Biomedical subjects

E Fujihira

Publications and source records attributed to E Fujihira.

At least 19 recordsLinked to original sources

PubMed2000

Excitatory amino acid release in the locus coeruleus during naloxone-precipitated morphine withdrawal in adjuvant arthritic rats.

OBJECTIVE AND DESIGN: Excitatory amino acid levels in the locus coeruleus (LC) and the behavioral signs during naloxone-precipitated withdrawal in arthritic rats treated with chronic morphine were investigated by in vivo microdialysis. METHODS: Increases in glutamate (Glu) and aspartate (Asp) were noted after naloxone (48 nmol/5 microl, LC)-precipitated withdrawal from normal and adjuvant arthritic rats which had been intracerebroventricularly infused for 3 days with morphine (26 nmol/l microl/h). RESULTS: The increases in Glu and Asp levels on morphine withdrawal in normal rats were attenuated following naloxone challenge in the morphine-dependent arthritic rats. Moreover, behavioral signs during morphine withdrawal were detected following the naloxone challenge in both the morphine-dependent normal and adjuvant arthritic rats, but not in the saline-infused controls. CONCLUSIONS: These results show that the attenuation of Glu and Asp release from the LC in the adjuvant arthritic rats might explain the anti-inflammatory and analgesic effects of mu-opioids in adjuvant arthritic rats.

Animals↗

PubMed2000

Molecular construction of a multidrug exporter system, AcrAB: molecular interaction between AcrA and AcrB, and cleavage of the N-terminal signal sequence of AcrA.

The AcrAB system of Escherichia coli is an intrinsic efflux protein with a broad substrate specificity. AcrA was thought to be localized in the periplasmic space, and to be linked to AcrB and TolC. The AcrAB-TolC system directly exports diverse substrates from the cell interior to the medium. In this study, we have determined the cellular localization of AcrA. By using the osmotic shock method, sucrose density gradient centrifugation, urea washing and Western blotting analysis, we reveal that AcrA is a peripheral inner membrane protein. A mutant plasmid encoding both the AcrA-TetBCt fusion protein and the AcrB-His fusion protein was constructed. Membrane vesicles prepared from cells expressing these fusion proteins were solubilized and AcrB-His was immunoprecipitated with an anti-polyhistidine antibody. After SDS-PAGE, Western blotting was performed with anti-TetBCt antiserum, resulting in the appearance of a 40 kDa band, indicating that AcrA co-precipitated with AcrB. Next we performed site-directed chemical labeling of Cys-introduced mutants of AcrA with [(14)C]N-ethylmaleimide. As judged from the labeling pattern and the molecular mass shift, the N-terminus of AcrA was removed and the mature protein is on the periplasmic surface. On the other hand, C25A mutants retained the N-terminal signal sequence on the cytoplasmic side of the membrane. We conclude that AcrA exists as a complex with AcrB on the periplasmic surface of the inner membrane after removal of the signal sequence.

Bacterial Proteins↗

PubMed2000

Effects of Aconitum alkaloid kobusine and pseudokobusine derivatives on cutaneous blood flow in mice; II.

Aconitum alkaloids of the C20-diterpenoid type, kobusine (1) and pseudokobusine (2), their anisoyl, veratroyl, p-nitrobenzoyl, nicotinoyl or pivaloyl derivatives, and dehydrokobusine and N,6-seco-6-dehydropseudokobusine derivatives were examined for their peripheral vaso-activities by laser-flowmetrical measurement of the cutaneous blood flow in the hind foot of mice after intravenous administration. Kobusine 15-anisoate (4), 11-veratroate (5), 15-veratroate (6), 11-pivaloate (9) and 15-pivaloate (10) were significantly effective at a low dose of 0.5 or 0.05 mg/kg. Pseudokobusine derivatives were all active at 1, 0.5 or 0.05 mg/kg, and the effects of pseudokobusine 15-anisoate (13), 15-veratroate (16) and 15-p-nitrobenzoate (19) at 0.1 mg/kg were remarkable. Yesoline (26) and alkaloid (28) were significantly effective at a low dose of 1 mg/kg, whereas yesonine (25) and N-acetyl-N,6-seco-6-dehydropseudokobusine (27) were inactive. Dehydrokobusine derivatives (29, 30) were significantly effective at a low dose of 0.5 or 0.1 mg/kg. It is thought that the hydroxyl groups of alkaloids, especially a free OH group of 2 at C-6, are important for action on the peripheral vasculature leading to dilatation, and the results indicated that esterification of the hydroxyl group at C-15 with either anisoate, veratroate orp-nitrobenzoate may contribute to enhancement of the activity of the parent alkaloids.

