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Biomedical subjects

Y Gotoh

Publications and source records attributed to Y Gotoh.

At least 109 records · Page 6Linked to original sources
PubMed1995

Activation of protein kinase cascades by osmotic shock.

Osmotic shock induces a variety of biochemical and physiological responses in vertebrate cells. By analyzing extracts obtained from rat 3Y1 fibroblastic cells exposed to hyper-osmolar media, we have found that mitogen-activated protein kinases (MAPKs) and stress-activated protein kinases (SAPKs, also known as JNKs) are both activated in response to osmotic shock. MAPKK1 (MEK1) was also activated markedly. Furthermore, Raf-1 and MEKK were activated strikingly by the osmotic shock. Activation of Raf-1 and MEKK in response to osmotic shock was detected also in PC12 cells, in which MEKK activation by the osmotic shock was much stronger than that by epidermal growth factor. Activation of SAPKs in PC12 cells by the osmotic shock was also more marked than that by epidermal growth factor. The activated MEKK phosphorylated not only MAPKKs but also XMEK2, which is distantly related to MAPKK. Recombinant wild-type XMEK2, but not kinase-negative XMEK2, was able to phosphorylate and activate recombinant SAPK alpha in vitro. In addition, this activity of XMEK2 was activated by the activated MEKK. These results suggest that the MAPK cascade consisting of Raf-1, MAPKK, and MAPK and the SAPK cascade consisting of MEKK, XMEK2, and SAPK are both activated in response to osmotic shock. Finally, it was found that XMEK2 is a good substrate for SAPK.

Animals↗
PubMed1995

Attachment and growth of fibroblast cells on silk fibroin.

The attachment and growth of fibroblast cells (L-929) on matrices of silk fibroin from Bombyx mori domestic silkworm (DSF) and Antheraea pernyi wild silkworm (WSF) were studied by a cell culture method. The performance of the two kinds of silk fibroin was compared to that of collagen. DSF exhibited as high a cell attachment and growth as collagen did. The cells attached to DSF were extensively spread out and their filopodia were visible in the SEM pictures. WSF, which contains the tripeptide sequence Arg-Gly-Asp (believed to be a specific interaction site for cell-attachment), displayed much higher cell attachment and growth compared to DSF. The cells attached on WSF became virtually flat and their filopodia could be seen, indicating that the cells were very strongly held on the surface.

Amino Acid Sequence↗
PubMed1995

Characterization of the major non-collagenous proteins of chicken bone: identification of a novel 60 kDa non-collagenous phosphoprotein.

Chicken bone powder, sequentially extracted with 4M guanidine HCl (NG), 0.3M HCl (H), 0.5 NaCl at neutral pH (NS), and 4M guanidine HCl pH 7.4 (G), preferentially solubilized osteocalcin (OC), osteopontin (OPN), alpha 2 HS-glycoprotein, bone sialoprotein (BSP), and osteonectin (ON). These studies provide the first amino acid sequences for a non-mammalian form of ON and both N-terminal and internal amino acid sequences obtained for ON demonstrated that the avian form of this protein is more than 80% conserved for all the peptides sequenced in this study. A novel approximately 60 kDa protein was identified with a unique N-terminal amino acid sequence and several internal amino acid sequences. Amino acid composition of this protein indicates that it is similar to other acidic glycosylated phosphoprotein of bone, and it is phosphorylated principally on serine residues, although a small amount of phosphorylated threonine residues were also detected. Solubility characteristics of this protein differed from those of ON, OPN and Decorin, and, it was not recognized by polyclonal antibodies to ON, BSP, OPN or by a monoclonal antibody to Decorin. This protein was not present in chicken serum and no comparable sequences were found in the GenBank or EMBL protein sequence database. These studies provide the first sequence for a non-mammalian form of ON and identify a novel acidic bone protein.

Amino Acid Sequence↗
PubMed1995

Histaminergic control of mucosal repair in the small intestine.

