Biomedical subjects

M Ui

Publications and source records attributed to M Ui.

At least 91 records · Page 5Linked to original sources

PubMed1996

A subtype of kappa-opioid receptor mediates inhibition of high-affinity GTPase inherent in Gi1 in guinea pig cerebellar membranes.

The kappa-opioid receptor agonists including U-50,488H and dynorphin A (1-17) in ranges of 0.1-100 nM inhibited the hydrolysis of GTP to GDP (P(i) release) inherent in GTP-binding proteins (G proteins) in guinea pig cerebellar membranes. U-50,488H inhibited only high-affinity GTPase activity, not low-affinity activity. The action of this agonist was found to be biphasic, and there was no inhibition at concentrations > 1 microM. The inhibition was abolished by pretreatment with preactivated pertussis toxin (PTX) at concentrations > 1 micrograms/ml but not with preactivated cholera toxin (30 micrograms/ml). Similar blockade of kappa-receptor-mediated inhibition was also observed when membranes were pretreated with a low concentration (8 microM) of N-ethylmaleimide (NEM) at low temperature (4 degrees C), which alkylates the cysteine residue to be ADP-ribosylated by PTX; but this treatment caused no significant change in kappa-agonist binding. When purified Gi1, but not G(o), was reconstituted into membranes pretreated with NEM, the kappa-receptor-mediated inhibition was recovered. These findings suggest that a subtype of kappa-opioid receptor is coupled to inhibition of intrinsic activity of Gi1.

Animals↗

PubMed1996

Fracture-separation of the medial humeral epicondyle caused by arm wrestling.

Ten cases with fracture-separation of the medial humeral epicondyle occurring during arm wrestling were studied to clarify the nature of injury and the mechanism involved. Patients were all male, aged 13 to 15 years. The following results were obtained: (1) the injury often occurred in children immediately before closure of the epiphyseal plate, (2) the injury occurred only when the arm wrestler took a posture in which his center of gravity could shift easily, (3) the injury developed only when one wrestler tried to force the end of the match and the other countered that move, and (4) no lateral instability of the elbow was observed in patients with this injury. These results suggest that the injury is caused by muscular violence. When the maximally contracted muscles attaching to the medial humeral epicondyle are suddenly and passively stretched by shifting of the patient's center of gravity and by the opposing wrestler's counterattack, a shift from concentric contraction to eccentric contraction could occur and apply a large muscle force to the medial humeral epicondyle.

Adolescent↗

PubMed1996

Characterization of the properties of a human homologue of Escherichia coli RecQ from xeroderma pigmentosum group C and from HeLa cells.

We showed that DNA-dependent ATPase Q1 (DNA helicase Q1) from xeroderma pigmentosum complementation group C (XP-C) cells elutes from FPLC Mono Q column at higher concentrations of KCl than that from other human cells (35). We purified DNA helicase Q1 from XP-C and HeLa cells. The purified fractions of both cells contained a major polypeptide with a molecular mass of 73 kDa and had the same enzymatic properties, including salt- and temperature-sensitivity. Characterization using an anti-DNA helicase Q1 antibody indicated that this enzyme localized in the nuclei and was not modified by incorporating phosphate groups through phosphorylation and ADP-ribosylation. No interactions of DNA helicase Q1 with other proteins were indicated by immunoprecipitation of the helicase from crude extracts. No difference was observed in XP-C cells in intracellular localization of DNA helicase Q1, phosphorylation, and the interaction with other proteins as compared to HeLa cells.

Adenosine Triphosphatases↗

PubMed1995

Radiolabeling of catalytic subunits of PI 3-kinases with 17 beta-hydroxy-16 alpha(-)[125I]iodowortmannin: identification of the G beta gamma-sensitive isoform as a complex composed of 46-kDa and 100-kDa subunits.

