Search PubMed⌕ Search

Biomedical subjects

S Ohno

Publications and source records attributed to S Ohno.

At least 271 records · Page 15Linked to original sources
PubMed1998

Atypical protein kinase Clambda binds and regulates p70 S6 kinase.

p70 S6 kinase (p70 S6K) has been implicated in the regulation of cell cycle progression. However, the mechanism of its activation is not fully understood. In the present work, evidence is provided that an atypical protein kinase C (PKC) isotype, PKClambda, is indispensable, but not sufficient, for the activation of p70 S6K. Both the regulatory and kinase domains of PKClambda associate directly with p70 S6K. Overexpression of the kinase domain without kinase activity or the regulatory domain of PKClambda results in the suppression of the serum-induced activation of p70 S6K. In addition, two types of dominant-negative mutants of PKClambda, as well as a kinase-deficient mutant of p70 S6K, suppress serum-induced DNA synthesis and E2F activation. The overexpresion of the active form of PKClambda, however, fails to activate p70 S6K. These results suggest that PKClambda is a mediator in the regulation of p70 S6K activity and plays an important role in cell cycle progression.

Amino Acid Sequence↗
PubMed1998

An atypical PKC directly associates and colocalizes at the epithelial tight junction with ASIP, a mammalian homologue of Caenorhabditis elegans polarity protein PAR-3.

Cell polarity is fundamental to differentiation and function of most cells. Studies in mammalian epithelial cells have revealed that the establishment and maintenance of cell polarity depends upon cell adhesion, signaling networks, the cytoskeleton, and protein transport. Atypical protein kinase C (PKC) isotypes PKCzeta and PKClambda have been implicated in signaling through lipid metabolites including phosphatidylinositol 3-phosphates, but their physiological role remains elusive. In the present study we report the identification of a protein, ASIP (atypical PKC isotype-specific interacting protein), that binds to aPKCs, and show that it colocalizes with PKClambda to the cell junctional complex in cultured epithelial MDCKII cells and rat intestinal epithelia. In addition, immunoelectron microscopy revealed that ASIP localizes to tight junctions in intestinal epithelial cells. Furthermore, ASIP shows significant sequence similarity to Caenorhabditis elegans PAR-3. PAR-3 protein is localized to the anterior periphery of the one-cell embryo, and is required for the establishment of cell polarity in early embryos. ASIP and PAR-3 share three PDZ domains, and can both bind to aPKCs. Taken together, our results suggest a role for a protein complex containing ASIP and aPKC in the establishment and/or maintenance of epithelial cell polarity. The evolutionary conservation of the protein complex and its asymmetric distribution in polarized cells from worm embryo to mammalian-differentiated cells may mean that the complex functions generally in the organization of cellular asymmetry.

3T3 Cells↗
PubMed1998

Mutation of tyrosine-194 and lysine-198 in the catalytic site of pig 3alpha/beta,20beta-hydroxysteroid dehydrogenase.

Pig 3alpha/beta,20beta-hydroxysteroid dehydrogenase is an NADPH-dependent enzyme that catalyses the reduction of ketones on steroids and aldehydes and ketones on various xenobiotics, like its homologue carbonyl reductase. 3alpha/beta,20beta-Hydroxysteroid dehydrogenase and carbonyl reductase are members of the short-chain dehydrogenases/reductase family, in which a tyrosine residue and a lysine residue have been identified as catalytically important. In pig 20beta-hydroxysteroid dehydrogenase these residues are tyrosine-194 and lysine-198. Here we report the effect on the reduction of two ketone and two aldehyde substrates by pig 3alpha/beta,20beta-hydroxysteroid dehydrogenase in which tyrosine-194 has been mutated to phenylalanine and cysteine, and lysine-198 has been mutated to isoleucine and arginine. Mutants with phenylalanine-194 or isoleucine-198 are inactive. Depending on the substrate, the mutant with cysteine-194 has a catalytic efficiency of 0.4-1% and the mutant with arginine-198 has a catalytic efficiency of 4-23% of the wild-type enzyme. We also mutated tyrosine-81 and tyrosine-253 to phenylalanine. Although both tyrosines are conserved in 3alpha/beta,20beta-hydroxysteroid dehydrogenase and carbonyl reductase, depending on the substrate, the mutant enzymes are as active as, or more active than, wild-type enzyme.