The aim of the present paper was to summarize histamine-mediated repair of rat intestinal mucosa. To evaluate intestinal repair, we examined lipid transport (an index of intestinal mucosal function) after 15 minutes occlusion of the superior mesenteric artery. Rats were pretreated with alpha-fluoromethylhistidine (a suicide inhibitor of histidine decarboxylase, a synthesizing enzyme of histamine), H1-receptor antagonist (chlorpheniramine maleate), H2-antagonist (cimetidine), or H3-antagonist (thioperamide) before ischemia-reperfusion (I/R). Lipid transport to rat mesenteric lymph decreased significantly 24 hours after I/R in all groups tested compared to sham-treated rats. Lipid transport was restored 48 hours after I/R in the vehicle-pretreated control group. Lipid transport was not restored to the control level 48 hours after I/R in rats pretreated with H1-antagonist and a suicide inhibitor of histidine decarboxylase. In contrast, intestinal function was restored to the control level 48 hours after I/R in rats pretreated with H2- and H3-antagonists. These results support our previous findings that newly formed histamine after I/R plays an important role in mucosal recovery through H1-receptors.

Animals↗
PubMed1995

Attachment and growth of cultured fibroblast cells on silk protein matrices.

The attachment and growth of L-929 cells on films made of Bombyx mori silk proteins--fibroin and sericin and their mixtures--was studied by a cell culture method. Both cell attachment and growth were dependent on a minimum of around 90% sericin in the mixture. The results from electron micrography as well as from the DSC measurements supported the notion that the mixture of the two proteins fibroin and sericin has a phase-separated structure in the solid state. The observed minimum of sericin in the cell attachment and growth is thought to be a result of this phase-separated structure. Films of pure component proteins (i.e., 100% fibroin or sericin) exhibited as high a cell attachment and growth as collagen, a widely used mammalian cell culture substrate. However, a morphological study of the attached cells revealed that the cells attached to silk fibroin were extended and had a spindle shape, just like the cells attached to collagen, while the cells attached to the silk sericin had a different shape. It is concluded, therefore, that the attachment condition on silk fibroin is ideal for the viability, growth and function of the cells.

Amino Acids↗
PubMed1995

Characterization of an avian bone sialoprotein (BSP) cDNA: comparisons to mammalian BSP and identification of conserved structural domains.

Bone sialoprotein (BSP) is one of the major noncollagenous proteins found in mineralized vertebrate tissue. It is an acidic glycoprotein containing a high sialic acid content and is phosphorylated on several of its Ser and Thr residues. While it has been extensively characterized from various mammalian species, little is known about its sequence or expression in lower vertebrates. The cloning and characterization of several cDNAs encoding the chicken bone sialoprotein are reported here. A partial cDNA clone encoding the carboxyl terminus of the protein was initially isolated from a lambda GT11 expression library using a polyclonal antibody gains BSP purified from chicken bone matrix. Subsequently, several additional clones were obtained by further screening and by reverse transcription polymerase chain reaction (RT-PCR). Three overlapping clones encompassing about 1 kb, which included the complete coding sequence for BSP, were analyzed. The deduced amino acid sequence revealed that chicken BSP contains 276 amino acid residues. Although the overall identity between chicken and mammalian BSP is only approximately 39%, the diversity in amino acid sequence occurs mostly between the major functional domains of this molecule. These domains include: (1) three acidic poly-Glu regions; (2) two tyrosine-rich domains, which may be sites for protein sulfation; (3) several casein kinase II phosphorylation sites; (4) an Asn glycosylation site; and (5) an RGD cell-binding motif. Of interest in the chicken BSP is the identification of two additional RGD motifs within the avian sequence, unlike the mammalian forms of BSP which has only one.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Activation mechanism and function of the MAP kinase cascade.