A fungal metabolite, wortmannin, is a potent inhibitor of phosphatidylinositol (PI) 3-kinases. In the present study, we prepared a radiolabeled derivative of wortmannin, 17 beta-hydroxy-16 alpha(-)[125I]iodowortmannin. The compound bound tightly to a 110-kDa subunit in the previously identified isoform of PI 3-kinase (p85/p110), and also to a 100-kDa peptide in a partially purified preparation of another isoform of PI 3-kinase whose activity was markedly stimulated by the beta gamma subunits of GTP-binding proteins (G beta gamma). The binding to both peptides was inhibited by non-radiolabeled wortmannin and also by LY294002, another inhibitor of PI 3-kinases. An antibody against p85 recognized a 46-kDa peptide in the G beta gamma-sensitive isozyme and precipitated the 100-kDa peptide specifically labeled with 17 beta-hydroxy-16 alpha(-)[125I]iodowortmannin. These results suggested that the newly found isozyme was a complex composed of 46-kDa and 100-kDa peptides.

Androstadienes↗

PubMed1995

Permissive effect of ceramide on growth factor-induced cell proliferation.

Addition of bacterial sphingomyelinase to quiescent Swiss 3T3 cells effectively potentiated the platelet-derived growth factor (PDGF)-stimulated cell proliferation, though the enzyme by itself had little effect on the cell proliferation. Such potentiation of the cell growth could also be observed by the addition of ceramide, a product of the sphingomyelinase-catalysed reaction. In contrast, phosphocholine, another product of the enzyme reaction, had no synergistic effect on the action of PDGF. Treatment of the cells with sphingomyelinase or ceramide increased the cellular activity of mitogen-activated protein kinases (MAP kinases), which have been implicated in the regulation of cell proliferation. However, the synergistic effect of sphingomyelinase on the PDGF-induced cell growth could still be observed even when the cellular MAP kinase activity was fully activated by the growth factor alone. These results indicate that a ceramide-mediated cellular event(s) other than the MAP kinase activation is potentially involved in the regulation of cell growth.

3T3 Cells↗

PubMed1995

Cyclic AMP-increasing agents interfere with chemoattractant-induced respiratory burst in neutrophils as a result of the inhibition of phosphatidylinositol 3-kinase rather than receptor-operated Ca2+ influx.

Superoxide anion and arachidonic acid were produced in guinea pig neutrophils in response to a chemotactic peptide formyl-methionyl-leucyl-phenylalanine (fMLP). Both responses were markedly, but the former response to a phorbol ester was not at all, inhibited when the cellular cAMP level was raised by prostaglandin E1 combined with a cAMP phosphodiesterase inhibitor. Increasing cAMP was also inhibitory to fMLP-induced activation of phosphatidylinositol (PI) 3-kinase and Ca2+ influx without any effect on the cation mobilization from intracellular stores. The fMLP-induced respiratory burst was abolished when PI 3-kinase was inhibited by wortmannin or LY294002, but was not affected when Ca2+ influx was inhibited. On the contrary, fMLP released arachidonic acid from the cells treated with the PI 3-kinase inhibitors as well as from non-treated cells, but it did not so when cellular Ca2+ uptake was prevented. The chemotactic peptide activated PI 3-kinase even in cells in which the receptor-mediated intracellular Ca2+ mobilization and respiratory burst were both abolished by exposure of the cells to a permeable Ca(2+)-chelating agent. Thus, stimulation of fMLP receptors gave rise to dual effects, activation of PI 3-kinase and intracellular Ca2+ mobilization; both effects were necessary for the fMLP-induced respiratory burst. Increasing cellular cAMP inhibited the respiratory burst and arachidonic acid release as a result of the inhibitions of PI 3-kinase and Ca2+ influx, respectively, in fMLP-treated neutrophils.

1-Methyl-3-isobutylxanthine↗

PubMed1995

A subtype of opioid kappa-receptor is coupled to inhibition of Gi1-mediated phospholipase C activity in the guinea pig cerebellum.