20-Hydroxysteroid Dehydrogenases↗
PubMed1998

Tumor necrosis factor signaling to stress-activated protein kinase (SAPK)/Jun NH2-terminal kinase (JNK) and p38. Germinal center kinase couples TRAF2 to mitogen-activated protein kinase/ERK kinase kinase 1 and SAPK while receptor interacting protein associates with a mitogen-activated protein kinase kinase kinase upstream of MKK6 and p38.

Tumor necrosis factor (TNF) elicits a diverse array of inflammatory responses through engagement of its type-1 receptor (TNFR1). Many of these responses require de novo gene expression mediated by the activator protein-1 (AP-1) transcription factor. We investigated the mechanism by which TNFR1 recruits the stress-activated protein kinases (SAPKs) and the p38s, two mitogen-activated protein kinase (MAPK) families that together regulate AP-1. We show that the human SPS1 homologue germinal center kinase (GCK) can interact in vivo with the TNFR1 signal transducer TNFR-associated factor-2 (TRAF2) and with MAPK/ERK kinase kinase 1 (MEKK1), a MAPK kinase kinase (MAPKKK) upstream of the SAPKs, thereby coupling TRAF2 to the SAPKs. Receptor interacting protein (RIP) is a second TNFR signal transducer which can bind TRAF2. We show that RIP activates both p38 and SAPK; and that TRAF2 activation of p38 requires RIP. We also demonstrate that the RIP noncatalytic intermediate domain associates in vivo with an endogenous MAPKKK that can activate the p38 pathway in vitro. Thus, TRAF2 initiates SAPK and p38 activation by binding two proximal protein kinases: GCK and RIP. GCK and RIP, in turn, signal by binding MAPKKKs upstream of the SAPKs and p38s.

Binding Sites↗
PubMed1998

Spatiotemporal measurement of free radical elimination in the abdomen using an in vivo ESR-CT imaging system.

Electron spin resonance (ESR) imaging can visualize the distribution of free radicals in living systems according to their concentrations. However, the application of ESR imaging to living animals has not been well established. Using a rapid field scan L-band ESR imaging system, we have successfully obtained two-dimensional ESR projection (xz-plane projection) and three-dimensional ESR-CT (trans-axial section along the y-axis) images of the abdomen of living mice after an injection of 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (carbamoyl-PROXYL) into the tail vein. The in vivo two-dimensional ESR projection imaging clearly visualized the carbamoyl-PROXYL distribution and the rapid decay process in the abdomen. Because among the viscera, the liver is most abundantly associated with a blood volume, the outline of the image can be composed mainly of this organ. We therefore attempted to find whether there will be a difference in spatiotemporal dynamics of carbamoyl-PROXYL in the abdomens between the control and the mice with liver damage by two-dimensional ESR projection. In the control mice, carbamoyl-PROXYL was almost completely eliminated from the abdomen within 5 minutes after administration. On the other hand, in mice with carbon tetrachloride-damaged livers, the decay of carbamoyl-PROXYL was markedly prolonged. Even at 5 min after administration, carbamoyl-PROXYL remained clearly visible in the abdomen. In vivo three-dimensional ESR-CT imaging showed an even distribution of carbamoyl-PROXYL throughout the whole liver, which corresponded well with the images of trans-axial sections of the murine abdomen. We have succeeded in displaying two-dimensional ESR projection and three-dimensional ESR-CT images of carbamoyl-PROXYL distribution and clearance in the abdomen of a living animal. The ESR-CT imaging technique is considered to be a powerful new tool for noninvasive investigations of the in vivo spatiotemporal dynamics of free radical distribution and elimination in the organs.

Abdomen↗
PubMed1998

Long-term survival of patients with stage IV gastric carcinoma.

