Biomedical subjects

S Ohno

Publications and source records attributed to S Ohno.

At least 343 records · Page 19Linked to original sources

PubMed1997

Interaction of Nck-associated protein 1 with activated GTP-binding protein Rac.

Bacterially expressed glutathione S-transferase fusion proteins containing Rac1 were used to identify binding proteins of this Rho family GTPase present in a bovine brain extract. Five proteins of 85, 110, 125, 140 and 170 kDa were detected, all of which were associated exclusively with guanosine 5'-[gamma-thio]triphosphate-bound Rac1, not with GDP-bound Rac1. The 85 and 110 kDa proteins were identified as the regulatory and catalytic subunits respectively of phosphatidylinositol 3-kinase. Several lines of evidence suggested that the 125 kDa protein is identical with Nck-associated protein 1 (Nap1). The mobilities of the 125 kDa protein and Nap1 on SDS/PAGE were indistinguishable, and the 125 kDa protein was depleted from brain extract by preincubation with the Src homology 3 domain of Nck to which Nap1 binds. Furthermore, antibodies to Nap1 reacted with the 125 kDa protein. Nap1 was co-immunoprecipitated with a constitutively active form of Rac expressed in Chinese hamster ovary cells. The observation that complex formation between activated Rac and PAK, but not that between Rac and Nap1, could be reproduced in vitro with recombinant proteins indicates that the interaction of Nap1 with Rac is indirect. The 140 kDa Rac-binding protein is a potential candidate for a link that connects Nap1 to Rac. The multimolecular complex comprising Rac, Nap1 and probably the 140 kDa protein might mediate some of the biological effects transmitted by the multipotent GTPase.

Animals↗

PubMed1997

A protein kinase Cdelta-binding protein SRBC whose expression is induced by serum starvation.

West-Western screening of a cDNA expression library using 32P-labeled, autophosphorylated protein kinase Cdelta (PKCdelta) as a probe, led us to identify cDNA clones encoding a PKCdelta-binding protein that contains a leucine zipper-like motif in its N-terminal region and two PEST sequences in its C-terminal region. This protein shows overall sequence similarity (43.3%) to the serum deprivation response (sdr) gene product, and we named it SRBC (sdr-related gene product that binds to c-kinase). PKCdelta binds to the C-terminal half of SRBC through the regulatory domain and phosphorylates it in vitro. In COS1 cells, the phosphorylation of over-expressed SRBC is stimulated by 12-O-tetradecanoylphorbol-13-acetate and further enhanced by the over-expression of PKCdelta. The mRNA for SRBC is detected in a wide variety of cultured cell lines and tissues and is strongly induced by serum starvation. Furthermore, SRBC mRNA is induced during retinoic acid-induced differentiation of P19 cells. These results suggest that SRBC serves as a substrate and/or receptor for PKC and might be involved in the control of cell growth mediated by PKC.

3T3 Cells↗

PubMed1997

A domain containing the Cdc42/Rac interactive binding (CRIB) region of p65PAK inhibits transcriptional activation and cell transformation mediated by the Ras-Rac pathway.

The molecular bases of the versatile functions of Rho-like GTPases are still unknown. Using luciferase assays with rat 3Y1 cells, we found that Rac1 is integrated downstream of Ras in the TRE (TPA response element) activation pathway. Coexpression of a mutant of p65PAK, PAK/RD, lacking the kinase domain but containing the Cdc42/Rac interactive binding (CRIB) region, suppressed the TRE activation and cell transformation caused by constitutively activated forms of Ras (RasV12) and Rac1 (Rac1V12). PAK/RD is a good tool to investigate the signaling pathways in which Rac and Cdc42 are involved.

Amino Acid Sequence↗

PubMed1997

Conformation-dependent activation of type II adenylyl cyclase by protein kinase C.