Animals↗

PubMed2000

Anti-type I allergic mechanisms of mao-bushi-saishin-to in mice.

We investigated the anti-allergic effect of mao-bushi-saishin-to (MBS) on the type I allergy model in mice. When MBS was administered orally at a dose of 0.5 or 1.0 g/kg, edema of the footpad, the amount of plasma IgE and the ratio of eosinophilic leukocytes in peritoneal exudate cells were all dose-relatedly suppressed. Moreover to investigate the anti-type I allergic mechanisms of MBS, enzyme-linked immunosorbent assay was performed to determine the interleukin (IL)-4, IL-5 and interferon (IFN)-gamma production from splenocytes that were stimulated by pokeweed mitogen for 48 h. In addition, we assayed IgE production from splenic B cells stimulated with the lipopolysaccharide and IL-4 for 7 days. MBS inhibited the IL-4 and IFN-gamma production, but IL-5 and IgE production were not affected. Thus possibly, the inhibition of IL-4 production may partially be involved in the expression of the anti-type I allergic effects of MBS.

Animals↗

PubMed1998

Membrane topology of the staphylococcal tetracycline efflux protein Tet(K) determined by antibacterial resistance gene fusion.

To determine the membrane topology of Tet(K), conventional antibiotic resistance genes were used as reporter molecules. A series of fusion genes comprising a resistance gene-beta-lactamase (amp) or chloramphenicol acetyl transferase (cat)-and one loop of the 14 putative transmembrane segments of Tet(K) was constructed. Escherichia coli TG1 with the tet(K)-amp fusion gene at the site of the putative periplasmic loop showed resistance to ampicillin, but the bacterium with the tet(K)-cat fusion gene at the site of the putative cytoplasmic loop showed resistance to chloramphenicol. These findings supported a topology of 14 membrane-spanning segments for Tet(K). Three exceptional cases were observed, which were apparently due to the presence of an acidic residue, Glu, in the preceding transmembrane segment. Mutants in which these acidic residues was substituted for alanine were also constructed, and the effect of glutamic acid in the transmembrane segment on the topology of the fusion proteins was examined.

Amino Acid Sequence↗

PubMed1998

Anti-inflammatory effects of mao-bushi-saishin-to in mice and rats.

Effects of Mao-Bushi-Saishin-to (MBS) on anti-inflammatory activities were examined in mice and rats. MBS significantly inhibited the increase in vascular permeability induced by acetic acid, the ear edema induced by arachidonic acid and phorbol ester, and the cutaneous extravasation induced by bradykinin and histamine. MBS, however, was not effective against the serotonin-induced cutaneous permeability increase in mice. MBS significantly inhibited carrageenin-induced hind foot edema and cotton pellet-induced granulation tissue growth in rats. These results show that MBS may exert anti-inflammatory effects through the underlying mechanism(s) of preventing mediator release from mast cells and macrophages.

Acetic Acid↗

PubMed1998

Effects of Aconitum alkaloid kobusine and pseudokobusine derivatives on cutaneous blood flow in mice.

Aconitum alkaloids of the C20-diterpenoid type, kobusine (1) and pseudokobusine (2), and their acetyl, benzoyl, propionyl or cinnamoyl derivatives are examined for their peripheral vaso-activities by laser-flowmetrical measurement of the cutaneous blood flow in the hind foot of mice after intravenous administration. A dose-relationship of maximally increased blood flow after the administration of either of the Aconitum alkaloids existed. Kobusine 15-acetate (4), 11-benzoate (6) and 15-benzoate (7) were significantly effective at a low dose of 1 mg/kg, whereas the other kobusine derivatives were all inactive. Alkaloid 2, alone, and the 11-acetate (14), 15-acetate (15), 15-propionate (22) and 15-cinnamoate (25) were all active at 1 mg/kg and the effect of 14 at 5 mg/kg was remarkable. The activity of 2 was abolished by esterification of the hydroxyl group at C-6. Alkaloid 15 at 5 mg/kg showed a pattern of time course of blood flow in which the increase was rapidly replaced with a decrease below the basal flow, probably suggesting the effect of excessive doses. Conclusively, it is considered that the hydroxyl groups of alkaloids, especially a free OH group of 2 at C-6, are important for action on the peripheral vasculature leading to dilatation, and these results indicated that esterification of the hydroxyl group at C-15 with either acetate or benzoate may contribute to enhancement of the activity of the parent alkaloids.

