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Biomedical subjects

T Ohba

Publications and source records attributed to T Ohba.

At least 55 records · Page 3Linked to original sources
PubMed1999

[Clinical examination to screen for laryngeal cancer: results, problems and prospects].

The Yokohama Municipal Cancer Detection Center, established as an affiliate of the Yokohama Municipal Citizen's Hospital in June, 1981, has performed clinical examinations to screen for laryngeal cancer in a total of 26,377 patients (12,205 men and 14,172 women) 40 years of age or older, in the past 15 years. Approximately 9.3% (2,398) of the patients required further examination, surgery, or observation. Thirty-one cases of malignant tumor were found, 26 of laryngeal cancer, 2 of hypopharyngeal cancer, and 1 each of thyroid cancer, tonsillar cancer and acute lymphoid leukemia. In addition, 51 cases of vocal fold leukoplakia and approximately 700 cases of vocal fold polyp and polypoid vocal fold were diagnosed. As mentioned in previous reports, examinations of this type present both definite benefits and deficiencies. Many problems still remain. Examples of these are the small absolute numbers of examinees, the rather heavy burden placed upon the examining doctor, and the low incidence of laryngeal cancer in female patients. Laryngeal cancer is predominantly a male disease, but female patients should not be excluded from receiving screening examinations. In recent years, many thyroid tumors have been detected in female patients. In the future, laryngeal cancer examination may develop into a head and neck cancer examination that includes screening for thyroid cancer.

Adult↗
PubMed1998

When overexpressed, a novel centrosomal protein, RanBPM, causes ectopic microtubule nucleation similar to gamma-tubulin.

A novel human protein with a molecular mass of 55 kD, designated RanBPM, was isolated with the two-hybrid method using Ran as a bait. Mouse and hamster RanBPM possessed a polypeptide identical to the human one. Furthermore, Saccharomyces cerevisiae was found to have a gene, YGL227w, the COOH-terminal half of which is 30% identical to RanBPM. Anti-RanBPM antibodies revealed that RanBPM was localized within the centrosome throughout the cell cycle. Overexpression of RanBPM produced multiple spots which were colocalized with gamma-tubulin and acted as ectopic microtubule nucleation sites, resulting in a reorganization of microtubule network. RanBPM cosedimented with the centrosomal fractions by sucrose- density gradient centrifugation. The formation of microtubule asters was inhibited not only by anti- RanBPM antibodies, but also by nonhydrolyzable GTP-Ran. Indeed, RanBPM specifically interacted with GTP-Ran in two-hybrid assay. The central part of asters stained by anti-RanBPM antibodies or by the mAb to gamma-tubulin was faded by the addition of GTPgammaS-Ran, but not by the addition of anti-RanBPM anti- bodies. These results provide evidence that the Ran-binding protein, RanBPM, is involved in microtubule nucleation, thereby suggesting that Ran regulates the centrosome through RanBPM.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Dot far-western blot analysis of relative binding affinities of the Src homology 3 domains of Efs and its related proteins.

The Src homology 3 (SH3) domains are a modular structure of about 60 amino acid residues found in many proteins important in signal transduction. Each SH3 domain has a binding specificity to sequences containing a PXXP motif in ligand proteins. We found that a focal adhesion kinase (FAK)-related protein, cell adhesion kinase beta (CAKbeta), was bound in vitro by the SH3 domain of embryonal Fyn-associated substrate (Efs), a docking protein structurally related to p130Cas (Cas) and HEF1. Here, we employed a dot far-Western blotting technique to evaluate the affinity and specificity of the binding by the SH3 domains of Efs and its related proteins. The SH3 domains and their ligands were prepared as glutathione S-transferase fusion proteins, and one of the binding components was immobilized on membranes while the other was labeled with 32P to use as a probe. The amount of the bound probe was determined by autoradiography using an imaging plate and a bioimaging analyzer. A competitive binding assay showed that Efs, compared with Cas and HEF1, had a SH3 domain with a lower relative affinity to CAKbeta and FAK and with a preference to interact with FAK rather than CAKbeta. Our assay based on dot far-Western blotting is a simple and sensitive method to evaluate fine differences in the binding affinity of SH3-mediated interactions.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Interaction of two proline-rich sequences of cell adhesion kinase beta with SH3 domains of p130Cas-related proteins and a GTPase-activating protein, Graf.