Phorbol ester treatment enhanced the catalytic activity of type II adenylyl cyclase overexpressed in insect cells. In cells coexpressing type II adenylyl cyclase and protein kinase C-alpha, type II adenylyl cyclase catalytic activity was higher even in the absence of phorbol ester treatment; phorbol ester treatment further and markedly enhanced type II adenylyl cyclase catalytic activity. However, this enhancement, either by phorbol ester treatment or by coexpression of protein kinase C-alpha, was lost following membrane solubilization with detergents. This attenuation was unaffected by phosphatase inhibitor or salts. In contrast, membrane solubilization did not affect forskolin-stimulated type II adenylyl cyclase catalytic activity. Purified type II adenylyl cyclase was stimulated by forskolin and Gs alpha, but not by protein kinase C-alpha. Therefore, a specific mammalian protein kinase C isoenzyme can activate type II adenylyl cyclase, but the mechanism clearly differs from that underlying either Gs alpha- or forskolin-mediated stimulation.

Adenylyl Cyclases↗

PubMed1997

Characterization of a cartilage-derived 66-kDa protein (RGD-CAP/beta ig-h3) that binds to collagen.

A 66-kDa collagen fiber-associated protein (RGD-CAP) was isolated from a fiber-rich fraction of pig cartilage by ultrafiltration and collagen-affinity chromatography. Amino acid sequencing and cDNA cloning indicated that the RGD-CAP is identical or closely related to beta ig-h3 protein which is induced in human adenocarcinoma cells by transforming growth factor-beta (TGF-beta) (Skonier, J., Neubauer, M., Madisen, L., Bennett, K., Plowman, G.D., and Purchio, A.F. (1992) DNA Cell. Biol. 11, 511-522). The RGD-CAP, as well as beta ig-h3, has the RGD sequence in the C-terminal region. The native RGD-CAP bound to type I, II, and IV collagens even in the presence of 1 M NaCl. A recombinant preparation of RGD-CAP expressed in Escherichia coli cells also bound to collagen but not to gelatin. The RGD-CAP mRNA was expressed in chondrocytes throughout all stages, although the expression level was highest during the prehypertrophic stage. In addition, TGF-beta increased the RGD-CAP mRNA level in chondrocyte cultures. Since RGD-CAP transcripts were found in most tissues, this novel collagen-binding protein may play an important role in cell-collagen interactions in various tissues including developing cartilage.

Amino Acid Sequence↗

PubMed1997

Triplet repeat polymorphism in the transmembrane region of the MICA gene: a strong association of six GCT repetitions with Behçet disease.

A member of a novel family of the human major histocompatibility complex (MHC) class I genes termed MIC (MHC class I chain-related genes), MICA, has been recently identified near the HLA-B gene on the short arm of human chromosome 6. The predicted amino acid sequence of the MICA chain suggests that it folds similarly to typical class I chains and may have the capacity to bind peptides or other short ligands. Therefore, MICA is predicted to have a specialized function in antigen presentation or T cell recognition. During nucleotide sequence analyses of the MICA genomic clone, we found a triplet repeat microsatellite polymorphism of (GCT/AGC)n in the transmembrane (TM) region of the MICA gene. In 68 HLA homozygous B cell lines, 5 distinct alleles of this microsatellite sequence were detected. One of them contained an additional one base insertion that created a frameshift mutation resulting in a premature termination codon in the TM region. This particular allele may encode a soluble, secreted form of the MICA molecule. In addition, we have investigated this microsatellite polymorphism in 77 Japanese patients with Behcet disease, which is known to be associated with HLA-B51. The microsatellite allele consisting of 6 repetitions of GCT/AGC was present at significantly higher frequency in the patient group (Pc = 0.00055) than in a control population. Furthermore, the (GCT/AGC)6 allele was present in all B51 positive patients and in an additional 13 B51 negative patients. These results suggest the possibility of a primary association of Behcet disease with MICA rather than HLA-B.

