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K Kariya

Publications and source records attributed to K Kariya.

At least 55 records · Page 3Linked to original sources
PubMed1997

Effect of association with adenylyl cyclase-associated protein on the interaction of yeast adenylyl cyclase with Ras protein.

Posttranslational modification of Ras protein has been shown to be critical for interaction with its effector molecules, including Saccharomyces cerevisiae adenylyl cyclase. However, the mechanism of its action was unknown. In this study, we used a reconstituted system with purified adenylyl cyclase and Ras proteins carrying various degrees of the modification to show that the posttranslational modification, especially the farnesylation step, is responsible for 5- to 10-fold increase in Ras-dependent activation of adenylyl cyclase activity even though it has no significant effect on their binding affinity. The stimulatory effect of farnesylation is found to depend on the association of adenylyl cyclase with 70-kDa adenylyl cyclase-associated protein (CAP), which was known to be required for proper in vivo response of adenylyl cyclase to Ras protein, by comparing the levels of Ras-dependent activation of purified adenylyl cyclase with and without bound CAP. The region of CAP required for this effect is mapped to its N-terminal segment of 168 amino acid residues, which coincides with the region required for the in vivo effect. Furthermore, the stimulatory effect is successfully reconstituted by in vitro association of CAP with the purified adenylyl cyclase molecule lacking the bound CAP. These results indicate that the association of adenylyl cyclase with CAP is responsible for the stimulatory effect of posttranslational modification of Ras on its activity and that this may be the mechanism underlying its requirement for the proper in vivo cyclic AMP response.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Perforin-like immunoreactivity in four cases of lymphoma of large granular lymphocytes in the cat.

Tumors composed of discrete round cells with large eosinophilic granules in their cytoplasm were observed in four cats. These cats were presented with severe gastrointestinal signs, including anorexia, vomiting, and diarrhea. The exploratory laparotomy in two cats and necropsy in the other two revealed thickening and perforation of the intestinal walls, enlargement of the mesenteric, ileocolic, and/or colic lymph nodes, and splenomegaly. Histologically, there was severe and disseminated infiltration of the tumor cells in the affected organs. In the two cats necropsied, the spleen and the jejunal mucosa appeared to be the primary organs involved. The tumor cells of all the cats showed the pore-forming protein (perforin)-like immunoreactivity unique to the cytotoxic lymphocytes but failed to show histamine immunoreactivity. These results indicate that the tumor cells were derived from large granular lymphocytes and that perforinlike immunoreactivity may be a useful marker for histologic diagnosis of feline large granular lymphoma.

Animals↗
PubMed1997

[Hematological characteristics of hypoplastic MDS patients with chromosomal abnormalities, who had been followed for aplastic anemia with normal karyotypes].

During 1990 to 1993, five patients who had been treated in our hospital for aplastic anemia (AA) with a normal karyotype, were found to have hypoplastic myelodysplastic syndromes (hMDS); three patients with RA, one RAEB, and one RAEB-T, and all of them have chromosomal abnormalities. The mean age of the hMDS patients (4 men, 1 woman) was 51.0 years (range 31-61). We retrospectively studied the hematological features of the 5 cases in the early stage without chromosomal abnormalities. They showed, 1) appearance of erythroblasts (3/5), 2) more reticulocyte counts than typical AA (mean 7.8 x 10(4)/microliter), 3) macrocytic anemia (mean MCV 106fl) in peripheral blood, 4) relative erythroid hyperplasia (M/E ratio 0.2-1.5), and 5) low lymphocyte counts (mean percentage of lymphocytes 32, 8%) in bone marrow. Considering about the differences or transformation between AA and hMDS, it is interesting that all five patients have initially had a normal karyotype and subsequently developed chromosomal abnormalities.

Adult↗
PubMed1996

Involvement of histone hyperacetylation in triggering DNA fragmentation of rat thymocytes undergoing apoptosis.