Cell adhesion kinase beta (CAKbeta) is a protein tyrosine kinase closely related to focal adhesion kinase (FAK) in structure. CAKbeta contains two proline-rich sequences within its C-terminal region. Since proline-rich sequences present in the corresponding region of FAK are known to mediate protein-protein interactions by binding to SH3 domains, we investigated binding of CAKbeta to a panel of SH3 domains. Affinity precipitation from rat brain lysate revealed selective interactions of CAKbeta with glutathione S-transferase (GST)-fused SH3 domains of p130(Cas)(Cas)-related proteins and Graf. Mutational analysis indicated that the proline-rich sequences of CAKbeta mediate this interaction. Each of the two proline-rich sequences fused to GST bound directly to these SH3 domains in dot blot analysis. A competitive binding assay revealed that the first proline-rich sequence of CAKbeta preferentially associated with the SH3 domain of Cas. The second proline-rich sequence of CAKbeta bound to the SH3 domain of Graf with higher specificity than the corresponding proline-rich sequence of FAK. Finally, we showed co-immunoprecipitation of CAKbeta with Graf from rat brain lysate. These results indicate that CAKbeta associates in vivo with Graf through its SH3 domain.

Amino Acid Sequence↗
PubMed1998

Cell adhesion kinase beta forms a complex with a new member, Hic-5, of proteins localized at focal adhesions.

Cell adhesion kinase beta (CAKbeta/PYK2) is the second protein-tyrosine kinase of the focal adhesion kinase subfamily. We identified a cDNA that encodes a CAKbeta-binding protein. This cDNA clone encodes the human homologue of Hic-5, the cDNA of which was cloned in 1994 as transforming growth factor beta1- and hydrogen peroxide-inducible mRNA. We found that Hic-5 exclusively localized at focal adhesions in a rat fibroblast line, WFB. This localization of Hic-5 was confirmed in WFB cells expressing Myc-tagged Hic-5. The amino acid sequence of Hic-5 is highly similar to that of paxillin in the four LD motifs as well as in the four contiguous LIM domains. The Hic-5 N-terminal domain directly associated in vitro with the extreme C-terminal region (residue 801 to the end) of CAKbeta. CAKbeta was coimmunoprecipitated with Hic-5 from the WFB cell lysate. The coimmunoprecipitation of CAKbeta with Hic-5 was markedly inhibited by the addition of the extreme C-terminal region of CAKbeta. Coimmunoprecipitation of Hic-5 with CAKbeta, which was shown in COS-7 cells doubly transfected with cDNA constructs of CAKbeta and Myc-tagged Hic-5, was lost when the CAKbeta amino acid residues 741-903 were deleted. Hic-5 was tyrosine-phosphorylated in Src-transformed 3Y1 cells and in cells treated with pervanadate. Hic-5 associated with CAKbeta was selectively tyrosine-phosphorylated in WFB cells exposed to hypertonic osmotic stress. These results indicate that Hic-5 is a paxillin-related component of focal adhesions and binds to CAKbeta, implying possible involvement of Hic-5 in the downstream signaling of CAKbeta.

Amino Acid Sequence↗
PubMed1998

Panoramic radiographic patterns of the infraorbital canal and anterior superior dental plexus.

OBJECTIVE: To describe and classify the presentations of the infra-orbital canal/groove (IOC/G) complex and anterior superior dental plexus (ASDP) on panoramic radiographs. METHODS: The frequency of occurrence and variations in appearance of the IOC/G were determined on 246 random panoramic radiographs. A classification system consisting of Types I, II and III was developed to describe the radiographic patterns of the IOC/G. RESULTS: Two hundred radiographs (81.3%) demonstrated evidence of the IOG/C. The most frequent appearance of the IOC was Type III (44.75%) closely followed by Type I (42%) and Type II (13.25%), with only minor differences in prevalence between right and left sides. Sixty one percent were bilateral. Radiologic evidence of the ASDP was noted in 29% of radiographs demonstrating an IOG/C with 12% of cases being bilateral. Three distinct radiographic patterns of the anterior superior dental plexus (ASDP) were also observed. CONCLUSION: A classification of the appearance of the IOC/G and ASDP on panoramic radiographs has been presented.