Asian People↗

PubMed1997

Direct binding of CRKL to BCR-ABL is not required for BCR-ABL transformation.

CRKL has previously been shown to be a major tyrosine phosphorylated protein in neutrophils of patients with BCR-ABL+ chronic myelogenous leukemia and in cell lines expressing BCR-ABL CRKL and BCR-ABL form a complex as demonstrated by coimmunoprecipitation and are capable of a direct interaction in a yeast two-hybrid assay. We have mapped the site of interaction of CRKL and BCR-ABL to the amino terminal SH3 domain of CRKL with a proline rich region in the C-terminus of ABL. The proline-rich region was mutated and the effect of this deletion on BCR-ABL transforming function was assayed. Our data show that this deletion does not impair the ability of BCR-ABL to render myeloid cells factor independent for growth. In cells expressing the proline deletion mutation of BCR-ABL, CRKL is still tyrosine phosphorylated and forms a complex with BCR-ABL as demonstrated by coimmunoprecipitation. Our data suggest that the interaction between CRKL and the proline deletion mutant of BCR-ABL is an indirect interaction as CRKL does not interact directly with the proline deletion mutant of BCR-ABL in a gel overlay assay or in a yeast two-hybrid assay. Thus, a direct interaction of CRKL and BCR-ABL is not required for CRKL to become tyrosine phosphorylated by BCR-ABL and suggests that CRKL function may still be required for BCR-ABL function through an indirect interaction.

Adaptor Proteins, Signal Transducing↗

PubMed1997

Prevention of adenocarcinoma colon 26-induced cachexia by interleukin 10 gene transfer.

A s.c. injection of a mouse colon adenocarcinoma cell line, colon 26 clone 20, induced cachexia, as evidenced by progressive weight loss and severe hypoglycemia. Several lines of evidence indicate that a pro-inflammatory cytokine, interleukin 6 (IL-6), plays a major role, albeit partially, in the establishment of cachexia in this model. Because IL-10 can potentially inhibit the production of pro-inflammatory cytokines including IL-6, we evaluated the effects of IL-10 gene transfer on the establishment of cachexia. IL-6 transcript was detected at tumor sites of mice inoculated with parental or control vector transfectant cells, and serum IL-6 levels were markedly increased in these mice. The injection of parental cells into IL-6-deficient mice induced cachexia with elevated serum IL-6 levels comparable to wild-type mice, indicating that tumor cells are a major source of IL-6. The inoculation of IL-10-transfectant cells kept IL-10 mRNA expression at tumor sites and induced the elevation in serum IL-10 levels without affecting the growth rates of colon 26 cells both in vitro and in vivo. However, the implantation with IL-10-transfectant cells reduced the expression of IL-6 mRNA at the tumor sites and the elevation in serum IL-6 levels. Concomitantly, mice inoculated with IL-10-transfectant cells did not exhibit progressive weight loss, a reduction in food intake, or severe hypoglycemia, which was observed in mice inoculated with parental or control vector-transfectant cells. Collectively, these results suggest that IL-10 gene transfer prevented the occurrence of cachexia with a concomitant inhibition of IL-6 production at the tumor sites.

Animals↗

PubMed1997

Polymorphonuclear leucocyte (PMN) inhibitory factor prevents PMN-dependent endothelial cell injury by an anti-adhesive mechanism.