BACKGROUND: Survival of patients with Stage IV (based on general rules established by the Japanese Research Society for Gastric Cancer) gastric carcinoma often is unfavorable. Among patients with a poor prognosis, a few do survive > 5 years. The authors examined pathologic and biologic features of tumors of long term survivors. METHODS: The authors analyzed data from 442 patients with Stage IV gastric carcinoma, including 20 surviving for > 5 years after gastrectomy (Group A) and 422 who died of gastric carcinoma within 5 years (Group B). Mutant p53 was immunohistochemically stained using the monoclonal antibody PAb1801. Proliferative activity was estimated by argyrophilic nuclear organizer region (AgNOR) staining and proliferating cell nuclear antigen (PCNA) staining. RESULTS: Group A had smaller and more localized tumors than Group B (P < 0.05 and P < 0.01, respectively). Lymphatic or venous invasion and peritoneal dissemination were less frequent in Group A than in Group B (P < 0.01). Abnormalities of p53 expression were found in 3 of the 14 tumors in Group A (21%), a value significantly lower than the 58 of 118 tumors in Group B (49%; P < 0.05). AgNOR count and percentage of PCNA labeling were not significantly different between Groups A and B. A multivariate analysis showed that lymph node dissection, liver metastasis, gastric resection, venous invasion, and tumor size were independent prognostic factors. CONCLUSIONS: Even in patients with Stage IV gastric carcinoma, radical gastrectomy and extensive lymph node dissection can lead to long term survival. The authors believe that combination analysis of pathologic features and p53 overexpression predict length of survival for patients with Stage IV gastric carcinoma.

Adult↗
PubMed1998

Unexpected homology between inducible cell wall protein QID74 of filamentous fungi and BR3 salivary protein of the insect Chironomus.

A gene, qid74, of mycoparasitic filamentous fungus Trichoderma harzianum and its allies encodes a cell wall protein that is induced by replacing glucose in the culture medium with chitin (simulated mycoparasitism conditions). Because no trace of this gene can be detected in related species such as Gibberella fujikuroi and Saccharomyces cerevisiae, the qid74 gene appears to have arisen de novo within the genus Trichoderma. Qid74 protein, 687 residues long, is now seen as highly conserved tandem repeats of the 59-residue-long unit. This unit itself, however, may have arisen as tandem repeats of the shorter 13-residue-long basic unit. Within the genus Trichoderma, the amino acid sequence of Qid74 proteins has been conserved in toto. The most striking is the fact that Qid74 shares 25.3% sequence identity with the carboxyl-terminal half of the 1,572-residue-long BR3 protein of the dipteran insect Chironomus tentans. BR3 protein is secreted by the salivary gland of each aquatic larva of Chironomus to form a tube to house itself. Furthermore, the consensus sequence derived from these 59-residue-long repeating units resembles those of epidermal growth factor-like domains found in divergent invertebrate and vertebrate proteins as to the positions of critical cysteine residues and homology of residues surrounding these cysteines.

Amino Acid Sequence↗
PubMed1998

Three-dimensional ultrastructure of apoptotic nuclei in rat prostatic epithelial cells revealed by a quick-freezing and deep-etching method.

BACKGROUND: To clarify intranuclear apoptotic changes, we have investigated chromatin organization in apoptotic nuclei of castrated rat prostatic cells by a quick-freezing and deep-etching (QF-DE) method. METHODS: The ventral prostates taken from intact and castrated adult male rats were investigated by light microscopy, in situ end-labeling (ISEL) technique, conventional electron microscopy, and the QF-DE method. RESULTS: In control nuclei, the chromatin fibers were uniformly distributed and formed a network structure. In apoptotic nuclei, destruction of such chromatin networks was detected, which was clearly seen by the QF-DE method. Although it first appeared spotty in the apoptotic nucleus, definite destruction of the intranuclear network occurred in the nuclear center at later stages, and broken fibrous structures were condensed along the nuclear margin. The ISEL technique was applied to the QF-DE method. Localization of damaged DNA fragments could three-dimensionally be detected on replica membranes. CONCLUSIONS: Intranuclear chromatin organization in apoptotic cell death of rat prostates was observed by the QF-DE method. We could examine early-stage apoptotic nuclei at an electron microscopic level, which would not be clarified by other conventional methods.

