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Biomedical subjects

Y Banno

Publications and source records attributed to Y Banno.

At least 73 records · Page 4Linked to original sources
PubMed1995

Deletion and site-directed mutagenesis of EF-hand domain of phospholipase C-delta 1: effects on its activity.

In order to elucidate a role of a putative EF-hand motif (144-172) in phospholipase C-delta 1 (PLC-delta 1), deletion and point mutation of the enzyme were performed and the mutated cDNAs were expressed in CHO cells and E. coli AD202 strain. Deletion of amino acid residues of 141-236 or 173-236 resulted in abolition of PLC activity. However, the decreased PLC activity to 15-20% by deletion of the EF-hand motif (144-172) was still Ca(2+)-dependent. Furthermore, mutants, in which conserved Asp153, Asp157, Glu164 or all these acidic amino acids in the EF-hand motif were replaced with alanine residues, showed nearly the same PLC activity and Ca(2+)-dependency as those of wild-type. These results suggest that the region containing the EF-hand motif may not play a role in regulation of Ca(2+)-sensitivity of PLC-delta 1, but is important for its activity.

Alanine↗
PubMed1995

Activation of membrane-bound phospholipase D by protein kinase C in HL60 cells: synergistic action of a small GTP-binding protein RhoA.

Regulation of phospholipase D (PLD) activation by protein kinase C (PKC) was studied in membranes isolated from human promyelocytic leukemia HL60 cells. The activation of membrane-bound PLD by PKC partially purified from rat brain was most effectively induced with phorbol 12-myristate 13-acetate (PMA) and Ca2+ (1 microM) which caused translocation of PKC to membranes. Ro31-8425, a potent inhibitor of PKC, suppressed the catalytic activity of PKC in a concentration-dependent manner, with complete inhibition at 5 microM. However, the PKC-mediated PLD activation was not affected by Ro31-8425. It was thus suggested that membrane-bound PLD of HL60 cells was activated by PKC translocation but probably via a phosphorylation-independent mechanism. Furthermore, addition by guanosine 5'-3-O-(thio)trisphosphate (GTP gamma S) potentiated the PKC-mediated PLD activation and this potentiating effect was abolished by Rho GTPase dissociation inhibitor (RhoGDI). The suppressed PLD activation by RhoGDI was completely restored by addition of recombinant RhoA. These results indicate that the PKC-mediated PLD activation can be synergistically potentiated by RhoA in HL60 membranes.

Animals↗
PubMed1995

Functions of [His321]gelsolin isolated from a flat revertant of ras-transformed cells.

A mutant gelsolin, [His321]gelsolin, was isolated from R1, a flat revertant of human activated c-Ha-ras oncogene-transformed NIH/3T3 cells (EJ-NIH/3T3) produced by ethylmethanesulfonate treatment. [His321]Gelsolin has a histidine instead of a proline residue at position 321 and suppresses the tumorigenicity of EJ-NIH/3T3 cells when it is constitutively expressed [Müllauer, L., Fujita, H., Ishizaki, A. & Kuzumaki, N. (1993) Oncogene 8, 2531-2536]. To investigate the biochemical consequences of the amino acid substitution of His321, we expressed the [His321]gelsolin and wild-type gelsolin in Escherichia coli, purified them, and analyzed their effects on actin, polyphosphoinositol lipids and phospholipase C. [His321]Gelsolin has decreased actin-filament-severing activity and increased nucleating activity compared with wild-type gelsolin in vitro. Furthermore, compared to wild-type gelsolin both nucleation and severing by [His321]gelsolin are inhibited more strongly by the phosphoinositol lipids phosphatidylinositol 4-phosphate (PtdInsP) and phosphatidylinositol 4,5-bisphosphate (PtdInsP2). In addition, [His321]gelsolin inhibits PtdInsP2 hydrolysis by phospholipase C gamma 1 more strongly than wild-type gelsolin in vitro because of its higher binding capacity for phosphoinositol lipid. Gelsolin has six homologous amino acid repeats called S1-S6. Our results suggest that the segment S3 which contains the mutation is functionally relevant for regulation of gelsolin's activities even though the relevant actin-binding domains are in segments 1, 2, and 4-6, and that the region around the residue 321 may contain a phosphoinositol-lipid-binding site. Altered functions of [His321]gelsolin might be important for the loss of tumorigenicity of the ras-transformed cells.

3T3 Cells↗
PubMed1995

Endogenous cleavage of phospholipase C-beta 3 by agonist-induced activation of calpain in human platelets.

