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Biomedical subjects

N Gotoh

Publications and source records attributed to N Gotoh.

At least 19 recordsLinked to original sources
PubMed2006

Unique role of SNT-2/FRS2beta/FRS3 docking/adaptor protein for negative regulation in EGF receptor tyrosine kinase signaling pathways.

The membrane-linked docking protein SNT-2/FRS2beta/FRS3 becomes tyrosine phosphorylated in response to fibroblast growth factors (FGFs) and neurotrophins and serves as a platform for recruitment of multiple signaling proteins, including Grb2 and Shp2, to FGF receptors or neurotrophin receptors. We previously reported that SNT-2 is not tyrosine phosphorylated significantly in response to epidermal growth factor (EGF) but that it inhibits ERK activation via EGF stimulation by forming a complex with ERK2. In the present report, we show that expression of SNT-2 suppressed EGF-induced cell transformation and proliferation, and expression level of SNT-2 is downregulated in cancer. The activities of the major signaling molecules in EGF receptor (EGFR) signal transduction pathways, including autophosphorylation of EGFR, were attenuated in cells expressing SNT-2 but not in cells expressing SNT-2 mutants lacking the ERK2-binding domain. Furthermore, SNT-2 constitutively bound to EGFR through the phosphotyrosine binding (PTB) domain both with and without EGF stimulation. Treatment of cells with MEK inhibitor U0126 partially restored the phosphorylation levels of MEK and EGFR in cells expressing SNT-2. On the basis of these findings, we propose a novel mechanism of negative control of EGFR tyrosine kinase activity with SNT-2 by recruiting ERK2, which is the site of negative-feedback loop from ERK, ultimately leading to inhibition of EGF-induced cell transformation and proliferation.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Novel approach on the risk assessment of oxidized fats and oils for perspectives of food safety and quality. I. Oxidized fats and oils induces neurotoxicity relating pica behavior and hypoactivity.

Food poisoning caused by deteriorated fat and oil in instant noodles was first reported in Japan approximately 40 years ago. In these cases, many people developed neurotoxic symptoms such as emesis and discomfort. The degree of oxidation of the fat and oil in the instant noodles that induced food poisoning was at least 100 meq/kg in peroxide value (PV). No general toxicity studies with animals, however, have examined the toxicity of fat and oil oxidized to that extent. In this study, pica behavior, a behavior characterized by eating a nonfood material such as kaolin and that relates to the degree of discomfort in animals, and alterations of locomotor activity of rats eating deteriorated fat and oil were measured. The groups fed fat and oil with at least 138.5 meq/kg PV consumed significantly more kaolin compared to the control group. Furthermore, rats that ate deteriorated fat and oil with at least 107.2 meq/kg PV had significantly decreased locomotor activity compared to control rats. These phenomena suggest that oxidized fat and oil with at least 100 meq/kg PV induce neurotoxicity. The toxicity of oxidized fat and oil has only been addressed using general toxicity tests, but the present results reveal the importance of evaluating toxicity by using other measures.

Animals↗
PubMed2005

Essential role of Shp2-binding sites on FRS2alpha for corticogenesis and for FGF2-dependent proliferation of neural progenitor cells.

Mammalian corticogenesis occurs through a complex process that includes neurogenesis, in which neural progenitor cells proliferate, differentiate, and migrate. It has been reported recently that neurogenesis occurs in the subventricular zone (SVZ), a region previously thought to be the primary site of gliogenesis. It has been recognized that in the SVZ, intermediate progenitor cells, derived from radial glial cells that are multipotent neural stem cells, produce only neurons. However, the molecular mechanisms underlying the regulation of neural stem cells and intermediate progenitor cells as well as their contribution to overall corticogenesis remain unknown. The docking protein FRS2alpha is a major mediator of signaling by means of FGFs and neurotrophins. FRS2alpha mediates many of its pleiotropic cellular responses by recruiting the adaptor protein Grb2 and the protein tyrosine phosphatase Shp2 upon ligand stimulation. Here, we report that targeted disruption of Shp2-binding sites in FRS2alpha leads to severe impairment in cerebral cortex development in mutant mice. The defect in corticogenesis appears to be due at least in part to abnormalities in intermediate progenitor cells. Genetic evidence is provided that FRS2alpha plays critical roles in the maintenance of intermediate progenitor cells and in neurogenesis in the cerebral cortex. Moreover, FGF2-responsive neurospheres, which are cell aggregates derived from neural stem/progenitor cells (NSPCs), from FRS2alpha mutant mice were smaller than those of WT mice. However, mutant NSPCs were able to self-renew, demonstrating that Shp2-binding sites on FRS2alpha play an important role in NSPC proliferation but are dispensable for NSPC self-renewing capacity after FGF2 stimulation.