PLC activity was stimulated either by 1-100 microM of GTP or by 100-3,000 microM Ca2+ in lysed synaptosomal membranes of the guinea pig cerebellum. The kappa-opioid receptor agonist selectively inhibited the PLC activity stimulated by 100 microM GTP, but not by 100-3,000 microM Ca2+. Pretreatment of membranes with PTX abolished such a kappa-agonist-induced inhibition of PLC activity. The reconstitution of Gi1, but not of Go purified from porcine brains with PTX-treated membranes showed a complete recovery of the kappa-agonist-inhibition of PLC activity. These findings suggest that a novel subtype kappa-receptor mediates inhibition of PLC through inhibiting the intrinsic activity of PTX-substrate G-proteins.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗

PubMed1995

Wortmannin as a unique probe for an intracellular signalling protein, phosphoinositide 3-kinase.

Wortmannin is a fungal metabolite that so far has been shown to act as a selective inhibitor of phosphoinositide 3-kinase. It can therefore be used to investigate the convergence between two major cellular signalling systems: those involving G-protein-coupled receptors and those involving receptor tyrosine kinases. Importantly, wortmannin can enter intact cells, making whole-cell studies of the above signalling pathways possible.

Androstadienes↗

PubMed1995

Efficient replication of polyomavirus DNA in a cell-free system supplemented with Escherichia coli single-stranded DNA binding protein, which exhibits species-specificity in the requirement for DNA polymerase alpha-primase.

We established a modified cell-free system for polyomavirus (PyV) DNA replication, which was supplemented with Escherichia coli single-stranded DNA binding protein (SSB). DNA synthesis in this system was enhanced by 1.4- to over 15-fold depending upon the amount of cell extracts contained in the reaction mixture. By supplementing with E. coli SSB, we were able to reduce the amount of cell extracts in the reaction mixture, and to lower the concentrations of creatine phosphate and Tris, rendering this system more resistant to salts than the conventional PyV DNA replication system. The modified system was characterized using mutant cell extracts which had heat-inactivated DNA polymerase alpha. DNA synthesis in the system was dependent on PyV T antigen, the PyV origin of DNA replication, mutant cell extracts, and DNA polymerase alpha-primase complex purified from wild-type cells. The DNA polymerase alpha-primase complex was not replaced by DNA polymerase alpha, indicating that this system requires a functional DNA polymerase alpha-primase complex. This system exhibited species-specificity in the requirement for DNA polymerase alpha-primase; only mouse DNA polymerase alpha-primase but not human DNA polymerase alpha-primase functioned in this system.

Animals↗

PubMed1995

Purification and characterization of the G203T mutant alpha i-2 subunit of GTP-binding protein expressed in baculovirus-infected Sf9 cells.

We expressed the Gly203-->Thr (G203T) mutant of Gi2alpha, which was expected to show a dominant-negative phenotype in Gi2-mediated signal transduction, in baculovirus-inefected Sf9 cells and purified the mutant alpha subunit for its characterization. The rate of dissociation of GDP from G203T Gi2alpha was 3- to 4-fold faster than that from wild type Gi2alpha, but their kappacat values for GTP hydrolysis were almost the same. The affinities of the two Gi2alpha proteins for the beta gamma subunits of G proteins to form alpha beta gamma timers, which served as substrates for pertussis toxin-catalyzed ADP-ribosylation, were the same. In marked contrast, G203T Gi2alpha was unable to form a tight complex with a non- hydrolyzable analog (GTP[gammaS) of GTP; bound GTP[gammaS] was readily released from the mutant Gi2alpha even in the presence of a high concentration of Mg2+. Its susceptibility to tryptic digestion also revealed that GTP[gammaS]-bound G203T Gi2alpha formed a conformation apparently different from that of the GTP[gammaS]-bound form of wild-type Gi2alpha. Both the G203T and wild-type Gi2alpha proteins were capable of coupling with membrane-bound alpha2-adrenergic receptors, resulting in the formation of receptor-G protein complexes with high affinity for agonists. However, GTP[gammaS]-dependent uncoupling from high-affinity receptors was markedly attenuated in the case of G203T Gi2alpha. Thus, G203T-mutated Gi2alpha had a unique property in terms of coupling to membrane receptors, in addition to the previously expected defect in the active conformation of the GTP-bound form of Gi2alpha.

