavage. Thus, xUPIII represents an egg raft-associated protein that is likely involved in sperm-egg interaction as well as subsequent Src-dependent intracellular events of egg activation in Xenopus.15 Apr. 2005, The Journal of biological chemistry, 280(15) (15), 15029 - 37, English, International magazine[Refereed]
- In a previous study, we presented evidence that the adaptor protein Shc interacts with and activates the tyrosine kinase c-Src without affecting the phosphorylation state of Tyr-527 in c-Src. Here we show that Shc-mediated c-Src activation occurs in mitotic NIH 3T3 cells. Co-immunoprecipitation studies demonstrate that the c-Src-p52Shc complex involves the activation segment/inter-DFG-APE (IDA) region of c-Src and the amino-terminal region of p52Shc. The complex formation contributes to the c-Src activation, because (i) specific activity of c-Src associated with p52Shc is higher than that of the total c-Src, and (ii) a recombinant protein containing the c-Src IDA sequence disrupts the complex and decreases the c-Src activity. Anti-Src IDA antibody can activate c-Src in vitro, and synthetic peptides that cover the carboxyl-terminal half of the Src IDA region interfere with the kinase-activating effect of anti-Src IDA antibody. These results support the idea that dephosphorylation-independent activation of c-Src by Shc is mediated by a molecular interaction involving the c-Src IDA region.Jan. 2005, Journal of biochemistry, 137(1) (1), 61 - 7, English, International magazine[Refereed]
- Dec. 2004, ZOOLOGICAL SCIENCE, 21(12) (12), 1285 - 1285, EnglishAnalysis of recognition site for SRC-SPECIFIC monoclonal antibodySummary international conference
- ACTIVATION MECHANISM OF SRC IN XENOPUS EGG FERTILIZATION(Developmental Biology,Abstracts of papers presented at the 74^
Annual Meeting of the Zoological Society of Japan) :Zoological Society of Japan, 2003, Zoological science, 20(12) (12), 1556 - 1556, English Zoological Society of Japan, 2002, Zoological science, 19(12) (12), 1407 - 1408, EnglishIn vitro reconstitution of fertilization signaling by using membrane rafts and cell-free extracts from Xenopus eggs(Symposium on the Mechanism of Signal Transduction in Fertilization: its Diversity, Universality and Evolution) :Elsevier Ltd, 2002, Cell Calcium, 32(1) (1), 11 - 20, EnglishFeb. 2001, DEVELOPMENT GROWTH & DIFFERENTIATION, 43(1) (1), 55 - 72, English[Refereed]Dec. 2000, MOLECULAR BIOLOGY OF THE CELL, 11, 406A - 406A, EnglishThe role of up-regulation and translocation of Src family tyrosine kinase at fertilization of Xenopus eggsSummary international conferenceAcademic Press Inc., 15 Aug. 2000, Developmental Biology, 224(2) (2), 453 - 469, English[Refereed]Wiley, 2000, Genes to Cells, 5(9) (9), 749 - 764, English01 Dec. 1998, 日本分子生物学会年会プログラム・講演要旨集, 21, 524 - 524, JapaneseSite-specific interaction of the adaptor protein Shc with c-Src01 Dec. 1998, 日本分子生物学会年会プログラム・講演要旨集, 21, 524 - 524, JapaneseIdentification of a MAPK-binding protein in Xenopus