Alkaloids↗

PubMed1997

Roles of acidic residues in the hydrophilic loop regions of metal-tetracycline/H+ antiporter Tet(K) of Staphylococcus aureus.

Three transmembrane glutamic acid residues play essential roles in the metal-tetracycline/H+ antiporter Tet(K) of Staphylococcus aureus [Fujihira et al., FEBS Lett. 391 (1996) 243-246]. In the putative hydrophilic loop region of the Tet(K) and Tet(L) proteins, six acidic residues are conserved. Asp74, Asp200, Asp318 and Glu381 are located on the putative cytoplasmic side, and Asp39 and Glu345 on the putative periplasmic side. These residues were replaced by a neutral amino acid residue or a charge-conserved one. In contrast to the transmembrane glutamic acid residues, the replacement of the two glutamic acid residues (Glu345 and Glu381) did not affect the tetracycline resistance level. Out of the other four aspartic acid residues, the only essential residue is Asp318, any replacement of which resulted in complete loss of the tetracycline resistance and transport activity. Asp318 is located in cytoplasmic loop 10-11 in the putative 14-transmembrane-segment topology of Tet(K). In the case of the tetracycline exporters of Gram-negative bacteria, the only essential acidic residue in the cytoplasmic loop region is located in loop 2-3 [Yamaguchi et al., Biochemistry 31 (1992) 8344-8348]. It may be a general role for tetracycline efflux proteins that three transmembrane and one cytoplasmic acidic residues are mandatory for the tetracycline transport function.

Amino Acid Sequence↗

PubMed1997

A novel compound, 1,1-dimethyl-5(1-hydroxypropyl)-4,6,7-trimethylindan, is an effective inhibitor of the tet(K) gene-encoded metal-tetracycline/H+ antiporter of Staphylococcus aureus.

A novel indan derivative, 1,1-dimethyl-5-(1-hydroxypropyl)-4,6,7-trimethylindan (Ro 07-3149), was found to be a strong inhibitor of the tet(K) gene-encoded tetracycline/H+ antiporter of Staphylococcus aureus. One micromole of this compound per mg membrane protein was enough for complete inhibition of the Tet(K)-mediated tetracycline transport and tetracycline-coupled proton transport, without the energy state of the membrane being affected. The mode of inhibition was non-competitive. Although this compound caused membrane de-energization at a high concentration, the IC50 value for de-energization (7.3 micromol/mg membrane protein) was about 17 times and 33 times higher than the values for Tet(K)-mediated proton/tetracycline antiport and [3H]tetracycline transport, respectively, indicating that the inhibitory action of Ro 07-3149 is not due to the uncoupling effect of the inhibitor.

Antiporters↗

PubMed1996

Transmembrane glutamic acid residues play essential roles in the metal-tetracycline/H+ antiporter of Staphylococcus aureus.

Three transmembrane aspartyl residues play essential roles in the transposon Tn10-encoded metal-tetracycline/H+ antiporter (Tet(B)) [Yamaguchi, A. et al. (1992) J. Biol. Chem. 267, 7490-7498]. The tetK gene-encoding tetracycline resistance protein (Tet(K)) of Staphylococcus aureus mediates metal-tetracycline/H+ antiport similarly to Tet(B); however, it has no transmembrane aspartyl residue. On the other hand, Tet(K) has three glutamyl residues, Glu-30, Glu-152 and Glu-397, in the putative transmembrane regions. In the present work, tet(K) gene was expressed in Escherichia coli and the transport activity was measured in everted membrane vesicles. When these glutamyl residues were replaced with Gln, the tetracycline transport activity was almost completely lost, indicating the important roles of these residues in Tet(K). In the case of Glu-397, even the charge-conserved mutation to Asp caused complete loss of the activity. On the other hand, the mutation of Glu-30 and Glu-152 to Asp resulted in significant retention of transport activity. These results are similar to those on the mutation of the three transmembrane aspartyl residues in Tet(B), indicating that the transmembrane glutamyl residues in Tet(K) play roles similar to those of the transmembrane aspartyl residues in Tet(B).

Amino Acid Sequence↗

PubMed1995

The tetracycline efflux protein encoded by the tet(K) gene from Staphylococcus aureus is a metal-tetracycline/H+ antiporter.