Animals↗

PubMed1995

Characterization of DNA synthesis and DNA-dependent ATPase activity at a restrictive temperature in temperature-sensitive tsFT848 cells with thermolabile DNA helicase B.

A temperature-sensitive mutant defective in DNA replication, tsFT848, was isolated from the mouse mammary carcinoma cell line FM3A. In mutant cells, the DNA-dependent ATPase activity of DNA helicase B, which is a major DNA-dependent ATPase in wild-type cells, decreased at the nonpermissive temperature of 39 degrees C. DNA synthesis in tsFT848 cells at the nonpermissive temperature was analyzed in detail. DNA synthesis measured by incorporation of [3H]thymidine decreased to about 50% and less than 10% of the initial level at 8 and 12 h, respectively. The decrease in the level of thymidine incorporation correlated with a decrease in the number of silver grains in individual nuclei but not with the number of cells with labeled nuclei. DNA fiber autoradiography revealed that the DNA chain elongation rate did not decrease even after an incubation for 10 h at 39 degrees C, suggesting that initiation of DNA replication at the origin of replicons is impaired in the mutant cells. The decrease in DNA-synthesizing ability coincided with a decrease in the level of the DNA-dependent ATPase activity of DNA helicase B. Partially purified DNA helicase B from tsFT848 cells was more heat sensitive than that from wild-type cells. Inactivation of DNA-dependent ATPase activity of DNA helicase B from mutant cells was considerably reduced by adding DNA to the medium used for preincubation, indicating that the DNA helicase of mutant cells is stabilized by binding to DNA.

Animals↗

PubMed1994

Possible involvement of phosphatidylinositol-specific phospholipase C related to pertussis toxin-sensitive GTP-binding proteins during adipocyte differentiation of 3T3-L1 fibroblasts: negative regulation of protein kinase C.

Insulin/dexamethasone/methylisobutylxanthine (hormones/IBMX) induce 3T3-L1 fibroblasts to differentiate into adipocytes. Our previous study suggested that pertussis toxin (IAP)-sensitive GTP-binding protein(s) (G-protein) is involved in the process of differentiation by hormones/IBMX, accompanied by c-fos induction. Northern blotting indicated that among the IAP-sensitive G-proteins, the levels of Gi2 alpha, Go alpha, and Gi3 alpha mRNA were decreased, increased and unchanged, respectively. Gi1 alpha was undetectable and IAP attenuated the decrease in Gi2 alpha mRNA level but did not affect the change in Go alpha mRNA level during the adipocyte differentiation. These results indicate that IAP-sensitive Gi2 alpha mRNA level is decreased during adipocyte differentiation. A combination of phosphatidylinositol-specific phospholipase C (PI-PLC) and IBMX induced c-fos expression in 3T3-L1 fibroblasts similar to that induced with hormones/IBMX. c-fos induced by both stimulators was also diminished by anti-inositolglycan antibody or anti-PI-PLC antiserum. Insulin stimulated the release of inositolproteoglycan and diacylglycerol from 3T3-L1 fibroblasts, which was suppressed by IAP treatment. These findings suggested that one of the pathways of adipocyte differentiation induced by hormones/IBMX occurs via the inositolglycan-specific PI-PLC cascade coupled to IAP-sensitive G-protein(s). Both activation of glycerophosphate dehydrogenase and stimulation of insulin-dependent 2-deoxyglucose uptake induced by hormones/IBMX were enhanced in protein kinase C-depleted cells exposed to phorbol 12-myristate 13-acetate (PMA), and attenuated in IAP-treated cells. The level of a 32P-labeled 52 kDa protein in plasma membrane fractions immunoprecipitated by anti-PI-PLC antiserum was increased by PMA stimulation, abolished in PMA-treated cells, and increased in IAP-treated cells. These findings suggest that protein kinase C phosphorylates PI-PLC, resulting in a decrease in PI-PLC activity related to the signal transduction pathway of adipocyte differentiation of 3T3-L1 fibroblasts.