oocytes01 Dec. 1998, 日本分子生物学会年会プログラム・講演要旨集, 21, 524 - 524, JapaneseImmunocytochemical analysis of c-Src and Shc proteins in NIH3T3 and PC12 cells01 Dec. 1998, 日本分子生物学会年会プログラム・講演要旨集, 21, 524 - 524, JapaneseTyrosine phosphorylation of the adaptor protein Shc in Xenopus fertilization01 Aug. 1996, 日本分子生物学会年会プログラム・講演要旨集, 19, 609 - 609, Japaneseゼノパス卵(母)細胞膜タンパク質の単離とその生化学的, 免疫化学的解析01 Aug. 1996, 日本分子生物学会年会プログラム・講演要旨集, 19, 610 - 610, Japaneseゼノパス卵を用いた受精シグナル伝達関連タンパク質の解析
■ Lectures, oral presentations, etc.- 生体膜国際研究拠点形成シンポジウム, Dec. 2024, EnglishSenescence-related gene EPN3 induces DNA damage via extracellular vesicles
- 第47回日本分子生物学会2024, Nov. 2024, Japaneseリジン特異的脱メチル化酵素LSD1の活性化を介した細胞老化抑制機構の解析
- 第47回 日本分子生物学会 2024, Nov. 2024, JapaneseTPAによる転移性メラノーマ増殖抑制におけるTC-TP/PTPN2及びSH-PTP2/PTPN11の機能解析Poster presentation
- 第47回 日本分子生物学会 2024, Nov. 2024, Japanese細胞老化抑制機構における脱メチル化酵素JMJD2Cの機能解析Poster presentation
- 第47回 日本分子生物学会 2024, Nov. 2024, Japanese自発老化細胞の形成機構とSASPを介した周辺がん細胞への影響Poster presentation
- 第47回 日本分子生物学会 2024, Nov. 2024, Japaneseアフリカツメガエル受精時の卵細胞膜マイクロドメインに着目したシグナル伝達に関するタンパク質の同定Poster presentation
- 第47回 日本基礎老化学会大会, Jun. 2024, JapaneseDNA damage induction by exosomes secreted from senescent cellsOral presentation
- 第47回日本基礎老化学会大会, Jun. 2024, JapaneseLysine Specific Demethylase 1 (LSD1) suppresses cellular senescence by epigenetically regulating Sirtuin-4 in a riboflavin uptake-dependent mannerOral presentation
- 若手フロンティア研究会 2023, Dec. 2023, Japanese, Domestic conferenceFAD依存性酵素LSD2によるSASP因子の発現制御Poster presentation
- 第46回 日本分子生物学会年会 2023, Dec. 2023, Japanese, Domestic conferenceSTEAP3は細胞内Fe2＋とFADに依存して老化細胞にアポトーシスを誘導するOral presentation
- 第46回 日本分子生物学会年会 2023, Dec. 2023, Japanese, Domestic conferenceFAD依存性リシン特異的脱メチル化酵素2によるSASP因子の発現制御Oral presentation
- 第46回 日本分子生物学会年会 2023, Dec. 2023, Japanese, Domestic conference老化細胞特異的な空胞形成を制御するLY6Dのタンパク質構造に基づく機能解析Poster presentation
- 第46回 日本分子生物学会年会 2023, Dec. 2023, Japanese, Domestic conferenceLY6Dに依存した老化細胞特異的空胞形成におけるATP1A1の機能解析Poster presentation
- 第46回 日本分子生物学会年会 2023, Dec. 2023, Japanese, Domestic conferenceメラノーマ細胞におけるZn2+が制御するSH-PTP1の機能解析Poster presentation
- 第46回 日本分子生物学会年会 2023, Dec. 2023, Domestic conference自発老化細胞の形成機構とがん悪性化への関与Poster presentation
- Cell Bio 2023, Dec. 2023, English, International conferenceSTAT3 stabilizes oocytes of Xenopus laevisPoster presentation
- Cell Bio 2023, Dec. 2023, English, International conferenceNectin-4 increases cell size and cell viability during cellular senescencePoster presentation
- Cell Bio 2023, Dec. 2023, English, International conferenceA novel senescence-associated gene, EPN3, induces DNA damage through extracellular vesiclesPoster presentation
- Cell Bio 2023, Dec. 2023, English, International conferenceLysine Specific Demethylase 1 (LSD1) contributes to the suppression of cellular senescence via epigenetic regulation of Sirtuin-4 in a riboflavin-dependent mannerPoster presentation
- Cell Bio 2023, Dec. 2023, English, International conferenceTyrosine phosphatases TC-PTP and SH-PTP2 inhibit proliferation of metastatic melanoma by phorbol ester TPAPoster presentation
