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E Yoshihara

Publications and source records attributed to E Yoshihara.

At least 37 records · Page 2Linked to original sources
PubMed1992

Nucleotide sequence of the protein D2 gene of Pseudomonas aeruginosa.

Protein D2 of the outer membrane of Pseudomonas aeruginosa was shown to form the imipenem-permeable pore. We cloned and sequenced the protein D2 gene. The protein D2 gene encodes a polypeptide with 443 amino acids consisting of 23 and 420 amino acid residues for the signal peptide and mature polypeptide (M(r), 46,010), respectively. Protein D2 contains the highest molar ratio of glycine and no cysteine. The polar amino acids are scattered throughout the sequence.

Amino Acid Sequence↗
PubMed1991

In vitro assembly of the functional porin trimer from dissociated monomers in Pseudomonas aeruginosa.

The molecular weights of monomeric and oligomeric forms of the newly identified porins, protein D2, of the outer membrane of Pseudomonas aeruginosa appeared to be 47,000 and 137,000, respectively, as determined by the light scattering technique. Presence of the trimeric aggregates of the homologous subunits in the intact outer membrane, the liposome membrane, and the non-ionic surfactant were confirmed through cross-linking experiments and immunoblotting techniques. The protein D2 monomers prepared in 0.1% of sodium dodecyl sulfate at 23 degrees C spontaneously reassembled into the trimeric aggregate when the surfactant dropped below critical concentration. The diffusion rates of saccharides and beta-lactam antibiotics through the liposome membranes reconstituted from the reassembled protein D2 trimers were indistinguishable from those of the native protein D2. This study shed some light on the porin trimer assembly as well as on the mechanism of carbapenem diffusion through the protein D2 pores.

Anti-Bacterial Agents↗
PubMed1990

Diffusion of beta-lactam antibiotics through liposome membranes reconstituted from purified porins of the outer membrane of Pseudomonas aeruginosa.

Determination of the rates of diffusion of beta-lactam antibiotics through purified Pseudomonas aeruginosa porins C, D2, and E in liposomes yielded the following results. (i) The rates of carbapenem (imipenem and meropenem) diffusion through the protein D2 pore were roughly 2 to 70 times higher than those through other porin pores. It is not clear why the protein D2 pore allowed rapid diffusion of carbapenems. The rates of diffusion of glucosamine and triglycine through the protein D2 pore were about 14 and 4 times higher, respectively, than that of an uncharged test solute with a similar Mr, glucose. (ii) The rates of diffusion of antipseudomonal anionic beta-lactams such as piperacillin, ceftazidime, cefsulodin, and aztreonam through the protein C pore were higher than those through other porin pores. This was probably due to the slightly larger pore size and the slight anion selectivity of protein C, since the apparent exclusion limit of the protein C pore for uncharged saccharides is higher than that of other porins and the rate of diffusion of gluconic acid through the protein C pore is about double that for glucose. (iii) The rates of diffusion of cefoperazone through all three species of porin were relatively high. These results indicate that the antipseudomonal beta-lactams permeate the P. aeruginosa outer membrane via newly identified porins.

Anti-Bacterial Agents↗
PubMed1990

Protection of Toxoplasma gondii-infected mice by stearylamine-bearing liposomes.

The cytotoxic activity of stearylamine-bearing liposomes against Toxoplasma gondii (RH strain) was examined. When tachyzoites were treated in vitro with liposomes consisting of 20 mol% stearylamine and 80 mol% phosphatidylcholine (130 micrograms/ml total lipids), more than 95% of the parasites were killed within 90 min. Intraperitoneal injection of 10 mg of 30 mol% stearylamine/70 mol% phosphatidylcholine-liposomes in mice shortly before or after T. gondii challenge afforded protection from death for more than 30 days to 70-80% of the treated mice, whereas all untreated mice succumbed within 9 days. The liposome-injected mice that survived remained symptom-free and behaved normally.

Amines↗
PubMed1989

Identification of porins in the outer membrane of Pseudomonas aeruginosa that form small diffusion pores.