The treatment of rat thymocytes with trichostatin A and sodium butyrate, which are inhibitors of histone deacetylase, resulted in an increase in DNA fragmentation in a concentration-dependent manner. A significant increase in DNA fragmentation induced by these compounds was observed after a lag time of 2 h. Analysis of the fragmented DNA revealed the production of approximately 50 kb DNA fragments and DNA ladders, the biochemical hallmarks of apoptotic cell death. Judging from a laser scanning microscopic analysis, the inhibitors of histone deacetylase induced nuclear condensation, the morphological feature of apoptosis. Biochemical and morphological analyses demonstrated that trichostatin A and sodium butyrate induced thymocyte apoptosis. Furthermore, hyperacetylation of nuclear histones was observed in thymocytes treated with the inhibitors of histone deacetylase. These effects of sodium butyrate and trichostatin A were seen 0.5 and 1 h, respectively, after incubation of the cells. These results thus indicate that hyperacetylation of nucleosomal histones precedes DNA fragmentation in thymocytes undergoing apoptosis induced by trichostatin A and sodium butyrate.

Acetylation↗
PubMed1996

Difference in the mechanism of interaction of Raf-1 and B-Raf with H-Ras.

Ras is known to possess multiple cellular targets including Raf-1. Here, we measured both direct binding of various H-Ras mutants to two representative mammalian Ras targets, Raf-1 and B-Raf, and the activity of the mutants to stimulate Raf-1 and B-Raf, and analysed the difference in their Ras-interaction mechanisms. B-Raf was shown to share almost the same H-Ras binding-specificity with Raf-1 by examining binding of the H-Ras mutants to Raf-1 and B-Raf in the yeast two-hybrid and in vitro binding assays. Mutants, Y32F, A59E, and V45E bound to Raf-1 in Sf9 cells coexpressing them, but failed to activate Raf-1. On the other hand, Y32F activated B-Raf in a cell-free system which consisted of rat brain cytosol and recombinant MEK. These results suggest that there is a subtle structural difference in requirements for the interaction of Ras with Raf-1 and B-Raf.

Amino Acid Sequence↗
PubMed1996

Differential structural requirements for interaction of Ras protein with its distinct downstream effectors.

Ras proteins have multiple effectors of distinct structures that do not share significant structural homology at their Ras interaction sites. To prove possible differences in their recognition mechanisms of Ras, we screened 44 human Ha-Ras proteins carrying mutations in the effector region and its flanking sequences for interaction with human Raf-1, Schizosaccharomyces pombe Byr2, and Saccharomyces cerevisiae adenylyl cyclase. The Ras binding specificities were largely shared between Raf-1 and Byr2 although Ras mutants, Y32F, T35S, and A59E, had their affinities for Byr2 selectively reduced. The only exception was Ras(D38N), which lost the ability to bind Raf-1 while retaining the activity to bind Byr2 and complement the Byr2- phenotype of S. pombe. On the other hand, adenylyl cyclase had quite distinct requirements for Ras residues; mutations P34G and T58A selectively abolished the ability to bind and activate it without considerably affecting the interaction with Raf-1 and Byr2. Y32F mutant, whereas losing the ability to activate Raf-1 and Byr2, could activate adenylyl cyclase efficiently. In addition, V45E mutation was found to impair the ability of Ras to activate both Raf-1 and adenylyl cyclase without significantly affecting the binding affinities for them. These results demonstrate that significant differences exist in the recognition mechanisms by which the three effector molecules associate with Ras and suggest that a region of Ras required for activation of the effectors in general may exist separately from that for binding the effectors.

Adenylyl Cyclases↗
PubMed1996

Post-translational modification of H-Ras is required for activation of, but not for association with, B-Raf.

B-Raf is regulated by Ras protein and acts as a mitogen-activated protein (MAP) kinase kinase kinase in PC12 cells and brain. Ras protein undergoes a series of post-translational modifications on its C-terminal CAAX motif, and the modifications are critical for its function. To elucidate the role of the post-translational modifications in interaction with, and activation of, B-Raf, we have analyzed a direct association between H-Ras and B-Raf, and constructed an in vitro system for B-Raf activation by H-Ras. By using methods based on inhibition of yeast adenylyl cyclase or RasGAP activity and by in vitro binding assays, we have shown that the segment of B-Raf corresponding to amino acid 1-326 binds directly to H-Ras with a dissociation constant (Kd) comparable to that of Raf-1 and that the binding is not significantly affected by the post-translational modifications. However, when the activity of B-Raf to stimulate MAP kinase was measured by using a cell-free system derived from rat brain cytosol, we observed that the unmodified form of H-Ras possesses an almost negligible activity to activate B-Raf in vitro compared to the fully modified form. H-RasSer-181,184 mutant, which was farnesylated but not palmitoylated, was equally active as the fully modified form. These results indicate that the post-translational modifications, especially farnesylation, are required for H-Ras to activate B-Raf even though they have no apparent effect on the binding properties of H-Ras to B-Raf.