- 神戸大学第12回サイエンスフロンティア研究会, Oct. 2023, Japanese, Domestic conferenceFAD依存性酵素LSD2によるSASP因子の発現制御Poster presentation
- 第１回 異分野融合若手研究者の会, Sep. 2023, Japanese, Domestic conference新規抗がん剤標的分子の発見：発がん促進物質が誘導するシグナル伝達Oral presentation
- 第１回 異分野融合若手研究者の会, Sep. 2023, Japanese, Domestic conferenceヒストン脱メチル化酵素LSD1の活性化を介した細胞老化抑制機構の解析Oral presentation
- 第１回 異分野融合若手研究者の会, Sep. 2023, Japanese, Domestic conference老化細胞はなぜ巨大化するのか？～その分子メカニズムと意義～Oral presentation
- 次世代研究者挑戦的研究プログラム 異分野共創発表会, Sep. 2023, English, Domestic conferenceビタミンB2により活性化される細胞老化抑制機構の解析Oral presentation
- サイズ生物学ワークショップ2023, Sep. 2023, Japanese, Domestic conference細胞サイズ増大を引き起こす細胞⽼化の分子機構解析Oral presentation
- 日本植物学会 第87回大会, Sep. 2023, Japanese, Domestic conference金属ナノ粒子の肥料転用における有効性の検証Oral presentation
- 日本研究皮膚科学会 第47回年次学術大会, Dec. 2022, English, Domestic conferenceZinc promotes cell proliferation thorough Akt activation in benign melanomaOral presentation
- 日本研究皮膚科学会 第47回年次学術大会, Dec. 2022, English, Domestic conferencePhorbol ester TPA inhibits the proliferation of metastatic melanoma via tyrosine phosphatases, TC-PTP and SH-PTP2.Oral presentation
- 第45回日本分子生物学会, Dec. 2022, Japanese, Domestic conference老化細胞における空胞形成に及ぼすLY6Dのタンパク質構造Poster presentation
- 第45回日本分子生物学会, Nov. 2022, Japanese, Domestic conferenceNectin-4は老化細胞の細胞面積増大を引き起こすことで細胞の生存を促進する[Invited]Public symposium
- 日本植物学会第86回大会, Sep. 2022, Japanese, Domestic conference酸化亜鉛ナノ粒子を褐虫藻が亜鉛源として利用する際の吸収機構の解析Poster presentation
- 第45回日本基礎老化学会, Jul. 2022, English, Domestic conferenceRiboflavin suppresses cellular senescence through LSD1-mediated downregulation of Sirtuin-4Oral presentation
- 第45回日本基礎老化学会, Jul. 2022, English, Domestic conferenceInduction of DNA damage by exosome derived from senescent cellsOral presentation
- 第44回日本分子生物学会, Dec. 2021, English, Domestic conferenceリボフラビントランスポーターSLC52A1による細胞老化抑制機構の解析Poster presentation
- 第44回日本分子生物学会, Dec. 2021, EnglishTPAによる転移性メラノーマ増殖抑制におけるTC-PTP/PTPN2およびSH-PTP2/PTPN11の分子機構Poster presentation
- 第44回日本分子生物学会, Dec. 2021, English自発老化メラノーマ細胞の形成機構と関連遺伝子の解析Poster presentation
- 日本サンゴ礁学会 第24回大会, Nov. 2021, Japanese金属ナノ粒子を曝露した褐虫藻の増殖挙動の評価・解析Oral presentation
- 第8回日本細胞外小胞学会学術集会, Oct. 2021, English老化細胞が分泌するエキソソームを介したDNA損傷誘導メカニズムの解明Oral presentation
- 第80回日本癌学会学術総会, Oct. 2021, EnglishNectin-4は老化細胞の巨大化に関与し、細胞生存を促進するPoster presentation
- 第80回日本癌学会学術総会, Oct. 2021, EnglishLY6DはIntegrin β1-FAK経路を介してマクロピノサイトーシスを誘導することで老化細胞の生存を促進する
- 第73回日本細胞生物学会大会, Jun. 2021, EnglishリボフラビントランスポーターSLC52A1による細胞老化抑制機構の解析Oral presentation
- 第44回⽇本基礎⽼化学会⼤会, Jun. 2021, EnglishLY6D-induced macropinocytosis as a survival mechanism of senescent cellsOral presentation
- 第44回⽇本基礎⽼化学会⼤会, Jun. 2021, EnglishNectin 4 is responsible for cellular senescence associated enlargement of cell sizeOral presentation
- 第24回バイオ治療法研究会, Dec. 2020, EnglishMAF化タンパク質によるマクロファージの迅速な貪食能活性化におけるAnnexin A2の関与Poster presentation
