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Biomedical subjects

H Tokuda

Publications and source records attributed to H Tokuda.

At least 217 records · Page 12Linked to original sources
PubMed1994

Targeted delivery of superoxide dismutase to macrophages via mannose receptor-mediated mechanism.

Human recombinant superoxide dismutase (SOD) was modified into a mannosylated form (Man-SOD), and its cellular uptake and inhibitory effect on superoxide anion release were studied in vitro, using cultured mouse peritoneal macrophages. [111In]Man-SOD was taken up by the macrophages to a great extent, whereas no significant uptake was observed for native and galactosylated SOD. The uptake of Man-SOD was inhibited significantly at a low temperature and by the presence of mannan, mannose and colchicine, demonstrating the targeted delivery of Man-SOD via mannose receptor-mediated endocytosis. Man-SOD exhibited a superior inhibitory effect on superoxide anion release from inflammatory macrophages stimulated by phorbol-myristate acetate. The present study suggested the potential of Man-SOD as a therapeutic agent for the inflammatory disease mediated by superoxide anions generated by macrophages.

Animals↗
PubMed1994

Prostaglandin F2 alpha activates phospholipase D independently from activation of protein kinase C in osteoblast-like cells.

We previously reported that prostaglandin F2 alpha (PGF2 alpha) receptor is coupled to pertussis toxin (PTX)-sensitive GTP-binding protein (G protein) in osteoblast-like MC3T3-E1 cells [Miwa et al. (1990): Biochem Biophys Res Commun 171:1229-1235]. In the present study, we examined the effect of PGF2 alpha on the activation of phosphatidylcholine-hydrolyzing phospholipase D in MC3T3-E1 cells. PGF2 alpha stimulated the formation of choline in a dose-dependent manner in the range between 10 nM and 10 microM. The formation of choline was stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C (PKC)-activating phorbol ester. 4 alpha-Phorbol 12, 13-didecanoate, a PKC-nonactivating phorbol ester, had little effect on choline formation. The formation of choline stimulated by a combination of PGF2 alpha and TPA was additive. Staurosporine, an inhibitor for protein kinases, which inhibited the effect of TPA on choline formation, dose-dependently enhanced the formation of choline induced by PGF2 alpha. NaF, an activator of G protein, stimulated the formation of choline. The formation of choline stimulated by a combination of PGF2 alpha and NaF was not additive. NaF-induced formation of choline was dose-dependently enhanced by staurosporine. PTX dose-dependently inhibited the PGF2 alpha-induced formation of choline. These results strongly suggest that PGF 2 alpha activates phospholipase D independently from the activation of PKC in osteoblast-like cells and PTX-sensitive G protein is involved in the PGF2 alpha-induced phospholipase D activation.

Alkaloids↗
PubMed1994

Effect of vitamin D3 on prostaglandin E2 synthesis in osteoblast-like cells.

We previously showed that prostaglandin (PG) F2 alpha stimulates PGE2 synthesis in osteoblast-like MC3T3-E1 cells, and that the activation of protein kinase C (PKC) amplifies the effect of PGF2 alpha through the potentiation of phospholipase A2 activity (H. Tokuda, Y. Oiso and O. Kozawa, J Cell Biochem. 48: 262-268, 1992). In the present study, we investigated the effects of vitamin D3 on PGF2 alpha-induced PGE2 synthesis in MC3T3-E1 cells. The pretreatment with 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], an active form of vitamin D3, significantly inhibited the PGF2 alpha-induced PGE2 synthesis in a dose-dependent manner in the range between 1 pM and 1 nM. This inhibitory effect of 1,25-(OH)2D3 was dependent on the time of pretreatment up to 8 h. On the contrary, the pretreatment with 24,25-dihydroxyvitamin D3, an inactive form of vitamin D3, had little effect on the PGF2 alpha-induced PGE2 synthesis in these cells. However, the pretreatment with 1,25-(OH)2D3 no longer affected the amplification of PGF2 alpha-induced PGE2 synthesis by 12-O-tetradecanoylphorbol-13-acetate, a PKC activator. These results strongly suggest that 1,25-(OH)2D3 inhibits PGF2 alpha-induced PGE2 synthesis in osteoblast-like cells, however, the activation of PKC reverses this inhibitory effect of 1,25-(OH)2D3.