The tet(K) gene from Staphylococcus aureus was highly expressed in Escherichia coli by an alteration of its initiation codon from TTG to ATG and its ribosome-binding sequence from GAGG to GGAGG [Noguchi, N. et al. (1994) Biol. Pharm. Bull. 17, 352-355]. The inverted membrane vesicles prepared from the tet(K)-expressing cells showed respiration-dependent [3H]tetracycline transport comparable to the vesicles from the tet(B)-expressing cells. The affinity of Tet(K) vesicles to tetracycline was the same as that of Tet(B) vesicles, whereas the former Vmax value was about 60% of the latter one. Contrary to Tet(B) vesicles, Tet(K) vesicles showed no significant minocycline uptake, which was consistent with the low minocycline resistance of the Tet(K)-producing cells. The tetracycline transport mediated by Tet(K) vesicles was coupled with proton transport and the translocation of 60Co2+ ions as well as in Tet(B) vesicles. This observation indicates that the class K tetracycline resistance determinant from Gram-positive bacteria also encodes a metal-tetracycline/H+ antiporter that is functionally similar to that encoded by tet(B), although there is a considerable difference in the primary sequences and the putative topologies of these Tet proteins.

Antiporters↗

PubMed1994

Biophysical parameters of glucocorticoid-binding proteins from rat inflammatory tissues.

The complexes of [3H]glucocorticoid and cytoplasmic proteins of the inflammatory tissue were characterized by the gel filtration and by the sucrose density gradient centrifugation. Protein-bound [3H]cortisol sedimented at about 4 S with a Stokes radius of about 4 nm and an apparent molecular weight (Mr) of about 70 kDa, irrespective with the presence of 0.4 M KCl. The complex of [3H]triamcinolone acetonide (TAA) and protein sedimented at about 7 S and showed about 170 kDa of Mr and about 6 nm of the Stokes radius in the absence of 0.4 M KCl. In the presence of 0.4 M KCl, the [3H]TAA-protein complex changed into a smaller form (4 S) with about 4 nm of the Stokes radius and about 60 kDa of Mr. This suggests that the so-called glucocorticoid receptor of the inflammatory tissue associates with a 110 kDa protein.

Animals↗

PubMed1993

Effects of H2-antihistamines in murine models of immediate hypersensitivity and chronic inflammation.

Effects of H2-antihistamines alone and in combination with H1-antihistamines on immediate hypersensitivity and chronic inflammation in mice and rats were investigated. The mouse PCA reaction was inhibited partially by a minimum effective dose of diphenhydramine and blocked completely when administered simultaneously with either famotidine, ranitidine or cimetidine. There was similar synergism between H1- and H2-antihistamines in inhibiting the PCA-like reaction in rat paw. Famotidine alone was able to inhibit those two reactions in a large oral dose of 100 mg/kg. A five-day course of therapy with H2-antihistamines alone or in combination with pyrilamine, suppressed effectively the chronic swelling induced in the hind paw of BUF rats. From these results, it is deduced that simultaneous blockage of both histamine H1- and H2-receptors may be necessary for sufficient inhibition of the microvascular permeability increase in some kinds of anaphylactic reactions, and that histamine, mainly interacting with H2-receptors, may take an important role for activation of a certain phase of chronic inflammation where bone destruction, blood capillary growth and mast cell degranulation involve.

Animals↗

PubMed1990

Characteristics of glucocorticoid-binding by inflammatory tissue of rats.

Glucocorticoid-binding activities of the granuloma cytosol were compared with those of the liver cytosol and of the serum in vitro. The granuloma cytosol bound cortisol (HC) about 4-fold higher than dexamethasone (DX) and triamcinolone acetonide (TA); the liver cytosol bound these two synthetic agonists more than HC. The kinetic parameters of the glucocorticoid-binding components of the granuloma and the liver cytosols were studied by the Scatchard method. The binding components of the granuloma cytosol had a single class of binding sites with high affinity for these three steroids, whereas the binding site of the liver cytosol had negative cooperativity or consisted of two distinct classes, because its Scatchard plot showed a hyperbolic curve. The granuloma glucocorticoid-binding components will be protein since their binding was prevented by a trypsin treatment and completely lost by heating at 60 C for 5 min. Heating at 25 or 37 degrees C for 30 min did not affect the HC-binding activity of the granuloma cytosol, regardless of prelabeling with the steroid. The binding activity for DX and TA were heat-labile and completely lost by heating the cytosol at 37 degrees C for 30 min without the respective steroid. The results of thermal inactivation and ammonium sulfate fractionation show the granuloma HC-binding protein closely resembles corticosteroid-binding globulin (CBG). From enzymatic determination of hemoglobin in tissue cytosols, attribution of the contaminating blood to the HC-binding activity of the cytosol is considered to be negligible.