Animals↗
PubMed2005

The docking protein FRS2alpha is an essential component of multiple fibroblast growth factor responses during early mouse development.

The docking protein FRS2alpha is a major mediator of fibroblast growth factor (FGF) signaling. However, the physiological role of FRS2alpha in vivo remains unknown. In this report, we show that Frs2alpha-null mouse embryos have a defect in anterior-posterior (A-P) axis formation and are developmentally retarded, resulting in embryonic lethality by embryonic day 8. We demonstrate that FRS2alpha is essential for the maintenance of self-renewing trophoblast stem (TS) cells in response to FGF4 in the extraembryonic ectoderm (ExE) that gives rise to tissues of the placenta. By analyzing chimeric embryos, we found that FRS2alpha also plays a role in cell movement through the primitive streak during gastrulation. In addition, experiments are presented demonstrating that Bmp4 expression in TS cells is controlled by mitogen-activated protein kinase-dependent FGF4 stimulation. Moreover, both the expression of Bmp4 in ExE and activation of Smad1/5 in epiblasts are reduced in Frs2alpha-null embryos. These experiments underscore the critical role of FRS2alpha in mediating multiple processes during embryonic development and reveal a potential new link between FGF and Bmp4 signaling pathways in early embryogenesis.

Animals↗
PubMed2004

Tyrosine phosphorylation sites on FRS2alpha responsible for Shp2 recruitment are critical for induction of lens and retina.

Early development of the lens and retina depends upon reciprocal inductive interactions between the embryonic surface ectoderm and the underlying neuroepithelium of the optic vesicle. FGF signaling has been implicated in this signal exchange. The docking protein FRS2alpha is a major mediator of FGF signaling by providing a link between FGF receptors (FGFRs) and a variety of intracellular signaling pathways. After FGF stimulation, tyrosine-phosphorylated FRS2alpha recruits four molecules of the adaptor protein Grb2 and two molecules of the protein tyrosine phosphatase Shp2, resulting in activation of the Ras/extracellular signal-regulated kinase (ERK) and phosphatidylinositol-3 kinase/Akt signaling pathways. In this report, we explore the role of signaling pathways downstream of FRS2alpha in eye development by analyzing the phenotypes of mice that carry point mutations in either the Grb2-(Frs2alpha(4F)) or the Shp2-binding sites (Frs2alpha(2F)) of FRS2alpha. Although Frs2alpha(4F/4F) mice exhibited normal early eye development, all Frs2alpha(2F/2F) embryos were defective in eye development and showed anophthalmia or microphthalmia. Consistent with the critical role of FRS2alpha in FGF signaling, the level of activated extracellular signal-regulated kinase in Frs2alpha(2F/2F) embryos was significantly lower than that observed in wild-type embryos. Furthermore, expression of Pax6 and Six3, molecular markers for lens induction, were decreased in the Frs2alpha(2F/2F) presumptive lens ectoderm. Similarly, the expression of Chx10 and Bmp4, genes required for retinal precursor proliferation and for lens development, respectively, was also decreased in the optic vesicles of Frs2alpha(2F/2F) mice. These experiments demonstrate that intracellular signals that depend on specific tyrosine residues in FRS2alpha lie upstream of gene products critical for induction of lens and retina.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Virulence of metallo-beta-lactamase-producing Pseudomonas aeruginosa in vitro and in vivo.