The purified outer membrane proteins of Pseudomonas aeruginosa were reconstituted with phosphatidylcholine and dicetylphosphate into membrane vesicles, and these were tested by the liposome swelling method for the diffusion of saccharides with different Mr. Proteins C (Mr, 70,000), D (Mr, 46,000), and E (Mr, 43,000) were found to confer the monosaccharide-permeable pores in the reconstituted liposome membranes. The membrane vesicles containing proteins F (Mr, 34,000), G (Mr, 25,000), or H (Mr, 19,000) showed no detectable pore-forming activity. The pores formed by proteins C, D, or E appeared to be smaller than that formed by the Escherichia coli porins. The size of the solutes that permeated through the newly identified porins is similar to that through the intact and purified outer membrane of P. aeruginosa (Yoneyama, H., and Nakae, T. (1986) Eur. J. Biochem. 157, 33-38; Yoshihara, E., Gotoh, N., and Nakae, T. (1988) Biochem. Biophys. Res. Commun. 156, 470-476).

Bacterial Outer Membrane Proteins↗
PubMed1989

[Type specific protein antigen of group B streptococci [II]. Diversity of the c alpha protein antigen].

The c protein antigen of group B streptococci has been reported to consist of at least two acid extractable antigens, c alpha and c beta. We previously reported that strain FZ301 contain the c alpha and novel protein antigen, named as S antigen by Denka Seiken, and the c alpha antigen of this strain exhibit a more complicated structure. In this report described the analysis of the diversity of the c alpha antigen by immunoelectrophoresis and immunodiffusion. Hot acid extract from strain A909 produced a single precipitin band against the anti-121424c alpha serum but the 121424 extract produced double precipitin bands by immunoelectrophoresis. These c alpha antigens showed anodic migration, but the 121424c alpha antigens tended to migrate slower than the A909c alpha antigen. Moreover, the anti-A909c alpha serum produced a single precipitin band when tested after electrophoresis of the 121424 extract. When examined by immunodiffusion, the anti-121424c alpha serum produced two distinct precipitin bands with the 121424 and FZ301 extract, and these were reactions of identity. However, the A909 extract produced only one band which joined one of the two bands produced with the 121424 extract. These results shown that the strain 121424 contain c alpha and S antigen as well as strain FZ301. The precipitin band of the c alpha antigen in the A909 extract against the anti-A909 serum formed the spur with the reaction of the c alpha antigen in the 121424 or FZ301 extract on immunodiffusion. Moreover, the c alpha antibody in the anti-A909 serum failed to removed completely by repeated adsorption with strain 121424.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigens, Bacterial↗
PubMed1989

Role of protein F in maintaining structural integrity of the Pseudomonas aeruginosa outer membrane.

To investigate the functional role of protein F of the outer membrane of Pseudomonas aeruginosa, we isolated mutants devoid of protein F, and the defective gene was transferred to a wild-type strain by plasmid FP5-mediated conjugation. Chemical analyses of the protein F-deficient outer membrane revealed that the amount of outer membrane protein was reduced to 72 to 74% of that of the protein F-sufficient strain and that lipopolysaccharides and phospholipids increased to 117 to 123% and 135 to 136%, respectively. The mutants and the transconjugant showed the following characteristics: (i) growth rates of protein F-deficient strains in low-osmolarity medium (e.g., L broth containing 0.1% NaCl) were less than 1/10 the rate of the protein F-sufficient strain; (ii) protein F-deficient cells were rounded, and the outer membrane formed large protruded blebs; and (iii) the outer membrane became physically fragile, since a significant amount of periplasmic proteins leaked out and the cells became highly sensitive to osmotic shock. The results suggested that protein F plays an important role in morphogenesis and in maintaining the integrity of the outer membrane. Determination of the diffusion rates of saccharides and beta-lactam antibiotics showed that the protein F-deficient outer membrane had no detectable transport defect compared with the protein F-sufficient outer membrane. The MICs of antibiotics for the protein F-deficient strains were nearly identical to those for the protein F-sufficient strain.

Bacterial Outer Membrane Proteins↗
PubMed1988

In vitro demonstration by the rate assay of the presence of small pore in the outer membrane of Pseudomonas aeruginosa.

