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Biomedical subjects

N Gotoh

Publications and source records attributed to N Gotoh.

At least 19 recordsLinked to original sources
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↗
PubMed2000

Substrate specificities of MexAB-OprM, MexCD-OprJ, and MexXY-oprM efflux pumps in Pseudomonas aeruginosa.

To find the exact substrate specificities of three species of tripartite efflux systems of Pseudomonas aeruginosa, MexAB-OprM, MexCD-OprJ, and MexXY-OprM, we constructed a series of isogenic mutants, each of which constitutively overproduced one of the three efflux systems and lacked the other two, and their isogenic mutants, which lacked all these systems. Comparison of the susceptibilities of the constructed mutants to 52 antimicrobial agents belonging to various groups suggested the following substrate specificities. All of the efflux systems extrude a wide variety of antimicrobial agent groups, i.e., quinolones, macrolides, tetracyclines, lincomycin, chloramphenicol, most penicillins (all but carbenicillin and sulbenicillin), most cephems (all but cefsulodin and ceftazidime), meropenem, and S-4661, but none of them extrude polymyxin B or imipenem. Extrusion of aminoglycosides is specific to MexXY-OprM, and extrusion of a group of the beta-lactams, i.e., carbenicillin, sulbenicillin, ceftazidime, moxalactam, and aztreonam, is specific to MexAB-OprM. Moreover, MexAB-OprM and MexCD-OprJ extrude novobiocin, cefsulodin, and flomoxef, while MexXY-OprM does not. These substrate specificities are distinct from those reported previously.

Bacterial Outer Membrane Proteins↗
PubMed2000

Molecular mechanisms of fluoroquinolone resistance in Pseudomonas aeruginosa isolates from cystic fibrosis patients.

Twenty P. aeruginosa isolates were collected from six cystic fibrosis (CF) patients, aged 27 to 33, in 1994 (9 isolates) and 1997 (11 isolates) at the CF Center, Copenhagen, Denmark, and were typed by pulse-field gel electrophoresis (PFGE) or ribotyping. Five of the patients had isolates with the same PFGE or ribotyping patterns in 1997 as in 1994, and ciprofloxacin had a two- to fourfold higher MIC for the isolates collected in 1997 than those from 1994. Genomic DNA was amplified for gyrA, parC, mexR, and nfxB by PCR and sequenced. Eleven isolates had mutations in gyrA, seven isolates had mutations at codon 83 (Thr to Ile), and four isolates had mutations at codon 87 (Asp to Asn or Tyr). Sixteen isolates had mutations in nfxB at codon 82 (Arg to Leu). Increased amounts of OprN were found in six isolates and OprJ in eight isolates as determined by immunoblotting. No isolates had mutations in parC or mexR. Six isolates had mutations in efflux pumps without gyrA mutations. The average number of mutations was higher in isolates from 1997 than in those from 1994. The results also suggested that efflux resistance mechanisms are more common in isolates from CF patients than in strains from urine and wounds from non-CF patients, in which mutations in gyrA and parC dominate (S. Jalal and B. Wretlind, Microb. Drug Resist. 4:257-261, 1998).

Adult↗
PubMed2000

Contribution of the MexX-MexY-oprM efflux system to intrinsic resistance in Pseudomonas aeruginosa.

To test the possibility that MexX-MexY, a new set of efflux system components, is associated with OprM and contributes to intrinsic resistance in Pseudomonas aeruginosa, we constructed a series of isogenic mutants lacking mexXY and/or mexAB and/or oprM from a laboratory strain PAO1, and examined their susceptibilities to ofloxacin, tetracycline, erythromycin, gentamicin, and streptomycin. Loss of either MexXY or OprM from the MexAB-deficient mutant increased susceptibility to all agents tested, whereas loss of MexXY from the MexAB-OprM-deficient mutant caused no change in susceptibility. Introduction of an OprM expression plasmid decreased the susceptibility of the mexAB-oprM-deficient-/mexXY-maintaining mutant, yet caused no change in the susceptibility of a mexAB-oprM- and mexXY-deficient double mutant. Immunoblot analysis using anti-MexX polyclonal rabbit serum generated against synthetic oligopeptides detected expression of MexX in the PAO1 cells grown in medium containing tetracycline, erythromycin, or gentamicin, although expression of MexX was undetectable in the cells incubated in medium without any agent. These results suggest that MexXY induced by these agents is functionally associated with spontaneously expressed OprM and contributes to the intrinsic resistance to these agents.

Anti-Bacterial Agents↗
PubMed2000

FRS2 proteins recruit intracellular signaling pathways by binding to diverse targets on fibroblast growth factor and nerve growth factor receptors.