Adolescent↗
PubMed1997

Identification of an Efs isoform that lacks the SH3 domain and chromosomal mapping of human Efs.

Efs was originally found by expression cloning of a mouse embryo cDNA library through its Fyn-SH3 binding capacity (Ishino et al., Oncogene 11, 2331-2338, 1995). Efs has characteristic regions important in intracellular signal transduction; these are an SH3 domain, a cluster of putative ligands for SH2 domains and proline-rich sequences with SH3-binding consensus. In this paper, we report cDNA cloning of human Efs and a variant of it from a hippocampal cDNA library. The human Efs gene was mapped to chromosome 14q11.2-q12 by fluorescence in situ hybridization. We identified two forms of human Efs, designated hEfs1 and hEfs2. hEfs1 represents the human counterpart of original mouse embryo Efs (mEfs1). hEfs2, the newly identified form, is identical to hEfs1, except for its lack of the SH3 domain. hEfs1 and mEfs1 are 80% identical in their amino acid sequences and 100% identical within the SH3 domain. Reverse transcription polymerase chain reaction analysis of adult mouse tissue RNA indicated expression of Efs2 and of Efs1 in various tissues. Evidence suggesting the presence of the Efs2 protein in human tissue was obtained by immunoprecipitation followed by immunoblotting with two different anti-Efs antibodies. Possible functions of Efs2 are discussed.

Adaptor Proteins, Signal Transducing↗
PubMed1997

The protein phosphatase inhibitors okadaic acid and calyculin A induce apoptosis in human osteoblastic cells.

To determine whether protein phosphorylation and dephosphorylation can affect apoptosis in osteoblastic cells, we examined the effects of okadaic acid (OA) and calyculin A (CA) on cultured human osteoblastic cells Saos-2 and MG63, and mouse osteoblastic MC3T3-E1 cells. After reaching confluence, these cells were exposed to varying concentrations of OA or CA. OA and CA induced cell death in all three cell lines in a dose- and time-dependent manner. Marked nuclear condensation and fragmentation of chromatin were also observed in these cells by using the Hoechst 33342 stain. DNA ladder formation, a hallmark of apoptosis, was detected in Saos-2 and MG63 cells, but not in MC3T3-E1 cells by treatment of OA or CA. In the Saos-2 cells, OA- and CA-induced DNA ladder formation was dose-dependent with maximal effect at concentrations of 10 and 2 nM, respectively, and was time-dependent from 14 to 48 h. DNA ladder formation in response to OA and CA was revealed by using conventional ethidium bromide staining of electrophoresed DNA without using autoradiography. Beyond the maximal effects at the respective concentrations, however, cell death did not indicate DNA laddering, suggesting that phosphatase activity may be required for ladder formation. Our results indicate that apoptosis in the cultured osteoblastic cells is induced by moderate inhibition of PP-1 or PP-2A based on the known selectivity of okadaic acid and of calyculin A.

Animals↗
PubMed1997

Advanced imaging of carcinoma of the mandibular gingiva.

OBJECTIVE: To evaluate the validity of MRI in detecting spread of mandibular gingival carcinoma within the bone marrow. SCHEME: Three patients with mandibular gingival carcinoma were examined by panoramic radiography, CT, MRI and radionuclide scanning. The resected mandibles were examined histologically for invasion of bone marrow by the tumor. RESULTS: With MRI the bone marrow in all cases had a low signal intensity on the T1-weighted images, a high signal intensity on the T2-weighted, and high signal intensity on the contrast-enhanced T1-weighted. In one case there was an obvious evidence of tumor invasion of the bone marrow histologically, but in two cases only dilatation of the blood vessels and evidence of hemorrhage associated with tumor. CONCLUSION: MRI is not specific for detecting tumor invasion of bone marrow in mandibular gingival carcinoma.

Aged↗
PubMed1996

Targeted mutagenesis of acyl-lipid desaturases in Synechocystis: evidence for the important roles of polyunsaturated membrane lipids in growth, respiration and photosynthesis.

