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K Okawa

Publications and source records attributed to K Okawa.

At least 37 records · Page 2Linked to original sources
PubMed1999

Identification of a novel phosphorylation site on histone H3 coupled with mitotic chromosome condensation.

Histone H3 (H3) phosphorylation at Ser(10) occurs during mitosis in eukaryotes and was recently shown to play an important role in chromosome condensation in Tetrahymena. When producing monoclonal antibodies that recognize glial fibrillary acidic protein phosphorylation at Thr(7), we obtained some monoclonal antibodies that cross-reacted with early mitotic chromosomes. They reacted with 15-kDa phosphoprotein specifically in mitotic cell lysate. With microsequencing, this phosphoprotein was proved to be H3. Mutational analysis revealed that they recognized H3 Ser(28) phosphorylation. Then we produced a monoclonal antibody, HTA28, using a phosphopeptide corresponding to phosphorylated H3 Ser(28). This antibody specifically recognized the phosphorylation of H3 Ser(28) but not that of glial fibrillary acidic protein Thr(7). Immunocytochemical studies with HTA28 revealed that Ser(28) phosphorylation occurred in chromosomes predominantly during early mitosis and coincided with the initiation of mitotic chromosome condensation. Biochemical analyses using (32)P-labeled mitotic cells also confirmed that H3 is phosphorylated at Ser(28) during early mitosis. In addition, we found that H3 is phosphorylated at Ser(28) as well as Ser(10) when premature chromosome condensation was induced in tsBN2 cells. These observations suggest that H3 phosphorylation at Ser(28), together with Ser(10), is a conserved event and is likely to be involved in mitotic chromosome condensation.

Amino Acid Sequence↗
PubMed1999

Cutting edge: combined treatment of TNF-alpha and IFN-gamma causes redistribution of junctional adhesion molecule in human endothelial cells.

Proinflammatory cytokines such as TNF-alpha and IFN-gamma induce cell adhesion molecules in endothelial cells and promote transmigration of leukocytes across endothelial cells. However, when those two were administered together, leukocyte transmigration paradoxically decreased. We cloned a human and bovine homologue of the junctional adhesion molecule (JAM), a novel molecule at the tight junction, and examined the effects of proinflammatory cytokines on JAM in HUVECs. The combined treatment of TNF-alpha plus IFN-gamma caused a disappearance of JAM from intercellular junctions. However, flow cytometry, cell ELISA, and subcellular fractionation analysis demonstrated that the amount of JAM was not reduced. This suggested that JAM changed its distribution in response to proinflammatory cytokines. This redistribution of JAM might be involved in a decrease in transendothelial migration of leukocytes at inflammatory sites.

Amino Acid Sequence↗
PubMed1999

Small G protein Ral and its downstream molecules regulate endocytosis of EGF and insulin receptors.

The involvement of Ral and its downstream molecules in receptor-mediated endocytosis was examined. Expression of either RalG23V or RalS28N, which are known to be constitutively active and dominantnegative forms, respectively, in A431 cells blocked internalization of epidermal growth factor (EGF). Stable expression of RalG23V or RalS28N in CHO-IR cells also inhibited internalization of insulin. Internalization of EGF and insulin was not affected by full-length RalBP1 which is an effector protein of Ral, but was inhibited by its C-terminal region which binds directly to Ral and POB1. POB1 is a binding protein of RalBP1 and has the Eps15 homology (EH) domain. Deletion mutants of POB1 inhibited internalization of EGF and insulin. However, internalization of transferrin was unaffected by Ral, RalBP1, POB1 and their mutants. Epsin and Eps15 have been reported to be involved in the regulation of endocytosis of the receptors for EGF and transferrin. The EH domain of POB1 bound directly to Epsin and Eps15. Taken together with the observation that EGF and insulin activate Ral, these results suggest that Ral, RalBP1 and POB1 transmit the signal from the receptors to Epsin and Eps15, thereby regulating ligand-dependent receptor-mediated endocytosis.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Rho-associated kinase of chicken gizzard smooth muscle.