Neutrophil inhibitory factor (NIF), a 41-kD glycoprotein isolated from the canine hookworm, inhibits CD11b/CD18-dependent neutrophil adhesion by binding to CD1lb. We studied the effects of NIF on neutrophil-dependent endothelial cell injury using bovine pulmonary microvessel endothelial cells grown on microporous filters. Endothelial injury was determined as an increase in the transendothelial 125I-albumin clearance rate (a measure of transendothelial permeability). Layering of neutrophils on the endothelial cell monolayer (ratio of 10 neutrophils: 1 endothelial cell) followed by activation of neutrophils with 500 nM of phorbol 12-myristate 13-acetate (PMA) increased transendothelial permeability of albumin by 3- to 4-fold over control monolayers. Pretreatment of neutrophils with NIF at concentrations of 100 nM and above prevented the increased permeability. Pretreatment of neutrophils with the anti-CD18 monoclonal antibody (mAb) IB4 similarly prevented the increase of permeability. Pretreatment of neutrophils with OKM-1, a control isotype-matched mAb directed against an irrelevant epitope on CD11b mAb, did not affect the neutrophil-dependent increase in permeability. NIF reduced the adhesion of neutrophils at concentrations of > or = 100 nM and this effect was abolished by an anti-NIF polyclonal Ab. However, NIF did not prevent the generation of superoxide anions following PMA-induced activation of neutrophils layered on endothelial cell. These findings indicate that NIF inhibits the neutrophil-dependent endothelial injury by preventing CD11b/CD18-mediated neutrophil adhesion, but without altering the oxidant generating capacity of neutrophils interacting with the endothelial cell monolayer.

Albumins↗

PubMed1997

Suppression of actively induced experimental autoimmune uveoretinitis by CD4+ T cells.

BACKGROUND: Helper/inducer T cells that exert an inhibitory effect on disease induction have been recently found in many experimental models. In order to clarify the mechanisms of spontaneous remission of experimental autoimmune uveoretinitis (EAU), we investigated the inhibitory effect and the phenotype of the post-recovery suppressor cells. METHODS: In a series of experiments, we separated spleen cells of rats that had recovered from EAU. Three groups of spleen cells, CD4+ T, CD8+ T and B cells, were each adoptively transferred into naive syngeneic rats before active immunization with retinal soluble antigen (S-Ag) and Freund's complete adjuvant or passive immunization with uveitogenic T cells from donor rats. Inflammation was examined clinically and histologically. RESULTS: The development of EAU could be significantly prevented by adoptive transfer of CD4+ T cells, whereas CD8+ T cells could not suppress the onset. However, post-recovery CD4+ T cells failed to inhibit EAU induced by passive immunization with uveitogenic T cells. CONCLUSION: These findings indicate that CD4+ post-recovery (suppressor) T cells may play an important role in the remission of EAU.

Adoptive Transfer↗

PubMed1997

Improved surgical results after combining preoperative hyperthermia with chemotherapy and radiotherapy for patients with carcinoma of the rectum.

PURPOSE: The aim of this study is to evaluate long-term results of preoperative hyperthermia combined with chemotherapy and irradiation (HCR therapy) in patients with carcinoma of the rectum. METHODS: Postoperative prognoses were compared among 36 patients with carcinoma of the rectum, who were given preoperative HCR therapy followed by surgery, and 52 patients undergoing surgery alone without any preoperative therapy. RESULTS: There were significant differences in the prognosis between patients given preoperative HCR therapy plus surgery and those having surgery alone, and five-year survival rates were 91.3 and 64 percent, respectively. Particularly, for patients with tumors invading beyond the muscularis propria and/or with positive lymph node metastasis, a significantly longer survival was obtained with HCR plus surgery than in surgery alone (86.5 vs. 50.9 percent and 92.9 vs. 51.7 percent, respectively). However, no significant differences were observed in the postoperative prognosis for cases with no lymph node metastasis and/or with tumors limited to the muscularis propria between these two groups. CONCLUSIONS: These data clearly demonstrated the effectiveness of preoperative HCR therapy for improving long-term results of patients with carcinoma of the rectum, especially those demonstrating an advanced stage of disease.

Aged↗

PubMed1997

The reason for as well as the consequence of the Cambrian explosion in animal evolution.