Acyl-lipid desaturases introduce double bonds (unsaturated bonds) at specifically defined positions in fatty acids that are esterified to the glycerol backbone of membrane glycerolipids. The desA, desB and desD genes of Synechocystis sp. PCC 6803 encode acyl-lipid desaturases that introduce double bonds at the delta12, omega3 and delta6 positions of C18 fatty acids respectively. The mutation of each of these genes by insertion of an antibiotic resistance gene cartridge completely eliminated the corresponding desaturation reaction. This system allowed us to manipulate the number of unsaturated bonds in membrane glycerolipids in this organism in a step-wise manner. Comparisons of the variously mutated cells revealed that the replacement of all polyunsaturated fatty acids by a monounsaturated fatty acid suppressed growth of the cells at low temperature and, moreover, it decreased the tolerance of the cells to photoinhibition of photosynthesis at low temperature by suppressing recovery of the photosystem II protein complex from photoinhibitory damage. However, the replacement of tri- and tetraunsaturated fatty acids by a diunsaturated fatty acid did not have such effects. These findings indicate that polyunsaturated fatty acids are important in protecting the photosynthetic machinery from photoinhibition at low temperatures.

Calorimetry, Differential Scanning↗
PubMed1996

Premature chromatin condensation induced by loss of RCC1 is inhibited by GTP- and GTPgammaS-Ran, but not GDP-Ran.

RCC1 is a guanine nucleotide exchanging factor acting on nuclear G protein Ran. Premature chromatin condensation occurs in the temperature-sensitive rcc1- mutant of the BHK21 cell line, tsBN2, at the restrictive temperature. This observation can be explained if the premature activation of MPF is normally inhibited by GTP-Ran. In the absence of RCC1, GDP-Ran predominates, resulting in MPF activation. However, experiments with Ran mutants to determine whether GTP- or GDP-Ran prevents activation of MPF have yielded conflicting results. In order to clarify this point, we have microinjected nucleotide-bound Ran, instead of mutated Ran, into the nuclei of tsBN2 cells treated to reduce RCC1-mediated guanine nucleotide exchange. GTP-Ran, GTPgammaS-Ran, and GDP-Ran all inhibited chromatin condensation. However, the inhibition of chromatin condensation by GDP-Ran could be completely abolished by co-injection with GDP, but not GTP. Thus, we conclude that GTP-Ran blocks the activation of MPF and that hydrolysis of GTP is not required to prevent MPF activation.

Animals↗
PubMed1996

Inhibitory mechanisms of H(+)-ATPase inhibitor bafilomycin A1 and carbonic anhydrase II inhibitor acetazolamide on experimental bone resorption.

The effects of the vacuolar-type H(+)-ATPase inhibitor bafilomycin A1 (baf.A1) and the carbonic anhydrase II inhibitor acetazolamide (AZ) on bone resorption and procathepsin L secretion of rat osteoclasts were investigated using the bone slice assay method, pit formation test. Baf.A1 completely suppressed osteoclastic bone resorption stimulated by parathyroid hormone (PTH), but did not affect procathepsin L secretion, while AZ suppressed both bone resorption and procathepsin L secretion. These findings suggest that bone resorption by procathepsin L secretion and its processing are regulated by proton production and proton secretion.

Acetazolamide↗
PubMed1996

Evaluation of endometriosis in uterosacral ligaments by transrectal ultrasonography.

Uterosacral ligaments are one of the common targets of pelvic endometriosis, which is usually clinically rather than surgically diagnosed. This study was performed to determine if uterosacral ligaments infiltrated by endometriosis could be detected by transrectal ultrasonography. Uterosacral ligaments in non-endometriosis subjects (n = 64) were observed as low echoic homogeneous arcs in both sides of the uterine cervix. Patients who had endometriosis (n = 29) on the ligaments showed thick and irregularly-shaped uterosacral ligaments by the transrectal ultrasound examination. The results also suggested that the thickness of uterosacral ligaments was associated with the clinical symptoms. Transrectal ultrasonography may provide quantitative information to manage patients with infiltrating endometriosis.

Adult↗
PubMed1996

Conserved histidine residues of RCC1 are essential for nucleotide exchange on Ran.