We evaluated the virulence of Pseudomonas aeruginosa carrying bla(IMP), a metallo-beta-lactamase gene, and the efficacy of ceftazidime, imipenem-cilastatin, and ciprofloxacin in the endogenous bacteremia model. The presence of bla(IMP) did not practically change the virulence of the parent strain, and ciprofloxacin was effective against infection with P. aeruginosa carrying bla(IMP).

Animals↗
PubMed2002

Early experiences of endoscopic procedures in general surgery assisted by a computer-enhanced surgical system.

We performed a variety of complete total endoscopic general surgical procedures, including colon resection, distal gastrectomy, and splenectomy, successfully with the assistance of the da Vinci computer-enhanced surgical system. The robotic system allowed us to manipulate the endoscopic instruments as effectively as during open surgery. It enhanced visualization of both the operative field and precision of the necessary techniques, as well as being less stressful for the endoscopic operating team. This technological innovation can therefore help surgeons overcome many of the difficulties associated with the endoscopic approach and thus has the potential to enable more precise, safer, and more minimally invasive surgery in the future.

Adult↗
PubMed2001

Molecular cloning and characterization of human PTB-like protein: a possible retinal autoantigen of cancer-associated retinopathy.

Human homologue of polypyrimidine tract binding protein (PTB), a possible autoantigen for cancer-associated retinopathy (CAR), was isolated from a human retinal cDNA library. This homologue, named PTB-like protein (PTBLP), encodes a 532 amino acid residue and has a 75% homology to the human PTB. The human PTBLP had four RNA recognition motifs (RRMs) and had a RNA binding ability. There are four splicing variants in PTBLP. The CAR serum recognized the full length form of PTBLP and the antigenic determinant was localized within 12 amino acids of the C-terminal region. The sequence was included in the fourth RRM sequence.

Amino Acid Sequence↗
PubMed2001

Unique phosphorylation mechanism of Gab1 using PI 3-kinase as an adaptor protein.

Grb2-associated binder-1 (Gab1) undergoes tyrosine phosphorylation in response to stimulation by growth factors and hormones including insulin, epidermal growth factor (EGF), nerve growth factor (NGF), and hepatocyte growth factor (HGF). However, the HGF receptor is the only one known to associate directly with Gab1. Herein, we explore the mechanism of Gab1 phosphorylation by other receptor protein-tyrosine kinases unable to bind to Gab1 directly. The Src homology 2 (SH2) domain of the phosphatidylinositol 3-kinase (PI3K) regulatory subunit binds Gab1 in a phosphorylation-independent manner. Moreover, the regulatory subunit of PI3K can mediate the association of Gab1 and receptor protein-tyrosine kinases including the insulin, EGF, and NGF receptors, all of which phosphorylate Gab1. Thus, it appears that the PI3K regulatory subunit acts as an adaptor protein via a phosphotyrosyl-independent SH2 interaction, allowing Gab1 to serve as a substrate for several tyrosine kinases. This is a new role for the PI3K regulatory subunit.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Critical role for the docking-protein FRS2 alpha in FGF receptor-mediated signal transduction pathways.

The docking protein FRS2 alpha has been implicated as a mediator of signaling via fibroblast growth factor receptors (FGFRs). We have demonstrated that targeted disruption of FRS2 alpha gene causes severe impairment in mouse development resulting in embryonal lethality at E7.0--E7.5. Experiments with FRS2 alpha-deficient fibroblasts demonstrate that FRS2 alpha plays a critical role in FGF-induced mitogen-activated protein (MAP) kinase stimulation, phosphatidylinositol-3 (PI-3) kinase activation, chemotactic response, and cell proliferation. Following FGF stimulation, tyrosine phosphorylated FRS2 alpha functions as a site for coordinated assembly of a multiprotein complex that includes Gab1 and the effector proteins that are recruited by this docking protein. Furthermore, we demonstrate that different tyrosine phosphorylation sites on FRS2 alpha are responsible for mediating different FGF-induced biological responses. These experiments establish the central role of FRS2 alpha in signaling via FGFRs and demonstrate that FRS2 alpha mediates multiple FGFR-dependent signaling pathways critical for embryonic development.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Stimulation of phosphatidylinositol 3-kinase by fibroblast growth factor receptors is mediated by coordinated recruitment of multiple docking proteins.