Adenylyl Cyclases↗
PubMed1996

A novel DNA cleaving agent, 2,2'-bis(2-aminoethyl)-4,4'-bithiazole, induces thymocyte apoptosis.

The incubation of rat immature thymocytes with 2,2-bis(2-aminoethyl)-4,4'-bithiazole, which possesses the cleaving activity toward plasmid DNA in the presence of Co2+, resulted in an increase in DNA fragmentation. No DNA fragmentation of the cells was induced by 2,2-bis(3-aminopropyl)-4,4'-bithiazole, which has little cleaving activity toward plasmid DNA. Analysis of fragmented DNA from thymocytes treated with 2,2-bis(2-aminoethyl)-4,4'-bithiazole by agarose gel electrophoresis revealed that the compound produced 50 kbp DNA fragments and caused internucleosomal DNA fragmentation. In addition, the confocal microscopic images of thymocytes treated with the compound showed condensation of chromatin. These results indicate that bithiazole derivative induces thymocyte apoptosis with the biochemical and morphological characteristics through triggering DNA scission.

Animals↗
PubMed1995

Cysteine-rich region of Raf-1 interacts with activator domain of post-translationally modified Ha-Ras.

The interaction between "switch I/effector domain" of Ha-Ras and the Ras-binding domain (RBD, amino acid 51-131) of Raf-1 is essential for signal transduction. However, the importance of the "activator domain" (approximately corresponding to amino acids 26-28 and 40-49) of Ha-Ras and of the "cysteine-rich region" (CRR, amino acids 152-184) of Raf-1 have also been proposed. Here, we found that Raf-1 CRR interacts directly with Ha-Ras independently of RBD and that participation of CRR is necessary for efficient Ras-Raf binding. Furthermore, Ha-Ras carrying mutations (N26G and V45E) in the activator domain failed to bind CRR, whereas they bound RBD normally. On the contrary, Ha-Ras carrying mutations in the switch I/effector domain exhibited severely reduced ability to bind RBD, whereas their ability to bind CRR was unaffected. Mutants that bound to either RBD or CRR alone failed to activate Raf-1. Ha-Ras without post-translational modifications, which lacks the ability to activate Raf-1, selectively lost the ability to bind CRR. These results suggest that the activator domain of Ha-Ras participates in activation of Raf-1 through interaction with CRR and that post-translational modifications of Ha-Ras are required for this interaction.

Animals↗
PubMed1995

Protein kinase Byr2 is a target of Ras1 in the fission yeast Schizosaccharomyces pombe.

Conservation of the structure and function of Ras proteins has been observed in a variety of eukaryotic organisms. However, the nature of their downstream effectors appears to be quite divergent; adenylyl cyclase and a protein kinase Raf-1, which do not share any structural homology with each other, are effectors of Ras in the budding yeast and in higher organisms, respectively. We show here that a protein kinase Byr2, which has been known to act downstream of Ras1 in a mating pheromone signal transduction system of Schizosaccharomyces pombe, binds directly to Ras proteins in a GTP-dependent manner. The region of Byr2 responsible for the Ras binding was mapped by a gene deletion analysis to its N-terminal segment of 206 amino acid residues, which does not possess any significant homology with the other effectors of Ras. The affinity of the Byr2 N terminus for Saccharomyces cerevisiae Ras2 was determined by measuring its activity to competitively inhibit Ras-dependent adenylyl cyclase activity and found to be comparable with those of yeast adenylyl cyclase and human Raf-1, with a dissociation constant (Kd) of about 1 nM. Furthermore, Byr2 inhibited a Ras GTPase-activating activity of Ira2, a S. cerevisiae homologue of neurofibromin. These results indicate that Byr2 is an immediate downstream target of Ras1 in S. pombe.