Ammonium Sulfate↗

PubMed1986

Prostaglandins: a possible mediator to inhibit hepatic drug metabolism in adjuvant arthritic rats.

In the liver of adjuvant arthritic rats perfused with a hemoglobin-free buffer solution, the rate of metabolism of a model drug, 2,6-dichloro-4-nitroanisole, was approximately half that of the control, while the bile flow rate was normal. Granulation tissue extracts and arthritic rat serum had no effect on the activity of CNA metabolism in normal rat liver preparations. In the perfused normal rat liver, the rate of CNA metabolism was inhibited by addition of prostaglandin (PG) E1, PGE2, and PGF2 alpha, respectively, in a final concentration of 0.5 microM. The inhibition by PGE1 was increased in the concentration range from 0.1 to 2.5 microM. The bile flow rate was not affected by the added PGs. However, these PGs had no direct effect on the CNA demethylating activity of the isolated hepatocytes from normal rat liver in a high concentration of 10 microM. Serotonin stimulated slightly CNA metabolism and bile production in the perfused livers by the intermittent infusion, but was without effect in the isolated hepatocytes. Epinephrine and histamine had no significant effect on CNA metabolism in both liver preparations. A similar pattern of the inhibition of CNA metabolism by PGs was reproduced in the normal rat liver perfused with the medium containing the supernatant of the hepatic nonparenchymal cells incubated in the presence of PGE1. The involvement of liver sinusoidal cells as secretory cells in depression of hepatic drug metabolism has been discussed.

Alprostadil↗

PubMed1986

Mouse IgE-mediated paw anaphylaxis in mice and rats.

Anaphylactic swelling is induced in the hind paws of mice and rats by intravenous challenge of antigen 72 h after subplantar injection of mouse IgE-rich antiserum into the paws, and can be quantified rapidly and accurately by measuring paw thickness. The edema model described may serve as an alternative to passive cutaneous anaphylaxis for studying immediate allergic reactions with mouse homocytotropic antibodies and screening potential antiallergic drugs.

Animals↗

PubMed1985

Hypersensitivity to endotoxin hepatotoxicity in rats with inflammatory lesions.

The ratio of sinusoidal nonparenchymal cells to hepatocytes in rat liver was significantly increased following induction of inflammation, and decreased after subsequent exposure to endotoxin, particularly in the region around the terminal portal venules. Rats with inflammatory lesions were more sensitive to endotoxin hepatocytotoxicity than normal controls, as judged from the dose-dependent increase in activity of serum transaminases and from the extent of liver tissue injury. In addition, these animals, which were already in a state of depletion of hepatic glycogen, demonstrated marked hyperglycemia 24 h after endotoxin administration in small doses of less than 2 mg/kg.

Alanine Transaminase↗

PubMed1984

Mouse paw anaphylaxis.

Anaphylactic swelling passively transferred to the hind paws of mice with homologous antibodies was investigated. The ascites fluid containing homocytotropic antibodies of the IgG1 class in a high concentration was obtained by repeated injections of bovine serum albumin in complete Freund's adjuvant into the peritoneal cavity of female ICR mice. The standard procedure for mouse paw anaphylaxis defined was as follows: female ICR mice received subcutaneously 25 microliters of a 1:20 diluted ascites in the hind footpad and 2 h later were given intravenously 1.0 mg of the antigen in saline, and subsequently at 5 min intervals paw thickness was assessed by means of a simple thickness gauge. Maximal paw edema occurred invariably 15 min after antigen challenge. Sex difference in response was not observed. Heating at 56 degrees C for 4 h resulted in a reduction of the activity of mouse ascites by approximately 40%. Histological examinations revealed that paw anaphylaxis was accompanied by mast cell degranulation and release of biologically active constituents from mast cell granules. Promethazine, pyrilamine, diphenhydramine, methysergide, cyproheptadine, ketotifen and phenoxybenzamine provided significant protection from the anaphylactic reaction in mice, while bromocriptine, dexamethasone and indomethacin were inactive. Relative drug effectiveness in paw anaphylaxis, passive cutaneous anaphylaxis and active and passive systemic anaphylaxis in mice was compared and discussed. Consequently, it was shown that mouse paw anaphylaxis was not only convenient for quantitative assessment of homocytotropic antibodies, but also useful in the routine work for screening of potential anti-allergic drugs.

Animals↗