- 第58回日本生物物理学会, Sep. 2020Cross-seeding of human and bovine insulin amyloid fibrils induces stepwise conformational transition via intermediate statesPoster presentation
- 第93回日本生化学会, Sep. 2020Multi-step conformational changes via intermediate states induced by cross seeding of human and bovine insulin amyloid fibrilsPoster presentation
- 第91回日本動物学会, Sep. 2020STAT3はアフリカツメガエル卵母細胞を安定化させるPoster presentation
- 第42回日本分子生物学会, Dec. 2019Nectin-4 regulates cell size in senescent cellsPoster presentation
- 第42回日本分子生物学会, Dec. 2019LY6D clustering induces macropinocytic vacuole formation through Src-Ras-PI3K pathwayPoster presentation
- 第41回日本分子生物学会, Nov. 2019Characterization of spontaneous senescent melanoma cellPoster presentation
- 第29回 色素細胞学会, Nov. 2019Tyrosine phosphatases TC-PTP and SH-PTP2 inhibit proliferation of metastatic melanoma by phorbol ester, TPA[Invited]Invited oral presentation
- 第13回 日本ツメガエル研究集会, Sep. 2019STAT3 はアフリカツメガエル卵母細胞を安定化させるOral presentation
- 第41回日本分子生物学会, Nov. 2018Functional characterization of protein tyrosine phosphatases, TC-PTP and SH-PTP2, in TPA-induced growth inhibition of metastatic melanomaPoster presentation
- 第41回日本分子生物学会, Nov. 2018Src/FAK-dependent tyrosine phosphorylation contributes to the cell proliferation accompanied by ant-iapoptotic mechanism and cell motility in human bladder cancer cellsPoster presentation
- 第41回日本分子生物学会年会, Nov. 2018, Japanese, Domestic conferenceRiboflavin (Vitamin B2) transporterとして機能するSLC52A1/GPR172Bの細胞老化制御機構の解析Poster presentation
- 第41回日本分子生物学会年会, Nov. 2018, Japanese, Domestic conferenceLY6Dは老化細胞の細胞膜脂質ラフト上でSrcやRasと会合することによりマクロピノサイトーシスを誘導するPoster presentation
- 第77回日本癌学会学術総会, Sep. 2018, Japanese, Domestic conference老化遺伝子EPN3の機能解析Oral presentation
- ConBio2017, Dec. 2017, Japanese, 神戸, Domestic conference新規老化関連遺伝子EPN3の機能解析Poster presentation
- 第21回 バイオ治療研究会, Dec. 2017, Japanese, 福岡, Domestic conferenceマクロファージの貪食活性化に対する血液成分の関与Poster presentation
- ConBio2017, Dec. 2017, Japanese, 神戸, Domestic conferenceTPAによる転移性メラノーマ増殖制御におけるSTAT3ホスファターゼの機能解析Poster presentation
- 研究皮膚科学会 第42回年次学術大会, Dec. 2017, Japanese, 高知, Domestic conferenceTPA-induced growth arrest of malignant melanoma is mediated by dephosphorylation of STAT3 through tyrosine phosphatases, PTPN11 and PTPN2Poster presentation
- ConBio2017, Dec. 2017, Japanese, 神戸, Domestic conferenceserum MAFによるマクロファージ活性化メカニズムの解析Poster presentation
- ConBio2017, Dec. 2017, Japanese, 神戸, Domestic conferenceLY6Dにより誘導されるマクロピノサイトーシスは老化細胞の生存促進に働くOral presentation
- The 2017 International Pigment Cell Conference, Aug. 2017, English, Denver, Colorado USA, International conferenceTyrosine phosphatase, TC-PTP and SH-PTP2 mediate dephosphorylation of STAT3 in TPA-induced growth inhibition of melanomaPoster presentation
- バイオ講演会, 2017, Japanese, Domestic conferenceSTAT3 によるメラノーマおよび色素細胞の増殖制御機構Public discourse
- 日本研究皮膚科学会 第41回年次学術大会, Dec. 2016, English, 仙台, Domestic conferenceTPA inhibits melanoma growth through inactivation of STAT3 through protein tyrosine phosphatases.Poster presentation