3T3 Cells↗

PubMed1994

Identification of the nature of modification that causes the shift of DNA topoisomerase II beta to apparent higher molecular weight forms in the M phase.

Human cell lines express two genetically distinct isoforms of DNA topoisomerase (topo II) II: topo II alpha (p170) and topo II beta (p180). We detected a higher molecular weight form with an apparent molecular mass of about 190 kDa in M phase-arrested HeLa cells (Kimura, K., Saijo, M., Ui, M., and Enomoto, T. (1994) J. Biol. Chem. 269, 1173-1176). In this study we confirmed, using anti-topo II alpha and topo II beta monoclonal antibodies, that this higher molecular weight form is topo II beta and consists of doublet bands around 190 kDa. We confirmed that the doublet bands constituted an M phase-specific phenomenon and were not an artifact of the procedure used to accumulate mitotic cells. Digesting the immunoprecipitated materials from mitotic cell extracts with alkaline phosphatase resulted in the disappearance of the doublet bands and the appearance of the 180-kDa band with the concomitant disappearance of 32P label in the region of the doublet bands. Neither heat-inactivated alkaline phosphatase nor phosphodiesterase affected the doublet bands and the 32P label. Topo II beta in interphase cells was also phosphorylated, but the shift in apparent molecular weight was very slight after alkaline phosphatase digestion. Analysis of the labeled phosphoamino acids present in topo II beta from M phase and logarithmically growing cells indicated that phosphorylation occurred mainly on serine and fairly on threonine residues in both topo II beta isoforms. These results indicated that topo II beta is phosphorylated at specific sites in M phase, resulting in the formation of the doublet bands.

Amino Acid Sequence↗

PubMed1994

Switching from alpha 1- to beta-subtypes in adrenergic response during primary culture of adult-rat hepatocytes as affected by the cell-to-cell interaction through plasma membranes.

The alpha 1-adrenergic response was predominant over the beta-adrenergic one in adult rat hepatocytes, when the responses were measured as the agonist-induced generations of Ins(1,4,5)P3 and cyclic AMP, respectively. During primary culture of the adult rat hepatocytes, the beta-adrenergic response developed rapidly, whereas the alpha 1-response decreased gradually. Such receptor-subtype switching did not occur unless the cells were cultured under conditions favourable for cell growth, i.e. at low cell density (10(4) cells/cm2). The switching was prevented progressively as the cell culture density was increased up to 20-fold or the low-density culture was achieved by addition of increasing amounts of liver plasma membranes. The gradual decrease in alpha 1-response was accounted for by a concurrent decrease in the receptor site density, whereas rapid development of the beta-response definitely preceded the increase in beta-ligand binding sites during the culture. This rapid development of the beta-response reflected enhanced coupling of the receptor to G-protein during the early stage of culture, as evidenced by the progressively developed ability of GTP to lower the affinity of beta-agonist binding to membranes prepared from these short-time-cultured hepatocytes.

Adrenergic beta-Antagonists↗

PubMed1994

Possible involvement of GTP-binding proteins in 1 alpha,25-dihydroxyvitamin D3 induction of tissue transglutaminase in mouse peritoneal macrophages.

Induction of transglutaminase was analyzed based on increases in the maximal enzymic activity and in the Northern blots of mRNA during culture of mouse resident peritoneal macrophages with active forms of hydrophobic vitamins and steroid hormones. The enzyme was induced by 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25-(OH)2D3) or retinoic acid but not by steroid hormones. The induction by 1 alpha,25-(OH)2D3 was characterized by its slow onset and marked synergism with retinoic acid induction. The induction was enhanced by protein kinase activators such as cholera toxin and phorbol 12-myristate 13-acetate but largely inhibited by pertussis toxin treatment of cells. It is suggested that pertussis toxin-linked and protein kinase-related signaling would mediate the 1 alpha,25-(OH)2D3-induced enzyme gene expression.