Rho-associated kinase (Rho-kinase) from chicken gizzard smooth muscle was purified to apparent homogeneity (160 kDa on SDS-polyacrylamide gel electrophoresis) and identified as the ROKalpha isoform. Several substrates were phosphorylated. Rates with myosin phosphatase target subunit 1 (MYPT1), myosin, and the 20-kDa myosin light chain were higher than other substrates. Thiophosphorylation of MYPT1 inhibited myosin phosphatase activity. Phosphorylation of myosin at serine 19 increased actin-activated Mg+-ATPase activity, i.e. similar to myosin light chain kinase. Myosin phosphorylation was increased at higher ionic strengths, possibly by formation of 6 S myosin. Phosphorylation of the isolated light chain and myosin phosphatase was decreased by increasing ionic strength. Rho-kinase was stimulated 1.5-2-fold by guanosine 5'-O-3-(thio)triphosphate.RhoA, whereas limited tryptic hydrolysis caused a 5-6-fold activation, independent of RhoA. Several kinase inhibitors were screened and most effective were Y-27632, staurosporine, and H-89. Several lipids caused slight activation of Rho-kinase, but arachidonic acid (30-50 microM) induced a 5-6-fold activation, independent of RhoA. These results suggest that Rho-kinase of smooth muscle may be involved in the contractile process via phosphorylation of MYPT1 and myosin. Activation by arachidonic acid presents a possible regulatory mechanism for Rho-kinase.

Amino Acid Sequence↗
PubMed1999

Hepatocyte growth inhibitory factor derived from HTLV-I(+) T-cell line is identical to IL-6.

We previously reported that the culture supernatant of the human T-cell leukemia virus (HTLV-I) infected-T-cell line--ATL-2--included factor(s), which had an inhibitory effect on epidermal growth factor (EGF)-stimulated proliferation of primary cultured rat hepatocytes. After crude purification, we arbitrarily named it hepatocyte growth inhibitory factor (HGI). In this study, we further purified HGI and determined its amino acid sequence. For purification, we used 4-steps column chromatography and SDS-PAGE. The purified proteins consisted of two bands of 20 and 27 kDa in SDS-PAGE analysis. Protein extracted from each band had an inhibitory effect on rat hepatocyte growth. Amino acid analysis of the purified 20 kDa band revealed that the 34 amino acids were identical to those of IL-6. The inhibitory effect of the factor was neutralized by an anti IL-6 neutralizing antibody. Using Western blot analysis of HGI, an anti IL-6 antibody recognized both 20 and 27 kDa bands. Consequently HGI was determined to be identical to IL-6, which occurred in higher levels in the sera of adult T-cell leukemia (ATL) patients.

Aged↗
PubMed1999

BLNK required for coupling Syk to PLC gamma 2 and Rac1-JNK in B cells.

Signaling through the B cell receptor (BCR) is essential for B cell function and development. Despite the key role of Syk in BCR signaling, little is known about the mechanism by which Syk transmits downstream effectors. BLNK (B cell LiNKer protein), a substrate for Syk, is now shown to be essential in activating phospholipase C (PLC)gamma 2 and JNK. The BCR-induced PLC gamma 2 activation, but not the JNK activation, was restored by introduction of PLC gamma 2 membrane-associated form into BLNK-deficient B cells. As JNK activation requires both Rac1 and PLC gamma 2, our results suggest that BLNK regulates the Rac1-JNK pathway, in addition to modulating PLC gamma 2 localization.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Mass mating method in combination with G418- and aureobasidin A-resistance markers for efficient selection of hybrids from homothallic strains in Saccharomyces cerevisiae.

We have developed a mass mating method using the spore suspensions of homothallic yeasts of Saccharomyces cerevisiae in combination with dominant selective drug resistance markers, Tn601(903) against geneticin and AUR1-C against aureobasidin A for the selection of the hybrids. To examine the effectiveness of these markers in the mass mating method, each marker was introduced into a homothallic wine yeast. Using a mixed culture of spore suspensions from the resultant transformants, many hybrids were screened by the drug resistance markers. This method is more practical than the spore-to-spore mating method because it does not require the use of a micromanipulator and many hybrids are obtained at one time. The resultant hybrids could be utilized for industrial brewing because plasmids, which are used to confer resistance markers, are easily eliminated from the hybrids by cultivation in a medium without drugs. We propose that the mass mating method using spore suspensions in combination with dominant selective geneticin- and aureobasidin A-resistance markers is useful for the selection of hybrids from industrial homothallic yeasts.