- 第20回バイオ治療法研究会学術集会, Dec. 2016, Japanese, 福岡, Domestic conferenceserum MAFによるマクロファージの貪食活性を向上させる要因の解明Poster presentation
- 日本分子生物学会, Nov. 2016, Japanese, 横浜, Domestic conference低濃度抗がん剤処理によって発現誘導されるSASP因子の同定とその機能解析Poster presentation
- 第27回日本色素細胞学会, Nov. 2016, Japanese, 岐阜, Domestic conferenceAnalysis of molecular mechanism of TPA-induced growth inhibition in melanoma cellsOral presentation
- 日本分子生物学会, Nov. 2016, Japanese, 横浜, Domestic conferencep53によるアミノ酸代謝経路の調節が細胞老化を誘導するPublic symposium
- American Association for Cancer Research, Cell Cycle Conference, Mar. 2016, English, Orland, Florida, US, During DNA synthesis, single-stranded DNA breaks (SSBs) are easily produced by mitogenic and oxidative stresses. Although SSBs are naturally converted to double-stranded DNA breaks (DSBs) at the collapse of replication forks, the integrity of chromosomal DNA is regularly maintained during DNA synthesis. Because DNA replication is a fundamental biological process involved in hom, International conferencec-MYC preserves genomic integrity during DNA replication: a paradigm shift of c-MYC[Invited]Invited oral presentation
- 日本動物学会 第８６回新潟大会, Sep. 2015, Japanese, Domestic conferenceApoptosis in Xenopus oocytes and eggsOral presentation
- 第３５回分子生物学会年会, Nov. 2014, English, 横浜, 日本, Domestic conferencePARP1 suppresses Chk2-dependent E2F1 phosphorylation and impedes E2F1-induced apoptosis, but not cell cyclePoster presentation
- THE 38th NAITO CONFERENCE ON Molecule-based biological systems, Oct. 2014, English, International conferenceNovel metabolome analyses of terpenoid indole alkaloids synthesis in Catharanthus roseusPoster presentation
- 第55回日本植物生理学会年会, Mar. 2014, Japanese, 富山大学・五福キャンパス, Domestic conferenceニチニチソウ組織におけるTerpenoid indole alkaloid合成機構の解明Poster presentation
- 日本植物学会第77回大会, Sep. 2013, Japanese, 札幌, Domestic conferenceニチニチソウ葉組織の単一細胞種を用いた二次代謝機構の解析Oral presentation
- 第54回日本植物生理学会年会, Mar. 2013, Japanese, 岡山, Domestic conferenceニチニチソウ細胞における二次代謝機能の解析Oral presentation
- 第54回日本植物生理学会年会, Mar. 2013, Japanese, 岡山, Domestic conferenceニチニチソウ細胞における二次代謝機構の解析Oral presentation
- Cold Spring Harbor Laboratory Symposium on Eukaryotic mRNA processing., 2013, English, Cold Spring Harbor, New York, International conferencemRNA degradation in apoptotic Xenopus eggs.Oral presentation
- 1st International Symposium on Profiling ISPROF2013, 2013, English, Costa da Caparica, Lisbon, Portugal, International conferenceGene expression profiling in single Xenopus oocytes and eggs.Oral presentation
- 第８６回日本生化学会年会, 2013, English, 横浜, Domestic conferenceGene expression profiling in living Xenopus oocytes.Oral presentation
- 第35回分子生物学会年会, Dec. 2012, English, 福岡, Domestic conferenceSingle-cell gene expression analysis corroborates apoptosis in post-meiotic Xenopus eggs（ポスター）Poster presentation
- 第35回分子生物学会年会, Dec. 2012, English, 福岡, Domestic conferenceSingle-cell gene expression analysis corroborates apoptosis in post-meiotic Xenopus eggsPublic symposium
- 第35回分子生物学会年会, Dec. 2012, English, 福岡, Domestic conferenceResistance to Src inhibition in malignant melanoma cells（ポスター）Poster presentation