Animals↗

PubMed1994

Involvement of phosphatidylinositol 3-kinase in Fc gamma receptor signaling.

Wortmannin, a potent and selective inhibitor of phosphatidylinositol (PI) 3-kinase (Okada, T., Sakuma, L., Fukui, Y., Hazeki, O., and Ui, M. (1994) J. Biol. Chem. 269, 3563-3567), prevented Fc receptor for IgG (Fc gamma R)-dependent phagocytosis of the human monocytic cell line U937 or guinea pig neutrophils. Cross-linking of Fc gamma R on the surface of U937 cells increased PI 3-kinase activity that was immunoprecipitated with antibody against phosphotyrosine or antibody against the 85-kDa regulatory subunit of PI 3-kinase. Specific cross-linking of Fc gamma R subclass Fc gamma RI or Fc gamma RII, using monoclonal antibodies against each receptor subclass and the F(ab')2 fragment of goat antibody against mouse IgG, increased anti-phosphotyrosine-precipitable PI 3-kinase activity. Treatment of cells with anti-Fc gamma RIII antibody plus the same F(ab')2 did not affect the activity, reflecting the lack of Fc gamma RIII in U937 cells. Fcy gamma R stimulation triggered prominent tyrosine phosphorylation of several proteins, among which the 115-kDa peptide showed strong association with PI 3-kinase. Thus, Fc gamma R appears to be coupled functionally, via a tyrosine kinase, to PI 3-kinase, which may regulate the phagocytotic activity of the cells.

Androstadienes↗

PubMed1994

Purification and cloning of a nucleotide excision repair complex involving the xeroderma pigmentosum group C protein and a human homologue of yeast RAD23.

Complementation group C of xeroderma pigmentosum (XP) represents one of the most common forms of this cancer-prone DNA repair syndrome. The primary defect is located in the subpathway of the nucleotide excision repair system, dealing with the removal of lesions from the non-transcribing sequences ('genome-overall' repair). Here we report the purification to homogeneity and subsequent cDNA cloning of a repair complex by in vitro complementation of the XP-C defect in a cell-free repair system containing UV-damaged SV40 minichromosomes. The complex has a high affinity for ssDNA and consists of two tightly associated proteins of 125 and 58 kDa. The 125 kDa subunit is an N-terminally extended version of previously reported XPCC gene product which is thought to represent the human homologue of the Saccharomyces cerevisiae repair gene RAD4. The 58 kDa species turned out to be a human homologue of yeast RAD23. Unexpectedly, a second human counterpart of RAD23 was identified. All RAD23 derivatives share a ubiquitin-like N-terminus. The nature of the XP-C defect implies that the complex exerts a unique function in the genome-overall repair pathway which is important for prevention of skin cancer.

Amino Acid Sequence↗

PubMed1994

Dual pertussis toxin-sensitive pathway of zymosan-induced activation in guinea pig macrophages. An anti-CR3 antibody-inhibitable stimulation of phagocytosis and -resistant stimulation of O2- production and arachidonate release.

Complement receptor type 3 (CR3)-mediated cellular responses in guinea pig macrophages were investigated by using zymosan and serum-opsonized zymosan (SOZ) as the multivalent ligand for CR3. The ingestion of zymosan and SOZ was accompanied by O2- generation and arachidonate release. These responses were suppressed by prior exposure of macrophages to pertussis toxin (PT). Opsonization of zymosan gave rise to more than 6-fold activation of the ingestion, whereas the magnitude of either arachidonate release or O2- generation was unchanged. The Fab' fragment of anti-Z-1, a monoclonal antibody specific for the alpha chain of guinea pig CR3, inhibited the ingestion of zymosan by 60% without affecting zymosan-induced arachidonate release and O2- generation. These data suggested that there might be at least two functionally distinct binding sites for zymosan. O2- generation and arachidonate release might be regulated through one site and phagocytosis another. Both sites should be coupled to PT-sensitive GTP binding protein.

Animals↗