MAP kinase (MAPK) and its activator, MAP kinase kinase (MAPKK), are commonly activated by a variety of extracellular stimuli in mammalian cells and in the process of Xenopus oocyte maturation. In order to investigate the function of the MAPK cascade in oocyte maturation, we produced an anti-Xenopus MAPKK which specifically reacts with MAPKK in vitro. When this antibody was microinjected into immature oocytes, MAPK activation induced by progesterone was prevented. Surprisingly, H1 kinase activation and germinal vesicle breakdown were also inhibited in the oocytes injected with this antibody. These results suggest that the MAPK cascade plays an important role in the maturation promoting factor (MPF) activation during the oocyte maturation process. When this antibody together with Mos was microinjected into Xenopus two-cell embryos, the Mos-induced metaphase arrest (CSF arrest) was prevented. Thus, the MAPK cascade may mediate CSF arrest. During Xenopus early embryogenesis, a low but significant level of MAPK remains active. Injection of mRNA encoding a constitutively active MAPKK resulted in mesoderm induction in animal cap explants. In addition, fibroblast growth-factor (FGF)-induced mesoderm induction was inhibited by expressing CL100 (a MAP kinase phosphatase) in animal cap explants. Thus the MAPK cascade may be involved in the mesoderm induction of Xenopus embryos. The activation pathways and roles of the MAPKK/MAPK cascade in various signaling processes will be discussed.

Amino Acid Sequence↗
PubMed1995

The MAP kinase cascade: its role in Xenopus oocytes, eggs and embryos.

Mitogen-activated protein kinase (MAPK) was originally identified as a serine/threonine kinase that is activated by mitogens. Now MAPK and its activator, MAPK kinase (MAPKK), are thought to function in a wide variety of intracellular signalling pathways from yeast to vertebrate. We describe here a brief summary of the dissection of the MAPK cascade and its possible functions, especially in Xenopus oocytes and embryos.

Animals↗
PubMed1995

Osteogenesis by human osteoblastic cells in diffusion chamber in vivo.

Osteogenic potential of osteoblastic cells isolated from human bone was evaluated by a diffusion chamber method. Cells placed in diffusion chambers were implanted intraperitoneally into the athymic mice. The diffusion chambers cultured in vivo were harvested and examined after implantation for 6-8 weeks. The content of the chamber was proved by a soft roentogenogram to contain the radioopaque area. Light microscopic study revealed that this area was made up of bone-like nodule. Within the diffusion chambers, the cell layers were observed alongside the interior surface of the membrane filters, and mineralized nodules were formed among the cell layers. The mineralized nodules were confirmed by staining with the von Kossa technique for calcium mineral deposits. In electron microscopic study, the osteoblastic cells had a relatively large nuclei, and an abundant rough endoplasmic reticulum produced numerous extracellular matrix. In the bone-like nodules, the osteoblastic cells were surrounded by a heavily mineralized matrix with nonmineralized matrix separating the osteoblastic cells from the mineralized matrix. The mineralized matrix contained well-banded collagen fibrils. Matrix vesicles and collagen fibrils which were closely associated with mineral deposition were observed at the mineralizing front. These results together with our previous report [13] indicate that isolated human osteoblastic cells possessed osteogenic potential in vivo as well as mineralization activity in vitro.

Animals↗
PubMed1995

Newly synthesized histamine accelerates ornithine decarboxylase activity in rat intestinal mucosa after ischemia-reperfusion.