- 第35回分子生物学会年会, Dec. 2012, English, 福岡, Domestic conferenceResistance to Src inhibition in malignant melanoma cellsOral presentation
- 第35回分子生物学会年会, Dec. 2012, English, 福岡, Domestic conferenceRegulation of Gene expression by STAT3 during Xenopus oocyte maturation（ポスター）Poster presentation
- 第35回分子生物学会年会, Dec. 2012, English, 福岡, Domestic conferenceRegulation of Gene expression by STAT3 during Xenopus oocyte maturationOral presentation
- 第35回分子生物学会年会, Dec. 2012, English, 福岡, Domestic conferenceFunctional analysis of Ser727-phosphorylated STAT3 in benign melanoma cells（ポスター）Poster presentation
- 第35回分子生物学会年会, Dec. 2012, English, 福岡, Domestic conferenceFunctional analysis of Ser727-phosphorylated STAT3 in benign melanoma cellsOral presentation
- 日本動物学会 第83回大阪大会, Sep. 2012, Japanese, 大阪, Domestic conferenceゼノパス卵成熟過程におけるSTAT3のリン酸化解析Oral presentation
- Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C), Grant-in-Aid for Scientific Research (C), Kobe University, Apr. 2021 - Mar. 2024, Principal investigatorEstablishment of a basis for the development of anti-aging medicine using spontaneous senescent cells本研究はメラノーマ細胞から自発的に形成される老化細胞（以下、自発老化細胞）を用いて、腫瘍組織などに出現する「老化細胞の特性を明らかにし(課題1)、老化細胞と周辺細胞との相互作用様式を解明する(課題2)とともに、抗老化薬の開発基盤(課題3)をつくることを目的としている。当該年度は上記目的のうち主に課題1と2に関する研究を行った。 まず自発老化細胞が形成される細胞株であるWM115 細胞とWM164 細胞を用いて、細胞内シグナル伝達経路に対する活性化剤や阻害剤を用いて解析を行った。その結果、自発老化細胞の出現にはMEK-ERK経路の活性化が必要であることが明らかになった。続いて自発老化細胞のタイムラプス観察を行った。その結果、自発老化細胞は細胞分裂の不全により形成されること、通常サイズのメラノーマ細胞より安定性が高く細胞死が起こりにくいことが明らかになった。さらにRNA-seq解析の結果から、自発老化細胞ではsenescence-associated secretory phenotype (SASP) 関連分泌因子の遺伝子発現上昇していることが明らかになった。また自発老化細胞の培養上清を用いた解析から、自発老化細胞は周辺細胞にSASP因子を介してEMT (epithelial-mesenchymal transition) を引き起こされている可能性が示された。 以上のことから自発老化細胞は、一部のメラノーマ細胞のMEK-ERK シグナルの過剰活性化に起因して形成が開始し、細胞質分裂が不完全に完了することで形成され、長期間組織中に滞在することが分かった。また自発老化細胞は腫瘍組織内の周辺細胞に分泌因子を介して EMTを誘導することでがんの悪性化を促進する可能性が高いことが示された。
- がん細胞から生じる老化細胞の形成機構とその除去方法の確立, 令和4年度 学術研究助成, Apr. 2022 - Mar. 2023, Principal investigatorがん細胞から生じる老化細胞の形成機構とその除去方法の確立
- Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C), Grant-in-Aid for Scientific Research (C), Kobe University, Apr. 2020 - Mar. 2023, CoinvestigatorBasic research for the development of novel anticancer drugs targeting senescent cells induced by cancer treatment本研究は、申請者らが同定した老化細胞特異的に発現上昇する遺伝子であるLY6Dについて、その生理機能を明らかにするとともに、LY6Dを標的とした新規抗がん剤開発に向けた分子基盤を確立することを目的としている。LY6DはGPIアンカー型の細胞膜タンパク質であり、高発現によって老化細胞に特徴的な巨大な空胞を形成することを見出していた。 本年度はLY6Dが空胞形成を誘導するシグナル伝達経路を解明するため、まず、LY6DのS-S結合形成に関与することが予想されるシステイン残基をセリン残基に置換したところ、空胞が形成されなかったことからS-S結合の重要性を検証できた。さらに、LY6Dは細胞膜ラフトに存在し、Src family kinase (SFK)と多量体を形成することが明らかとなった。また、LY6Dの下流ではRasが機能する可能性が示唆されていたため、Rasの活性に重要なGly12、及び、PI3K、Raf、Ral-GEF、それぞれの情報伝達に重要なTyr40、Thr35、Glu37の変異体を作製し細胞に高発現させたところ、LY6D誘導性空胞形成にはPI3Kが重要な働きをすることが明らかとなった。一方、細胞内に空胞を形成する機構の一つとしてオートファジーの関与が示唆されているが、オートファゴソームのマーカーであるLC3の発現や、オートリソソームなどの酸性オルガネラマーカーであるLysoTrackerを用いてLY6Dで誘導される空胞と局在が一致するかどうか調べたところ、LY6Dによって誘導される空胞はオートファジーによるのではなくマクロピノサイトーシス（エンドサイトーシスの一種）によって形成されることがわかった。そして、LY6Dによって誘導される空胞は、細胞外液の取り込みに関与し老化細胞の生存に寄与することが明らかとなった。