Adenylyl Cyclases↗
PubMed1995

M-CAT, CArG, and Sp1 elements are required for alpha 1-adrenergic induction of the skeletal alpha-actin promoter during cardiac myocyte hypertrophy. Transcriptional enhancer factor-1 and protein kinase C as conserved transducers of the fetal program in cardiac growth.

Induction of the fetal isogenes skeletal alpha-actin (skACT) and beta-myosin heavy chain (beta-MHC) is characteristic of cardiac growth in many models, suggesting a conserved signaling pathway. However, divergent regulation has also been observed. beta-Protein kinase C (PKC) and transcriptional enhancer factor-1 (TEF-1) are involved in induction of beta-MHC in alpha 1-adrenergic-stimulated hypertrophy of cultured cardiac myocytes (Kariya, K., Farrance, I.K. G., and Simpson, P.C. (1993) J. Biol. Chem. 268, 26658-26662; Kariya, K., Karns, L. R., and Simpson, P.C. (1994) J. Biol. Chem. 269, 3775-3782). In the present study, we asked whether the skACT promoter used the same mechanism. A mouse skACT promoter fragment (-113/-46) was induced by both alpha 1-adrenergic stimulation and co-transfection of activated beta-PKC, and contained three required DNA sequence elements: M-CAT, CArG, and Sp1. The skACT M-CAT element bound TEF-1 in cardiac myocytes. Thus the skACT and beta-MHC promoters both require a TEF-1 binding site for activation by alpha 1-adrenergic stimulation, but differ in that skACT also requires a CArG box. These results provide a potential molecular basis for divergent regulation of the fetal program, and also imply that PKC and TEF-1 are conserved transducers for this program during cardiac growth.

Actins↗
PubMed1995

Superoxide dismutase (SOD) activity with Fe-chlorin e6-Na and suppression of malignant tumor growth in rats.

Derivatives of porphyrin, specifically Fe-chlorin e6-Na (FeCNa), mimic superoxide dismutase (SOD). This SOD activity was determined by decrease in electron spin resonance (ESR) signals and increase in hydrogen peroxide (H2O2) as the intermediate of O2-. by the coloration using 4-aminoantipyrin. Chlorin e6-Na used for cancer photodynamic therapy (PDT)(1) does not show SOD mimicking activity. The specific activity of FeCNa, comparing with bovine RBC-SOD, was 1/7.5 as determined by ESR analysis. The iron element of Fe-chlorin e6-Na, being tightly encased in the molecule, did not participate in the Fenton reaction. The SOD mimetic activity of FeCNa was stable against physico-chemical treatment such as pH shock, heat and digestion by pronase. For cancer bearing rats with oxidative stress (OS), immediate relief of OS was possible by a single intraperitoneal injection of FeCNa and relief continued for 24 hours. The subsequent administration of FeCNa suppressed cancer growth in vivo.

Animals↗
PubMed1995

Dystrophic form of inherited epidermolysis bullosa in a dog (Akita Inu).

We report a dog with dystrophic epidermolysis bullosa. This 4-year-old female Akita Inu, a species of Canis familiaris var. japonicus Temminck, had a 3-year-history of ulcers and scars over the pressure areas on the limbs, and dystrophic nails, since the age of 1 year, which corresponds to early adulthood in humans. Electron microscopy of a blister revealed separation beneath the lamina densa, and a reduction in the number of anchoring fibrils. The NC-1 domain of type VII collagen was positively stained with monoclonal antibody LH7.2 at the basement membrane zone. These findings indicate that humans and dogs have a similar response to antibody LH7.2, which may aid the development of an animal model for this disease.

Animals↗
PubMed1995

Transport of nattokinase across the rat intestinal tract.

Intraduodenal administration of nattokinase (NK) at a dose of 80 mg/kg, resulted in the degradation of fibrinogen in plasma suggesting transport of NK across the intestinal tract in normal rats. The action of NK on the cleavage of fibrinogen in the plasma from blood samples drawn at intervals after intraduodenal administration of the enzyme was investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting analysis with an anti-fibrinogen gamma chain antibody. The 270 kDa fragment carrying antigenic sites for the binding of the anti-fibrinogen gamma chain antibody appeared within 0.5 h and was then degraded gradually to a 105 kDa fragment via a 200 kDa fragment. This suggests that fibrinogen was degraded to a 105 kDa fragment via several intermediates (270 and 200 kDa). In parallel with the degradation process, plasma recalcification times were remarkably prolonged NK was also detected in the plasma from blood samples drawn 3 and 5 h after administration of the enzyme by SDS-PAGE and Western blotting analysis with an anti-NK antibody. The results indicate that NK is absorbed from the rat intestinal tract and that NK cleaves fibrinogen in plasma after intraduodenal administration of the enzyme.