Two membrane-associated phosphoinositide-specific phospholipase Cs (mPI-PLC-1 and mPI-PLC-2) and a cytosolic enzyme (cPI-PLC) that were activated by brain G-protein beta gamma subunits have been isolated from human platelets. The truncation of mPI-PLC-1 that was mediated by mu-calpain induced much higher activation by beta gamma subunits (Banno, Y., Asano, T., and Nozawa, Y. (1994) FEBS Lett. 340, 185-188). On the basis of size and immunological cross-reactivity, mPI-PLC-1 (155 kDa) was PLC-beta 3, and mPI-PLC-2 (100 kDa) was its truncated form. The cPI-PLC (140 kDa) was recognized by the antibody selective for internal sequences of PLC-beta 3 but not by the antibody raised against its carboxyl terminus, indicating that it may be related to PLC-beta 3. Treatment of human platelets with A23187 and dibucaine, activators of calpain, caused cleavage of actin-binding protein and talin in a time-dependent manner. At the same time, decrease of PLC-beta 3 (155 and 140 kDa) and concomitant increase of the 100-kDa product of cleavage were observed on immunoblots with the antibody to internal sequences of PLC-beta 3. Furthermore, stimulation of platelets by natural agonists, thrombin and collagen, caused the cleavage of PLC-beta 3 (155 and 140 kDa) and an increase of 100 kDa PLC-beta 3 in a time- and dose-dependent manner. The cleavage of these PLC-beta 3 enzymes was completely blocked by calpain inhibitor, calpeptin, indicating that the PLC-beta 3 modification may be a consequence of platelet activation leading to activation of calpain. This is the first demonstration that PLC-beta 3 is indeed cleaved by calpain upon platelet activation by physiological agonists. The cleavage of PLC-beta 3 evoked by thrombin and collagen but not ADP was correlated with irreversible aggregation, suggesting that the PLC-beta 3 modification may play a role in secondary irreversible aggregation in agonist-stimulated human platelets.

Amino Acid Sequence↗
PubMed1995

Defective phospholipase D activation in Ki-ras-transformed NIH3T3 cells: evidence for downstream effector of PLC-gamma 1 in PDGF-mediated signal transduction.

Platelet-derived growth factor (PDGF), a potent mitogen for fibroblasts and many other cell types, was used to examine phosphatidylcholine-specific phospholipase D (PLD), phosphoinositide-specific phospholipase C (PI-PLC) and tyrosine phosphorylation in NIH3T3 fibroblast and its Ki-ras-transformed derivative, DT. When cells prelabeled with [3H] myristic acid were stimulated by 10 and 50 ng/ml of PDGF in presence of 0.3% butanol, formation of phosphatidylbutanol (PtdBut) was increased three to six fold in NIH3T3 fibroblasts whereas it was marginal in DT cells. Myo-[3H]inositol-labeled cells showed higher inositol phosphate production in nontransformed control fibroblasts, indicating higher phospholipase C activity compared to the transformed DT cells. PDGF caused increase in tyrosine phosphorylation of a group of proteins with 110-130 kDa, PLC-gamma 1 and PDGF receptor in NIH3T3 cells. There was no significant increase in tyrosine phosphorylation in both PDGF receptor and PLC-gamma 1 in DT cells. These results suggest that PLD acts as a downstream effector molecule of PLC-gamma 1 in the PDGF-mediated signal transduction pathway.

1-Butanol↗
PubMed1995

Evidence implicating phospholipase as a virulence factor of Candida albicans.

Three different approaches were used to investigate the role of extracellular phospholipases in the pathogenicity of Candida albicans. First, we compared 11 blood isolates of this yeast with an equal number of commensal strains isolated from the oral cavities of healthy volunteers. Blood isolates produced significantly more extracellular phospholipase activity than the commensal strains did. Second, two clinical isolates of C. albicans that differed in their levels of virulence in a newborn mouse model were compared for their ability to secrete phospholipases. The invasive strain produced significantly more extracellular phospholipase activity than the noninvasive strain did. Third, nine blood isolates were characterized for their phospholipase and proteinase production, germ tube formation, growth, and adherence to and damage of endothelial cells in vitro. These factors were analyzed subsequently to determine whether they predicted mortality in a mouse model of hematogenously disseminated candidiasis. By proportional hazard analysis, the relative risk of death was 5.6-fold higher (95% confidence interval, 1.672 to 18.84 [P < 0.005]) in the mice infected with the higher-phospholipase-secreting strains than in the low-phospholipase secretors. None of the other putative virulence factors predicted mortality. Characterization of phospholipases secreted by three of the blood isolates showed that these strains secreted both phospholipase B and lysophospholipase-transacylase activities. These results implicate extracellular phospholipase as a virulence factor in the pathogenesis of hematogenous infections caused by C. albicans.