Animals↗
PubMed1998

gp130 is involved in stretch-induced MAP kinase activation in cardiac myocytes.

We have recently reported that mitogen activated protein kinase (MAP kinase) is activated by the stretch of the cultured cardiac myocytes in the angiotensin II deficient state in the angiotensinogen-deficient mice (Atg-/-), suggesting that factors other than the cardiac renin-angiotensin system are involved in the stretch-induced MAP kinase activation. We examined the contribution of cytokines using RX435, an anti-gp130 antibody. Leukemia inhibitory factor, which is one of the cytokines and has the common receptor subunit gp130, activated MAP kinase and the response was completely blocked by pretreatment of the Atg-/- cardiac myocytes with RX435. RX435 pretreatment greatly reduced stretch-induced activation of MAP kinase in Atg-/- cardiac myocytes. Interestingly, the same results were obtained in the cardiac myocytes of control mice. These results suggest that cytokine-gp130 may play a role in the stretch-induced MAP kinase activation independently of Ang II in cardiac myocytes.

Angiotensinogen↗
PubMed1998

Differential activation of two JNK activators, MKK7 and SEK1, by MKN28-derived nonreceptor serine/threonine kinase/mixed lineage kinase 2.

MKN28-derived nonreceptor type of serine/threonine kinase/mixed lineage kinase 2 (MST/MLK2) directly phosphorylates and activates SEK1/MKK4/JNKK1/SKK1 in vitro, thereby acting as a mitogen-activated protein (MAP) kinase kinase kinase in the JNK/SAPK pathway (Hirai, S. -i., Katoh, M., Terada, M., Kyriakis, J. M., Zon, L. I., Rana, A., Avruch, J., and Ohno, S. (1997) J. Biol. Chem. 272, 15167-15173). The in vitro reconstitution system for the kinase cascade allowed us now to identify JNK/SAPK activators involved in the MST/MLK2-dependent activation of JNK/SAPK in vivo. We show that at least two distinct MST/MLK2-dependent JNK/SAPK activators are present in the fractionated COS-1 cell lysate, and that they appear to be SEK1/MKK4/JNKK1/SKK1 and MKK7/JNKK2/SKK4 by Western blot analysis. Notably, a majority of the MST/MLK2-dependent JNK/SAPK-activating activity is found in MKK7-containing fractions, whereas the MEKK1-dependent activity is comparably distributed in SEK1- and MKK7-containing fractions. Moreover, MST/MLK2 activates recombinant MKK7 more effectively than recombinant SEK1, whereas MEKK1 activates both to a similar extent. In addition, the deletion analysis on MST/MLK2 showed that the kinase domain is responsible for the determination of substrate specificity. These results provide a molecular aspect to the differential regulation of the two JNK activators by a variety of cellular stimuli.

Animals↗
PubMed1998

MKBP, a novel member of the small heat shock protein family, binds and activates the myotonic dystrophy protein kinase.

Muscle cells are frequently subjected to severe conditions caused by heat, oxidative, and mechanical stresses. The small heat shock proteins (sHSPs) such as alphaB-crystallin and HSP27, which are highly expressed in muscle cells, have been suggested to play roles in maintaining myofibrillar integrity against such stresses. Here, we identified a novel member of the sHSP family that associates specifically with myotonic dystrophy protein kinase (DMPK). This DMPK-binding protein, MKBP, shows a unique nature compared with other known sHSPs: (a) In muscle cytosol, MKBP exists as an oligomeric complex separate from the complex formed by alphaB-crystallin and HSP27. (b) The expression of MKBP is not induced by heat shock, although it shows the characteristic early response of redistribution to the insoluble fraction like other sHSPs. Immunohistochemical analysis of skeletal muscle cells shows that MKBP localizes to the cross sections of individual myofibrils at the Z-membrane as well as the neuromuscular junction, where DMPK has been suggested to be concentrated. In vitro, MKBP enhances the kinase activity of DMPK and protects it from heat-induced inactivation. These results suggest that MKBP constitutes a novel stress-responsive system independent of other known sHSPs in muscle cells and that DMPK may be involved in this system by being activated by MKBP. Importantly, since the amount of MKBP protein, but not that of other sHSP family member proteins, is selectively upregulated in skeletal muscle from DM patients, an interaction between DMPK and MKBP may be involved in the pathogenesis of DM.