We previously demonstrated that both histamine synthesis (histidine decarboxylase activity) and polyamine synthesis (ornithine decarboxylase activity) increased in the rat intestinal mucosa after ischemia-reperfusion, whereas the relationship between these two factors remains unclear. To elucidate this relationship, we performed the present study. The superior mesenteric artery was occluded for 15 min followed by reperfusion. After ischemia-reperfusion, histidine decarboxylase activity and ornithine decarboxylase activity in the rat jejunal mucosa were measured in a time-dependent manner. Histidine decarboxylase activity increased 1 hr after ischemia-reperfusion, although ornithine decarboxylase activity did not; however, its activity did increase 6 hr after. The increase of ornithine decarboxylase activity was attenuated when the increase of histamine synthesis was suppressed by the inhibition of histidine decarboxylase activity caused by pretreatment with alpha-fluoromethylhistidine, a suicide inhibitor of histidine decarboxylase. Pretreatment with H1-receptor antagonist attenuated the increase of ornithine decarboxylase activity after ischemia-reperfusion. These results indicate that the newly synthesized histamine, as indicated by an increase of histidine decarboxylase activity, increases ornithine decarboxylase activity after ischemia-reperfusion of the rat intestinal mucosa.

Animals↗
PubMed1995

Microtubule-severing activity in M phase.

The stable cytoplasmic microtubules that emanate from centrosomes in eukaryotic cells disappear at the onset of M phase and are replaced by the dynamic microtubules of the mitotic spindle. Microtubule-severing activity increases significantly under the control of maturation-promoting factor at the transition between G2 phase and M phase, and is thought to be involved in the microtubule reorganization. This review highlights three microtubule-severing factors that may be responsible for microtubule-severing activity in M phase.

Journal Article↗
PubMed1994

Analysis of the Ras p21/mitogen-activated protein kinase signaling in vitro and in Xenopus oocytes.

Ras p21 in the GTP-bound form was shown to act as an upstream activator for mitogen-activated protein (MAP) kinase kinase (MAPKK) and MAP kinase, and Raf-1 was reported to act as a MAPKK kinase. Further, physical association between Ras and Raf-1 was demonstrated. Here we have shown that incubation of Xenopus immature oocyte extracts with Ras enhances the ability of endogenous Raf-1 to activate MAPKK. Moreover, a dominant negative form of Raf-1 blocked the Ras-induced activation of MAPKK and MAP kinase in the extracts, but not the cyclin A-dependent activation of MAP kinase. When the extracts were depleted of 45-kDa MAPKK with polyclonal anti-MAPKK antibody, no activation of MAP kinase occurred even after incubation with Ras. These results suggest that Ras can activate the MAPKK kinase activity of Raf-1 in the extracts and that MAPKK is indispensable for the Ras-induced MAP kinase activation. It is well known that Ras can induce oocyte maturation when injected into immature Xenopus oocytes. Co-injection of Ras with an anti-MAPKK antibody that inhibits the MAPKK activity prevented the Ras-induced germinal vesicle breakdown, suggesting that MAPKK mediates, at least, one of cellular functions of Ras.

Animals↗
PubMed1994

Mitogen-activated protein kinase kinase is required for the mos-induced metaphase arrest.

The product of the c-mos proto-oncogene functions not only as an initiator of oocyte maturation but also as a component of cytostatic factor that causes the natural arrest of the unfertilized egg at the second meiotic metaphase. It has been shown that Mos can phosphorylate and activate mitogen-activated protein (MAP) kinase kinase (MAPKK) in vitro, leading to activation of MAP kinase. In this study, by using an anti-MAPKK antibody that can specifically inhibit Xenopus MAPKK activity, we have shown that MAPKK mediates the cytostatic factor activity of Mos. Coinjection of this anti-MAPKK antibody with the bacterially expressed Mos protein into a two-cell embryo prevented the Mos-induced cleavage arrest as well as the Mos-induced MAP kinase activation. The analysis of individual embryos indicated that the degree of the cleavage arrest was correlated with the extent of the MAP kinase activation in the Mos- and the Mos/antibody-injected embryos. These observations suggest the involvement of a signal transmission pathway consisting of Mos, MAPKK, and MAP kinase in the metaphase arrest.

Animals↗
PubMed1994

Microtubule severing by elongation factor 1 alpha.