Acyltransferases↗
PubMed1995

[The involvement of protein kinase C isozymes in activation of phospholipase D and secretion of serotonin in rat basophilic leukemia cells].

Western blot analysis shows the presence of protein kinase C (PKC) alpha, beta1, beta2, delta, epsilon, zeta isozymes in rat basophilic leukemia (RBL-2H3) cells. Antigen stimulation caused preferential translocation of protein kinase C (PKC) delta and epsilon to membranes. On the other hand, when the cells were stimulated by phorbol 12-myristate 13-acetate (PMA) or ionophore (A23187), PKCalpha and beta were predominantly translocated. Phospholipase D (PLD) was activated by three stimulants in order: A23187 > PMA >> antigen. Therefore, PKCalpha and beta seem to be involved in the activation of PLD in RBL cells. The translocation of all PKC isozymes in A23187 stimulation was weak. Therefore, PLD activation in A23187 stimulation may require some other factors than PKC which are associated with the increase of Ca2+ in the cells. The cells stimulated by antigen secreted serotonin to the same level as the cells stimulated by A23187. In both stimulation, PKCalpha and epsilon were translocated to almost similar level, suggesting that PKCalpha and epsilon are involved in the secretion. All PKC isozymes except zeta were markedly translocated in PMA stimulation, but secretion did not occur. These results indicate that translocation of PKC alpha and beta may be associated with PLD activation and also that both translocation of PKCalpha and epsilon and intracellular calcium increase are required for serotonin secretion in RBL cells.

Animals↗
PubMed1994

Thrombin-mediated phosphoinositide hydrolysis in Chinese hamster ovary cells overexpressing phospholipase C-delta 1.

The regulatory mechanism(s) of a phosphoinositide-specific phospholipase C, PLC-delta 1, was investigated using a clone of stably overexpressed PLC-delta 1 (PLC delta 30 cells) in Chinese hamster ovary cells. Thrombin stimulation of PLC delta 30 cells exhibited 6.5-fold increase in total inositol phosphates (InsP), which was significantly higher than that in the vector-transfected (V1) cells (2.0-fold). AIF-4 increased InsP accumulation in both V1 and PLC delta 30 cells, and pertussis toxin partially blocked InsP accumulation in thrombin-stimulated PLC delta 30 cells. Guanosine thiotriphosphate (GTP gamma S) markedly potentiated thrombin-stimulated InsP generation in permeabilized PLC delta 30 cells compared with V1 cells, suggesting possible involvement of a G-protein (s) in the activation of PLC-delta 1. In PLC delta 30 cells, ionomycin-induced significant InsP generation and thrombin-stimulated InsP generation were completely inhibited by addition of EGTA. Furthermore, the stimulatory effects of thrombin plus GTP gamma S in PLC delta 30 cells were more sensitive to change in free calcium concentration than in V1 cells. Suppression by 12-O-tetradecanoylphorbol 13-acetate of thrombin-stimulated InsP accumulation was not affected by increasing Ca2+ concentration. These results indicate that thrombin-induced PLC-delta 1 activation is regulated via both G-protein(s) and calcium.

Animals↗
PubMed1994

Proteolytic modification of membrane-associated phospholipase C-beta by mu-calpain enhances its activation by G-protein beta gamma subunits in human platelets.

Membrane-associated phosphoinositide-phospholipase C (PI-PLC)-beta (150 kDa) and its truncated forms (100 kDa and 45 kDa) were purified from human platelets. The 100 kDa PI-PLC-beta was found to be activated to a greater extent by brain G-protein beta gamma subunits compared to the intact 150 kDa enzyme. Furthermore, treatment with mu-calpain of the intact PI-PLC-beta (150 kDa) caused a marked augmentation of its activation by beta gamma subunits. This enhanced PLC activation by beta gamma subunits was due to truncation by mu-calpain, producing a 100 kDa PI-PLC, but not by another protease, thrombin.

Blood Platelets↗
PubMed1994

Reduced expression of phospholipase C-delta, a signal-transducing enzyme, in rat colon neoplasms induced by methylazoxymethanol acetate.