The docking protein FRS2 is a major downstream effector that links fibroblast growth factor (FGF) and nerve growth factor receptors with the Ras/mitogen-activated protein kinase signaling cascade. In this report, we demonstrate that FRS2 also plays a pivotal role in FGF-induced recruitment and activation of phosphatidylinositol 3-kinase (PI3-kinase). We demonstrate that tyrosine phosphorylation of FRS2alpha leads to Grb2-mediated complex formation with the docking protein Gab1 and its tyrosine phosphorylation, resulting in the recruitment and activation of PI3-kinase. Furthermore, Grb2 bound to tyrosine-phosphorylated FRS2 through its SH2 domain interacts primarily via its carboxyl-terminal SH3 domain with a proline-rich region in Gab1 and via its amino-terminal SH3 domain with the nucleotide exchange factor Sos1. Assembly of FRS2alpha:Grb2:Gab1 complex induced by FGF stimulation results in activation of PI3-kinase and downstream effector proteins such as the S/T kinase Akt, whose cellular localization and activity are regulated by products of PI3-kinase. These experiments reveal a unique mechanism for generation of signal diversity by growth factor-induced coordinated assembly of a multidocking protein complex that can activate the Ras/mitogen-activated protein kinase cascade to induce cell proliferation and differentiation, and PI3-kinase to activate a mediator of a cell survival pathway.

3T3 Cells↗
PubMed2001

Carbapenem resistance mechanisms in Pseudomonas aeruginosa clinical isolates.

In order to define the contributions of the mechanisms for carbapenem resistance in clinical strains of Pseudomonas aeruginosa, we investigated the presence of OprD, the expressions of the MexAB-OprM and MexEF-OprN systems, and the production of the beta-lactamases for 44 clinical strains. All of the carbapenem-resistant isolates showed the loss of or decreased levels of OprD. Three strains overexpressed the MexAB-OprM efflux system by carrying mutations in mexR. These three strains had the amino acid substitution in MexR protein, Arg (CGG) --> Gln (CAG), at the position of amino acid 70. None of the isolates, however, expressed the MexEF-OprN efflux system. For the characterization of beta-lactamases, at least 13 isolates were the depressed mutants, and 12 strains produced secondary beta-lactamases. Based on the above resistance mechanisms, the MICs of carbapenem for the isolates were analyzed. The MICs of carbapenem were mostly determined by the expression of OprD. The MICs of meropenem were two- to four-fold increased for the isolates which overexpressed MexAB-OprM in the background of OprD loss. However, the elevated MICs of meropenem for some individual isolates could not be explained. These findings suggested that other resistance mechanisms would play a role in meropenem resistance in clinical isolates of P. aeruginosa.

Bacterial Proteins↗
PubMed2001

Hypersusceptibility of the Pseudomonas aeruginosa nfxB mutant to beta-lactams due to reduced expression of the ampC beta-lactamase.

The Pseudomonas aeruginosa nfxB mutant lacking mexAB-oprM showed hypersusceptibility to 9 out of 24 beta-lactams tested. This hypersusceptibility was found for the nfxB mutant lacking mexAB-oprM-mexXY (N108) but not for the nfxB mutant lacking both mexAB-oprM-mexXY and ampC. The level of the AmpC beta-lactamase induction was reduced in N108. Thus, the reduced AmpC induction must be the cause of the hypersusceptibility.

Bacterial Outer Membrane Proteins↗
PubMed2001

Pseudomonas aeruginosa reveals high intrinsic resistance to penem antibiotics: penem resistance mechanisms and their interplay.