Charged amino acid residues of human RCC1 were converted to alanine and mutants which were unable to complement tsBN2 cells (a temperature-sensitive rcc1- mutant of the hamster BHK21 cell line) were selected. These RCC1 mutants were analyzed for the ability to inhibit premature chromatin condensation by microinjection into tsBN2 cells, and their steady-state kinetic parameters for guanine nucleotide exchange reaction were measured. Examined RCC1 mutants were unstable in tsBN2 cells at the restrictive temperature, yet they significantly inhibited premature chromatin condensation. Mutants located on the N-terminus of the RCC1 repeat showed an increased K(m), while their kcat values were comparable to that of wild-type RCC1. In contrast, mutants containing the conserved histidine residues in the C-terminus of the RCC1 repeat showed a value of K(m) similar to that of wild-type RCC1, while the kcat values of these mutants were reduced, depending upon the RCC1 repeats on which the mutation was located. These steady-state kinetic parameters of mutants indicate that the N-terminus and the C-terminus of RCC1 repeats play different roles in guanine nucleotide exchange on Ran. The comparison of kcat among the histidine mutants suggests that those histidine residues which are conserved in the RCC1 repeats and also through evolution comprise the catalytic site for the guanine nucleotide exchange reaction.

Alanine↗
PubMed1996

Thrombin in the synovial fluid of patients with rheumatoid arthritis mediates proliferation of synovial fibroblast-like cells by induction of platelet derived growth factor.

OBJECTIVE: To investigate the clotting and fibrinolytic activities in synovial fluid (SF) from patients with rheumatoid arthritis (RA) and to examine the role of thrombin in synovial hyperplasia. METHODS: We measured the amounts of thrombin-antithrombin-III complex (TAT), antithrombin-III (AT-III), thrombin, plasminogen, alpha 2-plasmin inhibitor (alpha 2-PI), and plasmin-alpha 2-antiplasmin complex (PAP) in SF of 20 patients with RA and 16 patients with osteoarthritis (OA). The proliferative response of synovial fibroblast-like cells to thrombin was measured using [3H] thymidine incorporation. Expression of platelet derived growth factors (PDGF) in conditioned medium was analyzed using a Western blot method, and expression of the mRNA of PDGF and their receptors was analyzed by reverse transcription polymerase chain reaction. RESULTS: The amounts of clotting factors (TAT, AT-III) and fibrinolytic factors (plasminogen, alpha 2-PI, and PAP) were significantly higher in the patients with RA than in patients with OA (p < 0.01). Moreover, SF thrombin concentrations of patients with RA correlated significantly with erythrocyte sedimentation rates (rs = 0.751, p < 0.01) and serum C-reactive protein concentrations (rs = 0.531, p < 0.05). Thrombin exhibits mitogenic activity toward synovial fibroblast-like cells in vitro, and this mitogenic activity is associated with an increase in the expression of mRNA of both PDGF-alpha receptor and PDGF-beta-receptor. CONCLUSION: The high levels of thrombin activity in the SF of patients with RA and strong mitogenic activity of thrombin toward the synovial fibroblast-like cells suggest that thrombin plays an important role in the pathogenesis of RA.

Adult↗
PubMed1995

Molecular cloning of a cDNA encoding a phosphoprotein, Efs, which contains a Src homology 3 domain and associates with Fyn.

Src homology 2 and 3 (SH2 and SH3) domains mediate protein-protein interactions in intracellular signaling by protein-tyrosine kinases (PTKs). We have isolated cDNA clones from mouse embryo cDNA expression library that encode a new signaling protein which we call Efs (Embryonal Fyn-associated Substrate). The deduced amino acid sequence of 560 residues in length revealed one SH3 domain at its amino-terminal region, two proline-rich motifs with the consensus sequences of binding to Src-family SH3s, and a cluster of YXXP motifs that are possibly tyrosine-phosphorylated to serve as ligands binding to SH2 domains. Structure and alignment of these characteristics sequences are homologous to those of p130Cas, but Efs and p130Cas are different proteins. Expression of the Efs gene was higher in placenta, embryo and brain than in other adult tissues. Transfection of COS-7 cells with a plasmid encoding an epitope-tagged Efs resulted in the expression of a 83 kDa protein. The epitope-tagged Efs was hyperphosphorylated when cotransfected with a vector expressing Fyn. In an in vitro kinase assay with the PCC4 cell lysate, Efs became phosphorylated on tyrosine residues and coprecipitated with p59fyn and p62yes; the result suggests that Efs is a physiological substrate of these PTKs.

Adaptor Proteins, Signal Transducing↗