Amino Acid Sequence↗
PubMed1998

Recent advances in preoperative hyperthermochemoradiotherapy for patients with esophageal cancer.

BACKGROUND AND OBJECTIVES: Hyperthermochemoradiotherapy (HCR) has been performed on numerous patients with esophageal cancer. The purpose of this study is to demonstrate the recent advances in HCR. METHODS: From 1965 to 1997, 294 patients given preoperative chemoradiotherapy (CR) or HCR were classified according to the anticancer agent that was administered (CR; group A given bleomycin (BLM); group B given cis-diamminedichloroplatinum (II) (CDDP), HCR; group C given BLM; and Group D given CDDP). The local response and the long-term results were investigated. RESULTS: The cases in which CR or HCR was evaluated to be effective numbered 44 (48.4%) in group A, 22 (73.3%) in group B, 79 (63.7%) in group C, and 36 (73.5%) in group D. A significant difference was observed between groups A and B (P < 0.05). The highest incidence of markedly effective cases was observed in group D. The 5-year survival rates for the group A and B patients were 17.2% and 43.9%, respectively (P < 0.01), while the same rates for those of groups C and D were 25.6% and 57.8%, respectively (P < 0.05). Our results thus showed CDDP to have a greater effect than BLM, while HCR had a greater effect than CR. CONCLUSIONS: Preoperative HCR has improved thanks to recent advances in anticancer agents.

Aged↗
PubMed1998

HSP-derived peptides inducing uveitis and IgG and IgA antibodies.

Heat shock protein (HSP) 65 kD-derived peptides, which specifically stimulate T cells from patients with Behçet's disease (BD), are capable of inducing uveitis in rats. Mycobacterial HSP 65 kD and BD-specific peptides were injected into Lewis rats and the development of uveitis was monitored clinically and histologically, and IgG and IgA antibodies were measured using an enzyme-linked immunosorbent assay (ELISA). Rats immunized with HSP peptides that developed uveitis showed significantly higher serum IgG antibody levels to peptide 311-326 (P < 0.05) and the corresponding homologous human peptide 336-351 (P < 0.01) than rats without uveitis. Significant increases in serum IgA antibodies in rats with uveitis were also observed in those immunized with peptides 111-125, 311-326 and 336-351 (P < 0.05). Rats injected with HSP 65 kD showed a rise in IgG antibody levels to peptides 111-125, 154-172 and 311-326 and to a lesser extent, a rise in IgA antibody level to peptide 311-326. HSP showed almost complete inhibition of binding of IgG antibodies to HSP 65 kD, but peptides 111-125, 154-172, 311-326 and 336-351 showed inhibition to a lesser extent in a competitive assay. These results suggest that increases in IgG and IgA antibody levels to specific peptides within HSP, develop in rats with uveitis. The T and B cell epitopes responsible for the development of ocular disease in rats immunized with HSP-derived peptides, appear to be similar or identical to those found in patients with the ocular type of BD.

Animals↗
PubMed1998

Seroprevalence of anti-Borrelia antibodies among patients with confirmed sarcoidosis in a region of Japan where Lyme borreliosis is endemic.