Journal Article↗
PubMed1999

Isolation and characterization of the glyceraldehyde-3-phosphate dehydrogenase gene of Lentinus edodes.

The glyceraldehyde-3-phosphate dehydrogenase (GPD) gene of Lentinus edodes was isolated from a genomic DNA library and cDNA corresponding to this gene was isolated from a mycelium cDNA library. The L. edodes GPD gene was found to encode a 337-aa protein. By comparison of the cDNA and genomic DNA sequences, the presence of eight introns in the GPD gene was confirmed. The putative amino acid sequence of the L. edodes GPD gene product showed high similarity to those of other basidiomycetes. The results of Southern blot analyses suggested that only one copy of the GPD gene is present in the genome of L. edodes. The promoter region was found to contain a CT-rich stretch, two CAAT boxes and a consensus TATA box. In addition, the transcript of the GPD gene was found to be expressed constitutively and strongly. These results suggest that the promoter of the L. edodes GPD gene may be very useful as a component of transformation vectors.

3' Untranslated Regions↗
PubMed1999

[A study of hypertriglyceridemia occurring in patients with chronic hepatitis C during administration of interferon beta].

The prevalence and risk factors of hypertriglyceridemia during the administration of interferon (IFN) were examined in 78 patients with chronic hepatitis C who were treated with 6 MU of IFN-beta once or 3 MU of IFN-beta twice a day for 6 weeks. Hypertriglyceridemia (serum triglyceride (TG) above 150 mg/dl) was found before the start of IFN treatment in 9% of the patients. During the administration of IFN, elevation of serum TG above 150 mg/dl was found in 82% of patients. In addition, serum TG level exceeded 500 mg/dl at least once during the administration of IFN in 13% of patients. On stepwise multiple regression analysis, three risk factors, high serum TG value before the administration of IFN, high ALT value before the administration of IFN, and divided administration of IFN-beta twice daily were found to be associated with hypertriglyceridemia during IFN administration.

Adult↗
PubMed1999

[Differential diagnosis of ulcerative colitis].

Important points during differential diagnosis of ulcerative colitis from other inflammatory disorders are endoscopic examination and microbial studies of stools. In acute phase of enterocolitis in which waterly diarrhea with bloody stool and abdominal pain appeared, infectious enterocolitis by Shigella, Salmonella, Campylobacter and Yersinia, which sometimes causes mucosal edema, hyperemia, erosions and ulceration should be distinguished carefully. Microbial studies of stool would bring helpful information in such situation. In chronic phase of inflammatory diseases of bowel, they often showed chronic diarrhea associated with mucobloody stools and abdominal pain. They often revealed mucosal inflammation mimicking ulcerative colitis during endoscopic evaluation. Among them, most important diseases are amebic colitis, ischemic colitis, radiation colitis and antibiotics associated hemorrhagic colitis.

Bacterial Infections↗
PubMed1999

[Glucocorticoid treatment of ulcerative colitis].

Glucocorticoid is an important drug for ulcerative colitis. In this paper, the action mechanism of glucocorticoid, particularly that at molecular level in the cell, was mentioned. Mechanism in anti-inflammatory action, influence on cytokine production, immunosuppressive action and glucocorticoid resistance were reviewed.

Arachidonic Acids↗
PubMed1999

[Appendiceal lesion in ulcerative colitis].

Recently, there has been a resurgence in the interest of the role of the appendix in ulcerative colitis. The lesion of appendix in ulcerative colitis is reviewed. Surgical studies were mainly reported in western countries and endoscopic studies were mainly reported in Japan. Epidemiological and experimental studies suggest a central role of the appendix in development of ulcerative colitis. It was considered that understanding of the significance of a skip lesion in the appendix would contribute to the elucidation of the pathogenesis of ulcerative colitis.

Animals↗
PubMed1998

Specific detection of phosphatidylinositol 3,4,5-trisphosphate binding proteins by the PIP3 analogue beads: an application for rapid purification of the PIP3 binding proteins.