24,25-Dihydroxyvitamin D 3↗
PubMed1994

Effect of retinoic acid on prostaglandin F2 alpha-induced prostaglandin E2 synthesis in osteoblast-like cells.

We previously reported that prostaglandin F2 alpha (PGF2 alpha) stimulates phosphoinositide hydrolysis via pertussis toxin-sensitive GTP-binding protein in osteoblast-like MC3T3-E1 cells (Miwa, Tokuda, Tsushita, Kotoyori, Takahashi, Ozaki, Kozawa and Oiso 1990) and that PGF2 alpha stimulates arachidonic acid release and prostaglandin E2 (PGE2) synthesis, and the activation of protein kinase C (PKC) amplifies the effect of PGF2 alpha in MC3T3-E1 cells (Tokuda, Oiso and Kozawa 1992). In the present study, we investigated the effect of retinoic acid (RA), a vitamin A (retinol) metabolite, on PGF2 alpha-induced PGE2 synthesis in MC3T3-E1 cells. The pretreatment with RA, which by itself had little effect on synthesis, significantly inhibited PGE2 synthesis induced by PGF2 alpha in a dose-dependent manner in the range between 1 nM and 0.1 microM. This effect of RA was dependent on the time of pretreatment up to 8 h. In addition, RA inhibited the amplification of PGF2 alpha-induced PGE2 synthesis by 12-O-tetradecanoylphorbol-13-acetate, known to be a PKC activator. However, RA had little effect on PGE2 synthesis induced by melittin, known as a phospholipase A2 activator. Moreover, pertussis toxin had little effect on arachidonic acid release induced by PGF2 alpha. These results strongly suggest that RA inhibits PGE2 synthesis induced by PGF2 alpha in osteoblast-like cells and the inhibitory effect is exerted at the point prior to the activation of phospholipase A2.

Cells, Cultured↗
PubMed1994

Inhibitory effects of cucurbitane triterpenoids on Epstein-Barr virus activation and two-stage carcinogenesis of skin tumors.

To search for possible anti-tumor-promoters, we carried out a primary screening of 21 cucurbitane triterpenoids using an in vitro assay system. Of these triterpenoids, scandenoside R6 (6), 23,24-dihydrocucurbitacin F (14), 25-acetyl-23,24-dihydrocucurbitacin F (15), 2-O-beta-D-glucopyranosyl-23,24-dihydrocucurbitacin F (17) and cucurbitacin F (18) exhibited significant inhibitory effects on Epstein-Barr virus (EBV) activation induced by the tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Further, compounds 14 and 17 exhibited remarkable anti-tumor-promotion effects on mouse skin tumor promotion in an in vivo two-stage carcinogenesis test.

Animals↗
PubMed1994

[Anti-tumor promoting activities of kampo prescriptions. II. Inhibitory effects of souseiryu-to on two-stage carcinogenesis of mouse skin tumors and mouse pulmonary tumors].

To search for possible anti-tumor promoters, we carried out a primary screening of fourteen kampo prescriptions utilizing their possible inhibitory effects on the Epstein-Barr virus early antigen (EBV-EA) activation which is induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). In these prescriptions, shouseiryu-to exhibited the most significant inhibitory effect on the EBV-EA activation. Furthermore, two-stage carcinogenesis of mouse skin tumors induced by 7,12-dimethylbenz[a]anthracene (DMBA) and TPA, and mouse pulmonary tumors induced by 4-nitroquinoline-N-oxide (4NQO) and glycerol were strongly inhibited to shouseiryu-to.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1994

Intracellular signaling mechanism of bradykinin in osteoblast-like cells: comparison with prostaglandin E2.