Phospholipase C (PLC), which hydrolyzes phosphoinositides, has been implicated as a key enzyme in signal transduction. We examined the expression of an isozyme of PLC, PLC-delta, in rat colon neoplasms induced by methylazoxymethanol (MAM) acetate. Large-bowel neoplasms were observed in five of 10 rats given MAM acetate (25 mg/kg body weight, by interperitoneal injection at 6 and 7 wk of age) 40 wk after treatment. Expression of PLC-delta in the neoplasms was not detected by northern blot analysis, and a low level of expression was detected by immunoblot analysis, although PLC-delta expression was apparent in the non-neoplastic colon mucosae of MAM acetate-treated rats as well as in the colon mucosae of control rats. Furthermore, analysis by reverse transcriptase-polymerase chain reaction revealed that the ratio of the expression of PLC-delta to that of beta-actin in the neoplasms was significantly lower than the ratios in the non-neoplastic colon mucosae of carcinogen-treated and control rats (P < 0.01). However, the ornithine decarboxylase (ODC) activity in the neoplasms was significantly greater than that of the non-neoplastic and control mucosae (P < 0.001). The differences in the levels of PLC-delta expression in neoplastic and non-neoplastic tissues and the inverse correlation of PLC-delta expression with ODC activity may suggest that PLC-delta has little effect on the PLC-mediated mitogenic signaling system, at least in MAM acetate-induced colon neoplasms in rats.

Animals↗
PubMed1994

Phosphoinositide hydrolysis in Ki-ras-transformed fibroblasts stimulated by platelet-derived growth factor and bradykinin.

Signal transduction in response to platelet-derived growth factor (PDGF)-BB and bradykinin (BK) have been examined by measuring inositol polyphosphate formation in NIH3T3 fibroblast and v-Ki-ras-transformed NIH3T3 fibroblast (DT). The PDGF-induced inositol polyphosphate formation in NIH3T3 was greater than that in DT cells, in which autophosphorylation of PDGF receptor and tyrosine phosphorylation of phospholipase C (PLC)-gamma 1 were suppressed when examined by immunoblotting with anti-phosphotyrosine antibody. On the other hand, BK-stimulation produced a much higher level of inositol polyphosphate in DT cells which have a greater number of BK receptors. These results indicate that in Ki-ras transformed cells the decrease (caused by PDGF) and the increase (caused by BK) in phosphoinositide hydrolysis are due to the defective autophosphorylation of PDGF receptors leading to a reduction in PLC-gamma 1 tyrosine phosphorylation and the overexpression of BK receptors, respectively.

3T3 Cells↗
PubMed1993

Potentiation by cholera toxin of bradykinin-induced inositol phosphate production in the osteoblast-like cell line MC3T3-E1.

Cells of the osteoblastic cell line MC3T3-E1 were shown to contain at least three phosphatidylinositol-specific phospholipase C (PI-PLC) isoenzymes (PLC-beta, PLC-gamma and PLC-delta) by Western blotting analysis with various anti-PLC antibodies. Stimulation of inositol phosphate production in MC3T3-E1 cells by bradykinin (BK) occurred via a GTP-binding protein. Inositol phosphate formation on stimulation by BK was not affected by pretreatment with pertussis toxin, whereas it was potentiated by cholera toxin pretreatment. Elevation of cellular cyclic AMP levels by brief pretreatment with dibutyryl cyclic AMP or forskolin failed to enhance the BK-mediated generation of inositol phosphates, but long-term preincubation with these agents partially mimicked the action of the cholera toxin. Cholera toxin also caused an increase in BK receptor number. Cycloheximide, a protein biosynthesis inhibitor, prevented the potentiating actions of the cholera toxin and the cyclic AMP-elevating agents on BK-induced inositol phosphate production, and also inhibited the increase in BK receptor number. The specific binding of [3H]BK to the whole MC3T3-E1 cells in the presence or absence of cholera toxin was completely inhibited by the B2 BK receptor antagonist D-Arg[Hyp3,Thi5,8,D-Phe7]BK, but not by the B1 BK receptor agonist des-Arg9-BK. These data suggest that the activation of PI-PLC induced by cholera toxin in BK-stimulated MC3T3-E1 cells was caused by an enhancement of the synthesis of BK receptor protein(s), at least part of which was mediated by a sustained increase in the intracellular level of cyclic AMP.