Phosphatidylinositol (PI) 3-kinase is known as one of the key molecules involved in the various biological events such as vesicle trafficking, cytoskeletal rearrangements and cell survival. T clarify the molecular basis underlying these events, we have tried to identify the proteins that can interact with phosphatidylinositol 3,4,5-trisphosphate (PIP3), the lipid product of PI3-kinase. Using a new PIP3 analogue, PIP3-APB, we synthesized an affinity column for PIP3 binding proteins. This enabled us to purify and identify several PIP3 binding proteins such as Tec tyrosine kinase, Gap1m, and Akt, as the candidates for the downstream molecules of PI3-kinase. All of these proteins contain PH domains, possible binding sites for phospholipids. Studies with various deletion mutants of Tec or Gap1m revealed that their PH domains are indeed the binding sites for PIP3. These results demonstrate that this PIP3-analogue binds various PIP3 binding proteins with high specificity and may be useful to elucidate the downstream mechanisms of PI3-kinases-mediated signaling pathways.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Katanin, a microtubule-severing protein, is a novel AAA ATPase that targets to the centrosome using a WD40-containing subunit.

Microtubule disassembly at centrosomes is involved in mitotic spindle function. The microtubule-severing protein katanin, a heterodimer of 60 and 80 kDa subunits, was previously purified and shown to localize to centrosomes in vivo. Here we report the sequences and activities of the katanin subunits. p60 is a new member of the AAA family of ATPases, and we show that expressed p60 has microtubule-stimulated ATPase and microtubule-severing activities in the absence of p80. p80 is a novel protein containing WD40 repeats, which are frequently involved in protein-protein interactions. The p80 WD40 domain does not participate in p60 dimerization, but localizes to centrosomes in transfected mammalian cells. These results indicate katanin's activities are segregated into a subunit (p60) that possesses enzymatic activity and a subunit (p80) that targets the enzyme to the centrosome.

Adenosine Triphosphatases↗
PubMed1998

Regulation of the association of adducin with actin filaments by Rho-associated kinase (Rho-kinase) and myosin phosphatase.

The small GTPase Rho is believed to regulate the actin cytoskeleton and cell adhesion through its specific targets. We previously identified the Rho targets: protein kinase N, Rho-associated kinase (Rho-kinase), and the myosin-binding subunit (MBS) of myosin phosphatase. Here we purified MBS-interacting proteins, identified them as adducin, and found that MBS specifically interacted with adducin in vitro and in vivo. Adducin is a membrane-skeletal protein that promotes the binding of spectrin to actin filaments and is concentrated at the cell-cell contact sites in epithelial cells. We also found that Rho-kinase phosphorylated alpha-adducin in vitro and in vivo and that the phosphorylation of alpha-adducin by Rho-kinase enhanced the interaction of alpha-adducin with actin filaments in vitro. Myosin phosphatase composed of the catalytic subunit and MBS showed phosphatase activity toward alpha-adducin, which was phosphorylated by Rho-kinase. This phosphatase activity was inhibited by the phosphorylation of MBS by Rho-kinase. These results suggest that Rho-kinase and myosin phosphatase regulate the phosphorylation state of adducin downstream of Rho and that the increased phosphorylation of adducin by Rho-kinase causes the interaction of adducin with actin filaments.

Actins↗
PubMed1998

A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD.

The homeostasis of animals is regulated not only by the growth and differentiation of cells, but also by cell death through a process known as apoptosis. Apoptosis is mediated by members of the caspase family of proteases, and eventually causes the degradation of chromosomal DNA. A caspase-activated deoxyribonuclease (CAD) and its inhibitor (ICAD) have now been identified in the cytoplasmic fraction of mouse lymphoma cells. CAD is a protein of 343 amino acids which carries a nuclear-localization signal; ICAD exists in a long and a short form. Recombinant ICAD specifically inhibits CAD-induced degradation of nuclear DNA and its DNase activity. When CAD is expressed with ICAD in COS cells or in a cell-free system, CAD is produced as a complex with ICAD: treatment with caspase 3 releases the DNase activity which causes DNA fragmentation in nuclei. ICAD therefore seems to function as a chaperone for CAD during its synthesis, remaining complexed with CAD to inhibit its DNase activity; caspases activated by apoptotic stimuli then cleave ICAD, allowing CAD to enter the nucleus and degrade chromosomal DNA.

Amino Acid Sequence↗