Bradykinin is recognized to be involved in the process of bone resorption in chronic inflammatory diseases. We previously reported that prostaglandin E2 (PGE2), known as a potent bone resorbing agent, induces phosphoinositide hydrolysis, cAMP production and Ca2+ influx in osteoblast-like MC3T3-E1 cells, and these dose-dependencies are different to one another. To clarify the signaling mechanism of bradykinin, we compared the intracellular signaling system of bradykinin with that of PGE2 in these cells. Bradykinin stimulated Ca2+ influx dose-dependently in the range between 0.1 nM and 0.1 microM even in the presence of nifedipine, a Ca2+ antagonist that inhibits the voltage-dependent L-type Ca2+ channel. The maximum effect of bradykinin (0.1 microM) on Ca2+ influx was almost as great as that of PGE2 (0.5 microM). Bradykinin had little effect on cAMP accumulation, while PGE2 significantly stimulated it. Bradykinin stimulated the formation of inositol phosphates much less strongly than PGE2. Bradykinin stimulated inositol 1, 4, 5-trisphosphate [Ins(1, 4, 5)P3] formation dose-dependently between 0.1 nM and 0.1 microM, and the dose-dependent curves of bradykinin-induced Ca2+ influx and Ins(1, 4, 5)P3 were similar. However, the maximum effect of PGE2 (10 microM) on Ins (1, 4, 5) P3 formation was about 2-fold higher than that of bradykinin (0.1 microM). These results suggest that bradykinin induces Ca2+ influx independent of the voltage-dependent L-type Ca2+ channel and phosphoinositide hydrolysis in a similar dose-dependent manner in osteoblast-like cells.

Bradykinin↗
PubMed1994

Effect of glucocorticoid on prostaglandin F2 alpha-induced prostaglandin E2 synthesis in osteoblast-like cells: inhibition of phosphoinositide hydrolysis by phospholipase C as well as phospholipase A2.

It is well known that osteoporosis is a common complication of patients with glucocorticoid excess. We showed previously that prostaglandin (PG) F2 alpha stimulates the synthesis of PGE2, a potent bone resorbing agent, and that the activation of protein kinase C amplifies the PGF2 alpha-induced PGE2 synthesis through the potentiation of phospholipase A2 activity in osteoblast-like MC3T3-E1 cells. In the present study, we examined the effect of dexamethasone on PGE2 synthesis induced by PGF2 alpha in MC3T3-E1 cells. The pretreatment with dexamethasone significantly inhibited the PGE2 synthesis in a dose-dependent manner in the range between 0.1 and 10 nmol/l in these cells. This effect of dexamethasone was dependent on the time of pretreatment up to 8 h. Dexamethasone also inhibited PGE2 synthesis induced by melittin, known as a phospholipase A2 activator. Furthermore, dexamethasone significantly inhibited the enhancement of PGF2 alpha- or melittin-induced PGE2 synthesis by 12-O-tetradecanoylphorbol-13-acetate, known as a protein kinase C activator. In addition, dexamethasone significantly inhibited PGF2 alpha-induced formation of inositol phosphates in a dose-dependent manner between 0.1 and 10 nmol/l in MC3T3-E1 cells. These results strongly suggest that glucocorticoid inhibits PGF2 alpha-induced PGE2 synthesis through the inhibition of phosphoinositide hydrolysis by phospholipase C as well as phospholipase A2 in osteoblast-like cells.

Animals↗
PubMed1994

[G-CSF and G-CSF receptor].

Relationship between leucocytosis and G-CSF blood concentration were examined. Increase in blood concentration of G-CSF of surgical patient and dialysis patient with leucocytosis were noticed. Determination of G-CSF receptor on leucocyte was performed by the flow cytometry using biotinylated G-CSF. Modulation of G-CSF receptor on leucocyte at surgery and dialysis was identified in vivo. Studies of G-CSF blood concentration and modulation of G-CSF receptor are useful in analysis of dynamics of leucocyte and analysis of infectious disease.

Granulocyte Colony-Stimulating Factor↗
PubMed1994

Dual effects of forskolin on glucose utilization in cultured fibroblasts, as observed with the use of glucose analogs.