BACKGROUND: Sarcoidosis is a multisystemic granulomatous disease of unknown etiology, while Lyme borreliosis is a multisystemic disorder caused by Borrelia burgdorferi. The purpose of this study is to evaluate the relationship between sarcoidosis and Lyme borreliosis in a region of Japan where Lyme borreliosis is endemic. METHODS: We determined the seroprevalence of anti-Borrelia burgdorferi antibodies as well as antibodies three Japanese Borrelia strains by enzyme-linked immunosorbent assay and dotblot assay using purified Borrelia-specific proteins in 46 patients with confirmed sarcoidosis and 150 controls (50 disease controls and 100 healthy controls) in Hokkaido, the affected region. RESULTS: Fifteen patients with sarcoidosis (32.6%) tested positive for Borrelia spirochete in both assays, compared with two disease controls (4.0%) and two healthy controls (2.0%). The seroprevalence of anti-Borrelia antibodies in patients with sarcoidosis was much higher in the affected region than in the region in our previous study were Lyme borreliosis is non-endemic. CONCLUSION: In a region where Lyme borreliosis is endemic, Borrelia infection may be partially associated with sarcoidosis.

Antibodies, Bacterial↗
PubMed1998

Localization and development of chromogranin A and luteinizing hormone immunoreactivities in the secretory-specific cells of the hypophyseal pars tuberalis of the chicken.

The pars tuberalis mainly consists of the secretory cells specific to this portion of the pituitary. We examined the localization and development of luteinizing hormone (LH) and chromogranin A in the chicken pars tuberalis by immunohistochemistry. The vast majority of the chicken pars tuberalis was occupied by cells immunoreactive for both LH and chromogranin A. Furthermore, immunoblot analysis of chicken pars tuberalis extracts with LH antiserum demonstrated that two bands, the large alpha-subunit and small beta-subunit of the LH molecule, were expressed in this tissue as well as in the pars distalis. A band for chromogranin A was also detected in pars tuberalis extracts with chromogranin A antiserum. In contrast to the cells of mammalian species that contain only a few small secretory granules, the specific cells of the chicken pars tuberalis were characterized by the presence of many secretory granules ranging from 90 to 400 nm in diameter. Postembedding immunogold labeling showed that gold particles representing immunoreactivity for LH were densely located on all secretory granules of the secretory-specific cells. Many secretory granules, especially the large ones, of the cells were also loaded with immunogold particles for chromogranin A. Double immunogold labeling confirmed that LH and chromogranin A were colocalized on the same secretory granules. During embryonic development, the primordium of the pars tuberalis was first detected at 8 days of incubation as a small group of cells containing LH- and chromogranin-immunoreactive cells. In the pars distalis, the onset of LH and chromogranin expression occurred earlier, at 6 days of incubation. At 10 days of incubation, the pars tuberalis primordium became large cell masses consisting of LH- and chromogranin-immunoreactive cells, which were located close to the median eminence. Subsequently, the primordium extended along the median eminence progressively with age. At 14 days of incubation, it reached to the rostral end and surrounded the median eminence as slender cell cords. These results indicate that specific cells of the chicken pars tuberalis synthesize a glycoprotein hormone related to the LH molecule, which is stored in the secretory granules together with chromogranin A. The pars tuberalis may be involved in the regulation of gonadal function in a different way from that of the pars distalis.

Animals↗
PubMed1998

Immunocytochemical study of lysosomal proteins with a new monoclonal antibody directed against epithelioid macrophages.

A monoclonal antibody, EPI-1, was produced against macrophages in epithelioid granulomas induced in rat foot pads by muramyl dipeptide. This EPI-1 antibody reacted to lysosome-like structures in epithelioid macrophages, peritoneal and pulmonary macrophages, and also in other tissues such as liver, testes, and kidneys. Western blot analysis of epithelioid granulomas, liver, testes, and kidneys revealed the same positive band of 62 kDa. Immunoelectron microscopic study of foot pad granulomas and hepatocytes demonstrated the EPI-1 antigen located in lysosomes and autophagic vesicles, preferentially along their membranes. These findings suggest that the EPI-1 antibody may recognize a novel antigen related to lysosomal membrane proteins in macrophages and other cells, which is useful for identifying lysosomes and their related structures.

Animals↗