Determination of the rates of saccharide diffusions by the proteoliposomes showed that the outer membrane of Pseudomonas aeruginosa only possesses small diffusion pores and that protein F might have not been involved in the pore formation. Proteoliposomes containing stachyose or Dextan T-10 showed the same relative diffusion rates as measured by the liposome swelling method. Slopes of the lines, diffusion rate vs saccharide Mr, in the liposomes made of the P. aeruginosa and E. coli B outer membranes appeared to be -7.4 and -3.5, respectively. Intercepts of the lines with x-axis in the liposomes containing the P. aeruginosa and E. coli B outer membrane appeared to be about Mr, 220 and 320, respectively. Relative diffusion rates of saccharides through the liposome membranes reconstituted from the protein F-deficient outer membrane were superimposable with that of the protein F-sufficient outer membrane.

Cell Membrane↗
PubMed1988

In vitro lysis of the bloodstream forms of Trypanosoma brucei gambiense by stearylamine-bearing liposomes.

Cytolytic activity of liposomes consisting of stearylamine and phosphatidylcholine (SA/PC-liposomes) was examined in vitro against the bloodstream forms of Trypanosoma brucei gambiense. More than 99% of the cells (2 X 10(6)/ml) were killed within 30 min by treatment with 15 mol% SA/PC-liposomes (100 microM total lipids). As few as 1.2 X 10(12) liposomes per ml (equivalent to 2 nM liposome) showed trypanocidal activity. Fluorescence microscopy of cells treated with the dansylated SA/PC-liposomes suggested that the liposomes bound to and accumulated on the cell surface, eventually damaging the plasma membrane. SA/PC-liposomes showed no significant hemolysis when incubated with human and mouse erythrocytes under conditions that killed greater than 99.9% of the T. b. gambiense trypomastigotes. Human leukocytes were also shown to be less susceptible to SA/PC-liposomes than T. b. gambiense. These results may point to a new direction in strategy for therapy of African trypanosomiasis.

Amines↗
PubMed1987

Trypanocidal activity of the stearylamine-bearing liposome in vitro.

Liposome made of stearylamine and phosphatidylcholine showed the trypanocidal activity in vitro. Cytotoxicity of the liposome against Trypanosoma cruzi appeared to be the strongest in trypomastigotes followed by amastigotes and epimastigotes. Lysis of the human erythrocyte was undetectably low under the conditions that the liposome kills more than 95% of trypomastigotes. The liposome seems to damage the plasma membrane.

Amines↗
PubMed1986

Cytolytic activity of liposomes containing stearylamine.

In order to develop the cytotoxic liposome, the cytolytic effect of polycationic liposome was examined. Upon incubation of the stearylamine-containing liposome (stearylamine-liposome) with rabbit erythrocyte, a significant extent of hemolysis was observed. Hemolytic activity of the liposome depends on the amount of stearylamine in the liposome membrane. The plots of the initial rate of hemolysis versus the concentration of stearylamine-liposome showed a sigmoidal curve, suggesting that stearylamine-liposomes act cooperatively on the erythrocyte membrane. Hemolytic activity of stearylamine-liposome was markedly influenced by the composition of hydrocarbon chains of the phospholipids in the liposome membrane, suggesting that the membrane fluidity of stearylamine-liposome is important to evoke the hemolysis. Since the liposomes containing acidic phospholipids inhibited markedly the stearylamine-liposome-caused hemolysis, it is likely that the primary target of stearylamine-liposome is the negatively charged component(s) such as acidic phospholipids on the erythrocyte membrane. Furthermore, stearylamine-liposome induced the release of the intravesicular contents from the liposome made of acidic phospholipids but not from the liposome made of phosphatidylcholine only. These results suggest that stearylamine-liposome interacted with the negative charges of the erythrocyte membrane and eventually damaged the cell. Erythrocytes from rabbit, horse and guinea pig are highly susceptible to stearylamine-liposome but those from man, sheep, cow and chicken are less so.

Amines↗
PubMed1984

Hepatic 7-alkoxycoumarin O-dealkylase in mice: induction by beta-naphthoflavone of hepatic enzyme activity.