It is well known that forskolin suppresses glucose transport in cell membranes. In the present study, however, we indicated a different effect of forskolin on the uptake of radioactive 2-deoxy-D-glucose (2-DG) by cultured fibroblasts between the values measured in the presence of unlabeled 2-DG and those measured in the presence of unlabeled glucose. In the former case, the uptake was inhibited by forskolin. On the contrary, [3H] 2-DG uptake in the presence of unlabeled glucose was increased by forskolin. These results suggest that the metabolic difference between the two sugars is involved in the above-mentioned difference in the effect of forskolin. This paper presents that forskolin has stimulatory effect on glycolysis in addition to an inhibitory effect on glucose transporter. The findings seem to be of another interest to indicate the relationship between membrane transport and subsequent intracellular metabolism of glucose.

Cell Line↗
PubMed1993

Specific uptake of succinylated proteins via a scavenger receptor-mediated mechanism in cultured brain microvessel endothelial cells.

Cellular uptake of succinylated catalase (Suc-CAT; Mw 227 kDa), bovine serum albumin (Suc-BSA; Mw 70 kDa), superoxide dismutase (Suc-SOD; Mw 34 kDa) and soybean trypsin inhibitor (Suc-STI; Mw 21 kDa) was studied using primary cultures of bovine brain microvessel endothelial cells (BMECs) developed as an in vitro model of the blood-brain barrier. Large succinylated proteins (Suc-CAT, Suc-BSA) were taken up by BMECs whereas significant uptake was not observed for native proteins and small succinylated proteins (Suc-SOD, Suc-STI). Uptake of Suc-BSA was significantly inhibited at 4 degrees C and in the presence of endocytosis inhibitors. Large succinylated proteins, maleylated BSA and dextran sulfate also showed competitive inhibition against Suc-BSA uptake while small succinylated proteins and carboxymethyl dextran did not show any effect. These results indicate that microvessel endothelial cells obtained from the brain endocytose succinylated proteins via a scavenger receptor-mediated mechanism for polyanions, and in addition, the importance of molecular weight or total numbers of anionic charges per one molecule of proteins is suggested. Usefulness of direct succinylation of proteins for their delivery to the brain capillary endothelium is thus demonstrated.

Animals↗
PubMed1993

Histidine residues are involved in translocation-coupled ATP hydrolysis by the Sec-A protein.

Treatment of SecA, an essential component of the protein translocation machinery of Escherichia coli, with a histidine-specific reagent, diethylpyrocarbonate, caused significant abolition of its translocation-coupled ATPase (translocation ATPase) activity, which requires a presecretory protein and membrane vesicles, whereas its endogenous ATPase (SecA-ATPase) activity was enhanced by a factor of 2. Diethylpyrocarbonate-treated SecA exhibited an absorption maximum at 240 nm due to the formation of N-carbethoxyhistidine. Upon the modification of about 5 of the total 22 histidine residues in the SecA molecule, both the abolition of its translocation ATPase activity and the enhancement of its SecA-ATPase activity occurred. Intact and modified SecA exhibited similar affinities for ATP, proOmpA and membranes, whereas Vmax of the translocation ATPase activity was significantly lower in the case of the modified SecA. ATP had no effect on the modification of SecA. Taken together, these results indicate that histidine residues susceptible to diethylpyrocarbonate are essential for the translocation ATPase, but not directly involved in the binding of ATP, proOmpA and membranes. A possible reason for the abolition of translocation ATPase is discussed.

Adenosine Triphosphatases↗
PubMed1993

Membrane vesicles containing overproduced SecY and SecE exhibit high translocation ATPase activity and countermovement of protons in a SecA- and presecretory protein-dependent manner.

Everted membrane vesicles were prepared from Escherichia coli cells containing either overproduced amounts (OP-membrane vesicles) or normal amounts (normal membrane vesicles) of SecY and SecE, both of which are essential components of the protein translocation apparatus. The rates of translocation of pro-OmpA were similar in the two types of membrane vesicles, whereas translocation ATPase activity, which requires SecA, a precursor protein (pro-OmpA), and membrane vesicles, was appreciably higher with OP-membrane vesicles than with normal membrane vesicles. Since ATP hydrolysis has been shown to take place at an earlier part of the translocation reaction, these results suggest that the overproduction of SecY and SecE enhanced the activity of the earlier process, but not the entire process, of the translocation reaction. The addition of pro-OmpA in the presence of SecA caused the partial collapse of delta pH (inside acidic) generated on OP-membrane vesicles, suggesting that protons come out from the inside of the membrane vesicles in a pro-OmpA-dependent manner. The collapse of delta pH caused by pro-OmpA required SecA, ATP, and SecY and was not detected when normal membrane vesicles were used. These results indicate that the early event of protein translocation, which requires the functioning of SecA, SecY, and SecE, causes the countermovement of protons.