An activity that severs stable microtubules is thought to be involved in microtubule reorganization during the cell cycle. Here, a 48-kilodalton microtubule-severing protein was purified from Xenopus eggs and identified as translational elongation factor 1 alpha (EF-1 alpha). Bacterially expressed human EF-1 alpha also displayed microtubule-severing activity in vitro and, when microinjected into fibroblasts, induced rapid and transient fragmentation of cytoplasmic microtubule arrays. Thus, EF-1 alpha, an essential component of the eukaryotic translational apparatus, appears to have a second role as a regulator of cytoskeletal rearrangements.

Adenosine Triphosphate↗
PubMed1994

Stimulatory effects of yeast and mammalian 14-3-3 proteins on the Raf protein kinase.

Intracellular signaling from receptor tyrosine kinases in mammalian cells results in activation of a signal cascade that includes the guanine nucleotide-binding protein Ras and the protein kinases Raf, MEK [mitogen-activated protein kinase (MAPK) or extracellular signal-regulated kinase (ERK) kinase], and MAPK. MAPK activation that is dependent on the coupling of Ras and Raf was reconstituted in yeast. Yeast genes were isolated that, when overexpressed, enhanced the function of Raf. One of them is identical to BMH1, which encodes a protein similar to members of the mammalian 14-3-3 family. Bacterially synthesized mammalian 14-3-3 protein stimulated the activity of Raf prepared from yeast cells expressing c-Raf-1. Thus, the 14-3-3 protein may participate in or be required for activation of Raf.

14-3-3 Proteins↗
PubMed1994

Feedback regulation of mitogen-activated protein kinase kinase kinase activity of c-Raf-1 by insulin and phorbol ester stimulation.

Recently, it has been reported that Raf-1 kinase (Raf-1) has mitogen-activated protein kinase kinase kinase (MAPKKK) activity in various cells, although Raf-1 and MAP kinase kinase (MAPKK) can be phosphorylated by MAP kinase (MAPK) in vitro. Here we show that the maximal hyperphosphorylation of Raf-1 and MAPKK (10 min) was substantially achieved after the maximal activation of MAPKKK of Raf-1, MAPKK (2-5 min), and MAPK in Chinese hamster ovary cells overexpressing human insulin receptor (CHO-HIR cells) treated with insulin or 12-O-tetradecanoylphorbol-13-acetate (TPA). Moreover, we show that overexpression of MAPK in CHO-HIR cells resulted in enhanced hyperphosphorylation of Raf-1, MAPKK, and mammalian homolog of son of sevenless (mSos) after insulin or TPA stimulation as compared with parental cells. Furthermore, the maximal hyperphosphorylation of Raf-1 appears to be accompanied by a significant decrease in MAPKKK activity. These results suggest that 1) signals initiated by insulin and TPA converge on Raf-1 and activate its MAPKKK activity and 2) Raf-1, MAPKK, and mSos not only lie upstream of MAPK but also are phosphorylated by MAPK, directly or indirectly, and at least Raf-1 kinase activity might be down-regulated by this feedback mechanism.

Amino Acid Sequence↗
PubMed1994

Requirement for the MAP kinase kinase/MAP kinase cascade in Xenopus oocyte maturation.

MAP kinase kinase (MAPKK) has been identified as a protein factor that can induce phosphorylation and activation of inactive MAP kinase in vitro. In this study, we produced an anti-Xenopus MAPKK antibody that can specifically inhibit Xenopus MAPKK activity in vitro. Microinjection of this antibody into immature oocytes prevented progesterone-induced MAP kinase activation. Moreover, progesterone-induced histone H1 kinase activation and germinal vesicle breakdown (GVBD) were inhibited in the oocytes injected previously with this antibody. Furthermore, when a bacterially expressed Mos was introduced into immature oocytes, Mos-induced MAP kinase activation and GVBD were blocked in the oocytes injected with the anti-MAPKK antibody. These results show that MAPKK is responsible for the activation of MAP kinase in vivo and that the MAPKK/MAP kinase cascade plays a pivotal role in the MPF activation during the oocyte maturation process.

Animals↗