The first 1 billion years of our 4.5-billion-year-old planet were extremely violent, characterized by constant meteorite bombardment. Therefore, it is with a great surprise that we note that cellular life flourished 3.5 billion years ago. It appears that the cellular life came into being as soon as the earth's environment became hospitable. Because the main ingredient of the Archean sea was sodium bicarbonate, neither archeobacteria nor eubacteria but rather photosynthesizing organisms dominated-initially, prokaryotic cyanobacteria, soon joined by eukaryotic blue-green algae. These consumers of carbon dioxide were also releasers of molecular oxygen. The toil of 3 billion years by these releasers of molecular oxygen finally triggered the Cambrian animal explosion. With exceptions of two animal phyla, Porifera and Coelenterata, which amde slightly earlier appearances, nearly all other extant animal phyla sprang into almost simultaneous existence within 6 to 10 million years. The notion of the Cambrian pananimalia genome was advanced to explain various evolutionary consequences of this Cambrian explosion.

Animal Population Groups↗

PubMed1997

Magnetic resonance imaging with fluid-attenuated inversion recovery pulse sequences in MELAS syndrome.

We report the brain magnetic resonance imaging findings in the case of a 5-year-old boy with mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS). Fluid-attenuation inversion recovery (FLAIR) imaging revealed multifocal abnormal cortical signals which were difficult to see on conventional T1- and T2-weighted images. Although the patient was in an advanced clinical stage of disease and had severe cortical atrophy, FLAIR imaging demonstrated characteristic findings of MELAS.

Atrophy↗

PubMed1997

Allelic variants of the human MHC class I chain-related B gene (MICB).

The human major histocompatibility complex (MHC) is located within a 4 megabase segment on chromosome 6p21.3. Recently, a highly divergent MHC class I chain-related gene family, MIC was identified within the class I region. The MICA and MICB genes in this family have unique patterns of tissue expression. The MICA gene is highly polymorphic, with more than 20 alleles identified to date. To elucidate the extent of MICB allelic variations, we sequenced exons 2 (alpha 1), 3 (alpha 2), 4 (alpha 3), and 5 (transmembrane) as well as introns 2 and 4 of this gene in 46 HLA homozygous B-cell lines. We report the identification of eleven alleles based on seven non-synonymous, two synonymous, and four intronic nucleotide variations. Interestingly, one allele has a nonsense mutation resulting in a premature termination codon in the alpha 2 domain. Thus, MICB appears to have fewer alleles than MICA, not unlike the allelic ratio between the HLA-C and -B loci. A preliminary linkage analysis of the MICB alleles with those of the closely located MICA and HLA-B genes revealed no conspicuous linkage disequilibrium between them, implying the presence of a potential recombination hotspot between the MICB and MICA genes.

Alleles↗

PubMed1997

Dystrophin in rod spherules; submembranous dense regions facing bipolar cell processes.

It is known that the retina contains the protein dystrophin in the ribbon synapse, but the ultrastructural analysis is not yet fully elucidated. Our previous study reported that dystrophin is localized under the rod cell membranes in rat retinas. In the present study, we have investigated the relationship between dystrophin-rich regions of rod cell membranes and other neuronal processes in mouse retinas with a monoclonal antibody raised against the human dystrophin C-terminus. Immunoblotting, immunofluorescence stainings, and immunoelectron microscopy were employed. Immunoblotting analysis indicated that mouse retinas possessed some of the dystrophin isoforms of approximately 260 kDa, 140 kDa, and 70 kDa molecular weight. Confocal images showed a punctate appearance in the outer plexiform layer, as previously described. Immunoelectron microscopy showed that dystrophin immunoreactive products were always observed at submembranous dense regions of the rod spherule abutting bipolar processes. These results suggest that retinal dystrophin may be closely involved in signal transmission from rods to bipolar cells.

Animals↗

PubMed1997

Colour Doppler sonography of breast masses: a multiparameter analysis.