Animals↗
PubMed1995

Thrombolytic effect of nattokinase on a chemically induced thrombosis model in rat.

Nattokinase is a new fibrinolytic enzyme which cleaves directly cross-linked fibrin in vitro. In this study, we investigated the thrombolytic effect of nattokinase on a thrombus in the common carotid artery of rat in which the endothelial cells of the vessel wall were injured by acetic acid. When a section of occluded vessel was stained for CD61 antigen by immunofluorescence utilizing a monoclonal antibody, the antigen was localized around the surface of the occluded blood vessels. This result suggests that the occlusive thrombosis was caused by platelet aggregation. In addition, thrombolysis with urokinase (UK; 50000 IU/kg, i.v.) or tissue plasminogen activator (tPA; 13300 IU/kg, i.v.) in our model was observed to restore the blood flow over a 60 min monitoring period. The results indicate that our chemically induced model is useful for screening and evaluating a thrombolytic agent. We evaluated the thrombolytic activity of nattokinase using this model and compared it with fibrino(geno)lytic enzyme, plasmin or elastase. On a molar basis, the recovery of the arterial blood flow with nattokinase, plasmin and elastase were 62.0 +/- 5.3%, 15.8 +/- 0.7% and 0%, respectively. The results indicate that the thrombolytic activity of nattokinase is stronger than that of plasmin or elastase in vivo.

Animals↗
PubMed1994

CCK activates excitatory ventral tegmental pathways to the posterior nucleus accumbens.

In urethane-anaesthetized male rats, electrical stimulation of the ventral tegmental area (VTA) produced either excitation (n = 26) or inhibition (n = 5) in the activity of neurones in the posterior nucleus accumbens (PNAc). Local administration of sulfatedcholecystokinin octapeptide (CCK8, 0.03 microgram) into the stimulation site enhanced the neuronal excitability in 10 out of 21 PNAc neurones that demonstrated the excitation to VTA stimulation, but did not affect the remaining 11 neurones. Similar administration, on the other hand, was without effect in any of PNAc neurones (n = 5) that displayed the inhibition to VTA stimulation. These results suggest that CCK8 may act at the VTA to elicit an activation of the excitatory pathways from the VTA to the PNAc.

Animals↗
PubMed1994

Systemic administration of CCK-8S, but not CCK-4, enhances dopamine turnover in the posterior nucleus accumbens: a microdialysis study in freely moving rats.

The present study was carried out to examine the effects of peripheral administration of sulfatedcholecystokinin octapeptide (CCK-8S) on dopamine (DA) turnover in the posterior nucleus accumbens (PNAc) and the caudate-putamen (CP) in awake rats. Microdialysis was used to quantify the extracellular concentrations of DA and its two metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA). Intraperitoneal injections of CCK-8S (0.3 mg/kg b.wt.) caused a significant increase in DOPAC and HVA concentrations in the PNAc, but did not affect the DA level. Such increases in the metabolite contents were not found in the CP. Similar injections of vehicle (1% NaHCO3 solution, 1 ml/kg b.wt.) did not have an effect in either brain region. In an attempt to determine the type of receptor involved in the CCK-8S-induced changes, CCK tetrapeptide (CCK-4, 0.3 mg/kg b.wt.) known to have high affinity for CCKB subtype or vehicle (10% DMSO-saline, 1 ml/kg b.wt.) was administered intraperitoneally. Neither CCK-4 nor vehicle caused significant changes in any of extracellular DA, DOPAC and HVA contents in the PNAc. These results suggest that peripherally administered CCK-8S has stimulatory effects on the dopaminergic system in the PNAc, and raise the possibility that the effect appears to be mediated via CCKA receptors.

3,4-Dihydroxyphenylacetic Acid↗