Adenosine Diphosphate Ribose↗
PubMed1993

Phospholipase C activity in cerebrospinal fluid following subarachnoid hemorrhage related to brain damage.

Phosphoinositide-specific phospholipase C (PLC) activities were measured in CSF from patients after subarachnoid hemorrhage (SAH). Their PLC activities were significantly higher than those in control CSF. Moreover, there was an obvious correlation between the PLC activity in CSF collected on day 3 and the preoperative clinical grade. The PLC activity was also closely correlated with the level of neuron-specific enolase as a marker of brain damage. Furthermore, the PLC activities were partially purified from CSF of patients after SAH and were immunologically identified to be PLC beta, PLC gamma, and PLC delta. These results suggest that PLCs are released into the CSF from brain tissue in conjunction with the initial hemorrhage and that their activity may reflect the extent of brain damage.

Aneurysm, Ruptured↗
PubMed1992

Effects of gelsolin on human platelet cytosolic phosphoinositide-phospholipase C isozymes.

The effective resolution of human platelet cytosolic phosphoinositide-phospholipase C (PLC) revealed five distinct activity peaks by Q-Sepharose and heparin-Sepharose column chromatographies when assayed using phosphatidylinositol (PI) and phosphatidylinositol 4,5-bisphosphate (PIP2). The results of Western blotting analysis with various antibodies against PLC isozymes showed that peak-Ia (PLC-delta type), peak-Ib (PLC-gamma 1 type), and peak-IIc (PLC-beta type) and two unidentified activity peaks (PLC-IIa and PLC-IIb) were present in human platelet cytosol. A protein with guanosine 5'-3-O-(thio)triphosphate-binding activity was coeluted with the PLC-IIa and was purified to homogeneity. It exhibited 86- and 42-kDa polypeptide bands upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis which were identified as gelsolin and actin by immunostaining, respectively. Large amounts of gelsolin/actin (1:1) complex "gelsolin complex" were detected in the PLC-delta and PLC-gamma 1 fractions. The PLC-gamma 1 and the gelsolin complex were co-immunoprecipitated by the antibody raised against PLC-gamma 1. Furthermore, the partially purified bovine brain PLC-gamma 1 fraction also was found to be associated with the gelsolin complex and the association was released by the addition of 1% sodium cholate. This finding has prompted us to examine effects of the gelsolin complex and the free gelsolin on activities of the above PLC isoforms from platelet cytosol. The gelsolin complex did not affect the PIP2 hydrolyzing activities of all PLC isoforms. In contrast, the purified gelsolin inhibited distinctly PIP2 hydrolyses by PLC-Ia (delta), PLC-Ib (gamma 1), and PLC-IIa (unidentified), whereas the inhibitory effects for PLC-IIb (unidentified) and PLC-IIc (beta) were moderate. The inhibitory effect of gelsolin on PIP2-hydrolysis by PLC-gamma 1 was diminished by a large amount of PIP2 substrate. These results suggested that the inhibition of PLC by gelsolin is due to sequestration of substrate PIP2 by its competitive binding.

Animals↗
PubMed1992

Metabolic fate of [14C]triglyceride-entrapped lactosylceramide-bearing liposomes after intravenous injection into mouse.

We investigated the distribution and fate of liposomes after their intravenous injection into a mouse. Liposomes were composed of dimyristoylphosphatidylcholine, cholesterol and dicetylphosphate (7:2:1, molar ratio) with or without lactosylceramide. They were characterized as small unilamellar vesicles, approximately 100 nm in diameter, using gel-exclusion chromatography on a Sephacryl S-1000, freeze-fracture electron microscope and dynamic light scattering method. Liposomes were very stable in serum as seen by the results of leakage of the entrapped marker and electrophoresis experiments. We demonstrated that liposomes were internalized by way of an endocytotic process via coated vesicles detected in the electron microscope. The increase in liver uptake of lactosylceramide-bearing liposomes was mostly accounted for by enhanced uptake in the parenchymal cells, while uptake by non-parenchymal cells was only slightly increased. This observation supported the notion that a galactose-specific receptor was involved in liver uptake of lactosylceramide liposomes. The lactosylceramide-bearing liposomes were preferentially recovered in the liver and were found to first be predominantly localized in the mitochondria-lysosomal fraction. They were then decomposed by lysosomal enzymes, and the hydrolyzed components were reincorporated into membrane phospholipids in the microsomal fraction. At the same time, a rapid and reversible exchange of phosphatidylcholine between microsomes and mitochondria was demonstrated.

Absorption↗