Pseudomonas aeruginosa exhibits high intrinsic resistance to penem antibiotics such as faropenem, ritipenem, AMA3176, sulopenem, Sch29482, and Sch34343. To investigate the mechanisms contributing to penem resistance, we used the laboratory strain PAO1 to construct a series of isogenic mutants with an impaired multidrug efflux system MexAB-OprM and/or impaired chromosomal AmpC beta-lactamase. The outer membrane barrier of PAO1 was partially eliminated by inducing the expression of the plasmid-encoded Escherichia coli major porin OmpF. Susceptibility tests using the mutants and the OmpF expression plasmid showed that MexAB-OprM and the outer membrane barrier, but not AmpC beta-lactamase, are the main mechanisms involved in the high intrinsic penem resistance of PAO1. However, reducing the high intrinsic penem resistance of PAO1 to the same level as that of penem-susceptible gram-negative bacteria such as E. coli required the loss of either both MexAB-OprM and AmpC beta-lactamase or both MexAB-OprM and the outer membrane barrier. Competition experiments for penicillin-binding proteins (PBPs) revealed that the affinity of PBP 1b and PBP 2 for faropenem were about 1.8- and 1.5-fold lower, than the respective affinity for imipenem. Loss of the outer membrane barrier, MexAB, and AmpC beta-lactamase increased the susceptibility of PAO1 to almost all penems tested compared to the susceptibility of the AmpC-deficient PAO1 mutants to imipenem. Thus, it is suggested that the high intrinsic penem resistance of P. aeruginosa is generated from the interplay among the outer membrane barrier, the active efflux system, and AmpC beta-lactamase but not from the lower affinity of PBPs for penems.

Anti-Bacterial Agents↗
PubMed2001

Mesodermal patterning defect in mice lacking the Ste20 NCK interacting kinase (NIK).

We have previously shown that the Drosophila Ste20 kinase encoded by misshapen (msn) is an essential gene in Drosophila development. msn function is required to activate the Drosophila c-Jun N-terminal kinase (JNK), basket (Bsk), to promote dorsal closure of the Drosophila embryo. Later in development, msn expression is required in photoreceptors in order for their axons to project normally. A mammalian homolog of msn, the NCK-interacting kinase (NIK) (recently renamed to mitogen-activated protein kinase kinase kinase kinase 4; Map4k4), has been shown to activate JNK and to bind the SH3 domains of the SH2/SH3 adapter NCK. To determine whether NIK also plays an essential role in mammalian development, we created mice deficient in NIK by homologous recombination at the Nik gene. Nik(-/-) mice die postgastrulation between embryonic day (E) 9.5 and E10.5. The most striking phenotype in Nik(-/-) embryos is the failure of mesodermal and endodermal cells that arise from the anterior end of the primitive streak (PS) to migrate to their correct location. As a result Nik(-/- )embryos fail to develop somites or a hindgut and are truncated posteriorly. Interestingly, chimeric analysis demonstrated that NIK has a cell nonautonomous function in stimulating migration of presomitic mesodermal cells away from the PS and a second cell autonomous function in stimulating the differentiation of presomitic mesoderm into dermomyotome. These findings indicate that despite the large number of Ste20 kinases in mammalian cells, members of this family play essential nonredundant function in regulating specific signaling pathways. In addition, these studies provide evidence that the signaling pathways regulated by these kinases are diverse and not limited to the activation of JNK because mesodermal and somite development are not perturbed in JNK1-, and JNK2-deficient mice.

Animals↗
PubMed2001

[Antibiotic resistance caused by membrane impermeability and multidrug efflux systems].

Multiple antibiotic resistance(MDR) in bacteria was at first thought to be caused exclusively by the combination of several resistance mechanisms including membrane impermeability, the outer membrane barrier in gram-negative bacteria. More recently, it became clear that MDR are often achieved by interplay between impermeability and multidrug efflux pumps. Streptococcus pneumoniae MefA, Staphylococcus aureus NorA and Pseudomonas aeruginosa Mex of these pumps are significant, because those bacteria are more frequently isolated in clinical. In this review, we described on characteristics and clinical significance of P. aeruginosa Mex systems, MexA-MexB-OprM, MexC-MexD-OprJ, MexE-MexF-OprN and MexX-MexY-OprM, and furthermore, on interplay between the efflux systems, the outer membrane barrier, hydrolyzing enzyme and mutated target.

Bacteria↗