- 産学が連携した研究開発成果の展開 研究成果展開事業 研究成果最適展開支援プログラム(A-STEP) 産学共同(育成型), 大阪府立大学, 2020 - 2022, Coinvestigator植物の細胞培養や水耕栽培では、無機塩類を供給する。多様な無機塩類が溶解した養液の体積は大きく、循環送液のエネルギー消費がコストを逼迫するため、養液量を最小化する技術革新が望まれる。そこで、粒子がナノ化すると同一質量で大きな界面積を有し、高い反応性と凝集性を得ることに着目した。低溶解性の無機塩ナノ粒子を植物細胞に暴露すると、粒子が表面に局在し、溶解したミネラルが細胞に高吸収されることから、粒子の溶解速度の制御で細胞の吸収量が増加することを見出した。従って、無機塩類を微細粒子化して徐放性を最適化すると、養液の最小化が達成できる。本課題は、無機塩ナノ粒子を用途拡大するためのメカニズム解析を取り扱う。
- 学術研究助成基金助成金／挑戦的萌芽研究, Apr. 2015 - Mar. 2018Competitive research funding
- Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research, Grant-in-Aid for Challenging Exploratory Research, Nara Medical University, Apr. 2014 - Mar. 2017, OthersIn order to investigate the mechanism of steroid hormone production, we developed the direct visualization methods of steroid hormones on mammalian adrenal tissue sections by imaging mass spectroscopy. Since the ionization efficiency of steroid hormones are low when common organic matrices such as CHCA or DHB are used, we explored various SALDI/Nano-PALDI ionization methods and various derivatization reagents to improve the ionization efficiency. Among these methods, Girard-T (GirT) efficiently derivatized steroid hormones. We used GirT as an on-tissue derivatization reagent on adrenal tissue sections and detected m/z peaks of major steroid hormones (corticosterone, progesterone, cortisol, aldosterone, etc.) on adrenal cortex. Using FT-ICR MS with ultrahigh mass resolution, precise m/z peak signals of steroid hormones within +/-0.001 were obtained.
- ひょうご科学技術協会, 平成21年度 奨励研究助成, Apr. 2009 - Mar. 2010, Principal investigatorアフリカツメガエル初期発生における遺伝子翻訳制御機構の解明
- Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (B), Grant-in-Aid for Young Scientists (B), Kobe University, 2009 - 2010, Principal investigatorWe have previously demonstrated that Src tyrosine kinase plays an important role in the Xenopus egg activation. However, the molecular mechanism of egg activation is still unclear. Here, we investigated the activation mechanism and the physiological function of Src kinase in Xenopus egg fertilization. Biochemical and immunochemical analyses have revealed the involvement of heterotrimeric G proteins in sperm-induced Src activation. Moreover, we analyzed hnRNP K protein, which is known as a substrate of Src kinase in Xenopus egg fertilization. Our molecular biological and biochemical analyses showed that the hnRNP K binds to specific mRNAs in unfertilized egg, and that Src-mediated tyrosine phosphorylation acts as a trigger for their dissociation at egg activation.
- Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Encouragement of Scientists, Grant-in-Aid for Encouragement of Scientists, 神戸大, 2003 - 2003, Principal investigator卵活性化における分子メカニズムの解明
- Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Encouragement of Young Scientists (B), Grant-in-Aid for Encouragement of Young Scientists (B), 神戸大学遺伝子実験施設, 2001 - 2001受精のシグナル伝達におけるアダプター蛋白質Shcの機能解析