Bacterial Outer Membrane Proteins↗
PubMed1993

Inhibitory effects of 3-nitrophloroglucinecarboxylic acid derivatives on Epstein-Barr virus early antigen induction.

The inhibitory effects of two series of 3-nitrophloroglucinecarboxylic acid derivatives, 3-nitro-2,4,6-trihydroxythiobenzamides (II) and 3-nitro-phloroglucinecarboxylates (III), on Epstein-Barr virus early antigen (EBV-EA) induction were examined using Raji cells. Some of them strongly inhibited the induction, N-nonyl and O-decyl derivatives being the most potent inhibitors among the thioamides and esters, respectively. These results suggest the possibility that these two 3-nitrophloroglucinecarboxylic acid derivatives may be listed as novel inhibitors of tumor promotion.

Antigens, Viral↗
PubMed1993

A novel membrane protein involved in protein translocation across the cytoplasmic membrane of Escherichia coli.

A novel factor, which is a membrane component of the protein translocation machinery of Escherichia coli, was discovered. This factor was found in the trichloracetic acid-soluble fraction of solubilized cytoplasmic membrane. The factor was purified to homogeneity by ion exchange column chromatographies and found to be a hydrophobic protein with a molecular mass of approximately 12 kDa. The factor caused > 20-fold stimulation of the protein translocation when it was reconstituted into proteoliposomes together with SecE and SecY. SecE, SecY, SecA and ATP were essential for the factor-dependent stimulation of the activity. The factor stimulated the translocation of all three precursor proteins examined, including authentic proOmpA. Stimulation of the translocation of proOmpF-Lpp, a model presecretory protein, was especially remarkable, since no translocation was observed unless proteoliposomes were reconstituted with the factor. Partial amino acid sequence of the purified factor was determined. An antibody raised against a synthetic peptide of this sequence inhibited the protein translocation into everted membrane vesicles, indicating that the factor is playing an important role in protein translocation into membrane vesicles. The partial amino acid sequence was found to coincide with that deduced from the reported DNA sequence of the upstream region of the leuU gene. Cloning and sequencing of the upstream region revealed the presence of a new open reading frame, which encodes a hydrophobic protein of 11.4 kDa. We propose that the factor is a general component of the protein translocation machinery of E. coli.

Adenosine Triphosphatases↗
PubMed1993

Effects of vitamin D3 on signaling by prostaglandin E2 in osteoblast-like cells.

We investigated the effects of vitamin D3 on the signaling pathways by prostaglandin E2 (PGE2) in osteoblast-like MC3T3-E1 cells. The pretreatment with 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3), an active form of vitamin D3, significantly inhibited cAMP accumulation induced by 10 microM PGE2 in a dose-dependent manner in the range between 1 pM and 1 nM. This effect of 1,25-(OH)2D3 was dependent on the time of pretreatment up to 8h. 1,25-(OH)2D3 also inhibited the cAMP accumulation induced by NaF, a GTP-binding protein activator, or forskolin which directly activates adenylate cyclase. On the other hand, 1,25-(OH)2D3 significantly inhibited PGE2-induced IP3 formation in a dose-dependent manner between 10 pM and 1 nM. However, 1,25-(OH)2D3 had little effect on NaF-induced IP3 formation. The pretreatment with 24,25-dihydroxyvitamin D3, an inactive form of vitamin D3, affected neither cAMP accumulation nor IP3 formation induced by PGE2. These results strongly suggest that 1,25-(OH)2D3 modulates the signaling by PGE2 in osteoblast-like cells as follows: the inhibitory effect on the cAMP production is exerted at a point downstream from adenylate cyclase and the inhibitory effect on the phosphoinositide hydrolysis is exerted at the point between the PGE2 receptor and GTP-binding protein, probably Gi2.

Animals↗