The docking protein FRS2 was implicated in the transmission of extracellular signals from the fibroblast growth factor (FGF) or nerve growth factor (NGF) receptors to the Ras/mitogen-activated protein kinase signaling cascade. The two members of the FRS2 family, FRS2alpha and FRS2beta, are structurally very similar. Each is composed of an N-terminal myristylation signal, a phosphotyrosine-binding (PTB) domain, and a C-terminal tail containing multiple binding sites for the SH2 domains of the adapter protein Grb2 and the protein tyrosine phosphatase Shp2. Here we show that the PTB domains of both the alpha and beta isoforms of FRS2 bind directly to the FGF or NGF receptors. The PTB domains of the FRS2 proteins bind to a highly conserved sequence in the juxtamembrane region of FGFR1. While FGFR1 interacts with FRS2 constitutively, independent of ligand stimulation and tyrosine phosphorylation, NGF receptor (TrkA) binding to FRS2 is strongly dependent on receptor activation. Complex formation with TrkA is dependent on phosphorylation of Y490, a canonical PTB domain binding site that also functions as a binding site for Shc (NPXpY). Using deletion and alanine scanning mutagenesis as well as peptide competition assays, we demonstrate that the PTB domains of the FRS2 proteins specifically recognize two different primary structures in two different receptors in a phosphorylation-dependent or -independent manner. In addition, NGF-induced tyrosine phosphorylation of FRS2alpha is diminished in cells that overexpress a kinase-inactive mutant of FGFR1. This experiment suggests that FGFR1 may regulate signaling via NGF receptors by sequestering a common key element which both receptors utilize for transmitting their signals. The multiple interactions mediated by FRS2 appear to play an important role in target selection and in defining the specificity of several families of receptor tyrosine kinases.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Apoptosis in microvascular endothelial cells of perfused rabbit lungs with acute hydrostatic edema.

We test the hypothesis that microvascular endothelial cells may undergo apoptosis in response to acute pulmonary venous hypertension. The isolated rabbit lungs were perfused in situ for 4 h with left atrial pressure of 0, 10, or 20 mmHg at a constant blood flow. Edema formation was monitored by lung weight gain. To assay for apoptosis, we performed agarose gel electrophoresis of DNA, in situ nick end labeling of DNA strand breaks, and electron microscopy. We also examined the levels of expression of Bcl-2, a suppressor of apoptosis, in microvascular endothelial cells using an immunohistochemical technique. In a vascular pressure-dependent fashion, we found apoptosis in endothelial cells of alveolar septal capillaries, as well as expression of Bcl-2 in arteriolar and venular endothelial cells. We conclude that acute pulmonary venous hypertension induces apoptosis in capillary endothelial cells but not in arteriolar and venular endothelial cells, suggesting that microvascular endothelial cell apoptosis is dependent on the levels of Bcl-2 expression and influences the formation or resolution of acute hydrostatic lung edema.

Acute Disease↗
PubMed1999

Topological analysis of an RND family transporter, MexD of Pseudomonas aeruginosa.

The membrane topology of a resistance-nodulation-division (RND) family transporter, MexD of Pseudomonas aeruginosa, was determined. Although it had been predicted previously that most RND proteins contain 12 transmembrane helices, three independent computer programs used in the present study predicted that MexD possessed 11, 14 or 17 transmembrane segments. To investigate the topology of MexD more thoroughly, 25 MexD-PhoA (alkaline phosphatase) and 18 MexD-Bla (beta-lactamase) fusion plasmids were constructed and analyzed. The resulting topological model had just 12 transmembrane helices and two periplasmic loops of about 300 residues between helices 1 and 2 and helices 7 and 8. It is therefore proposed that the N- and C-termini are located in the cytoplasm and the predicted orientation is consistent with the 'positive-inside rule'. This topological model can be applied to other RND proteins.

Alkaline Phosphatase↗
PubMed1999

Shc regulates epidermal growth factor-induced activation of the JNK signaling pathway.

Two adaptor molecules, Grb2 and Shc, have been implicated in the extracellular signal-regulated kinase (ERK) activation by receptor tyrosine kinases such as the epidermal growth factor receptor (EGFR). Here we show that the EGF-mediated ERK activation is abolished by loss of Grb2, whereas this response is not affected by loss of Shc. Conversely, the EGF-mediated c-Jun N-terminal kinase (JNK) activation is dependent on Shc, but not Grb2. These findings strongly support distinct roles for Grb2 and Shc in controlling ERK and JNK activation after EGF stimulation.

Adaptor Proteins, Signal Transducing↗