OBJECTIVE: To evaluate signals from breast lesions on both colour and spectral Doppler US, and to correlate findings with angiographic and histopathological features including microvessel density. MATERIALS AND METHODS: One hundred and sixteen breast lesions in 113 patients were evaluated with colour Doppler ultrasonography. Subjective and semi-quantitative assessment of colour signals as well as spectral Doppler analysis were performed and compared. Comparison of colour Doppler features with angiographic and histopathological findings were also carried out. RESULTS: Subjective evaluation revealed that colour signals were more commonly found in malignant (89%) than benign (56%) lesions. Malignant lesions demonstrated a more elaborate vascular network (51%) compared to 12.5% in benign lesions. Spectral Doppler analysis revealed slightly higher specificity values for the pulsatility and resistivity indices, respectively, but lower sensitivity and accuracy values compared to qualitative assessment. Colour Doppler patterns corresponded well with angiograhic images, however, the correlation between colour Doppler parameters and microvessel density was not significant. CONCLUSION: Despite the increased examination time required, spectral analysis does not appear to contribute significantly to the differentiation between malignant and benign breast tumours. Features such as the density, pattern and predominant site of colour signals may be more useful for the evaluation of breast lesions on colour Doppler imaging.

Adolescent↗

PubMed1997

Rapid diagnosis of adenoviral conjunctivitis on conjunctival swabs by 10-minute immunochromatography.

PURPOSE: Several methods are available for the diagnosis of acute conjunctivitis, all of which are time-consuming or require the use of a well-equipped laboratory. A new method, immunochromatography (IC), for detecting the presence of adenovirus (Ad) has been developed. Two direct rapid tests to detect Ad antigen, IC and enzyme immunoassay (EIA), were compared with regard to sensitivity, specificity, and technical complexity. METHODS: The study materials consisted of 130 swabs from patients with conjunctivitis (95 samples of adenoviral conjunctivitis proven by positive virus DNA on polymerase chain reaction [PCR], 35 samples of nonadenoviral conjunctivitis proven by PCR). IC is a one-step procedure that detects the presence of adenoviral antigen by sandwich EIA on a paper disc. RESULTS: In 95 adenoviral DNA-positive samples by PCR, the sensitivity and specificity of IC were 54.7% and 97.1%, respectively, whereas those of EIA were 50.5% and 100%, respectively. By IC, PCR-positive Ad type 3 was recognized in 31%; Ad4 in 100%; Ad7 in 60%; Ad8 in 67%; and Ad37 in 59%, showing similar positivity rates for different serotypes (except Ad7) to those using EIA. Visual determination of the presence of Ad took an average of 10 minutes by IC compared with 70 minutes by EIA. CONCLUSIONS: These results indicate that IC is a more rapid and easier test compared with EIA, and it has high specificity. Detection of Ad antigen by this simple and rapid method will serve physicians as a useful tool for early diagnosis and prevention of adenoviral conjunctivitis.

Adenoviridae↗

PubMed1997

A histochemical study of anionic sites in the intermediate layer of rat femoral cartilage using polyethyleneimine at different pH levels.

Anionic sites in the intermediate layer of young rat hyaline cartilages were examined using a cationic dye, polyethyleneimine (PEI), at different pH levels. Femoral heads were resected and fixed in 2.5% glutaraldehyde and treated with 0.5% PEI at pH 7.4, pH 2.5 or pH 1.0. Some cartilage samples were first digested with chondroitinase ABC or hyaluronidase. The PEI deposits at pH 7.4 appeared to be irregular shapes. Their sizes seemed to be larger than those at pH 2.5 or pH 1.0. The PEI deposits were also found on the surface of collagen fibrils at both pH 7.4 and pH 2.5 even after the chondroitinase ABC digestion, but were not found at pH 1.0. Moreover, they disappeared after hyaluronidase digestion. Accordingly, it is suggested that PEI-positive structures varied depending on pH levels. In addition, hyaluronan may be localized near collagen fibrils, but most sulphated proteoglycans may not.

Animals↗