Hepatic 7-alkoxycoumarin O-dealkylation activities in control and beta-naphthoflavone-pretreated mice were determined. The O-demethylation and O-deethylation activities of 7-alkoxycoumarin in control mice were almost the same values, while the O-depropylation activity was lower than those of the other reactions. The O-dealkylase activity varied markedly among the 18 strains of mice surveyed, and strain-dependent differences in the cytochrome P-450 content were also detected among the strains. beta-Naphthoflavone induced O-dealkylation activity, especially O-deethylation and O-depropylation activities, only in ddY, DS and its substrains (A2-3, A3-1, C1-2 and Nh/+). C3H/He and C57BL/6J strains, but not in DBA/2, BALB/c and KYF/2 strains. The former strains of mice are thus classified as "responsive strains" to beta-naphthoflavone and the latter as "non-responsive strains". The O-dealkylase activity in other strains of mice were not clear in responsiveness to beta-naphthoflavone. The hepatic cytochrome P-450 content in responsive strains also increased upon pretreatment of the animals with beta-haphthoflavone. The results indicate marked strain differences in basal and beta-naphthoflavone-induced activity of hepatic 7-alkoxycoumarin O-dealkylase in mice.

7-Alkoxycoumarin O-Dealkylase↗
PubMed1983

Liver microsomal cytochrome P-450-dependent O-dealkylation reaction in various animals.

Liver microsomal O-dealkylation activity was determined using O-methyl, O-ethyl and O-propyl derivatives of p-nitrophenol, 7-hydroxycoumarin (umbelliferon) and 7-hydroxyphenoxazone (resorufin) as substrates. Microsomal O-dealkylation activities of p-nitrophenol and 7-hydroxycoumarin O-alkyl derivatives were of similar levels, but the activities of 7-hydroxyphenoxazone O-alkyl derivatives were very low compared with those of other substrates. Pretreatment of rats with beta-naphthoflavone resulted in the preferential increase of O-deethylation and O-depropylation activities regardless of the ring structure of the substrates, and the ratio of O-deethylation and O-depropylation activities to that of O-demethylation increased markedly. On the other hand, the O-dealkylase activity of all substrates increased generally upon pretreatment of the rats with phenobarbital, but the ratio of O-deethylase or O-depropylase activity to that of O-demethylase in the pretreated rats was not very different from that of the untreated animals. Hexobarbital inhibited competitively the O-dealkylation activity in control and phenobarbital-pretreated rat microsomes. On the other hand, the O-dealkylase activity in microsomes obtained from beta-naphthoflavone-pretreated rats was inhibited remarkably by alpha-naphthoflavone, but not in microsomes prepared from untreated and phenobarbital-pretreated rats. Based on these results, this report discusses the relationship between the alteration of O-dealkylation activity and the composition change of cytochrome P-450 in microsomal membrane. Species differences in the substrate specificity of the O-dealkylation reaction and in the responsiveness of the animals to typical inducers were also observed using liver microsomes obtained from several animals under various conditions.

Animals↗
PubMed1983

Biotransformation of coumarin derivatives. (2). Oxidative metabolism of 7-alkoxycoumarin by microsomal enzymes and a simple assay procedure for 7-alkoxycoumarin O-dealkylase.

The in vitro biotransformation of 7-alkoxycoumarin by rat liver microsomes was studied to develop a simple and accurate assay procedure for 7-alkoxycoumarin O-dealkylase. 7-Alkoxycoumarin was converted to the O-dealkylated metabolite, 7-hydroxycoumarin, by aerobic incubation of the parent compound with microsomes and NADPH, but the decreased amount of 7-alkoxycoumarin in the reaction mixture was several times higher than that of the 7-hydroxycoumarin produced during the incubation. The thin-layer chromatogram of the ether extractable metabolites in the reaction mixture showed the existence of several fluorescent metabolites including 7-hydroxycoumarin. Fluorescent properties of the parent compound, 7-alkoxycoumarin, and most of the metabolites differed from that of 7-hydroxycoumarin, but the reaction cofactor, NADPH, showed similar properties. Treatment of the reaction mixture with perchloric acid resulted in conversion of NADPH to the non-fluorescent form without any effect upon the fluorescent properties of 7-hydroxycoumarin and its related compounds. Based on these properties, an improved and simple in vitro fluorometric assay of the O-dealkylation of 7-alkoxycoumarin was developed. The method is applicable to routine determination of O-dealkylase activity in both isolated microsomes and whole homogenate. Species differences in the substrate specificity of the O-dealkylation reaction and in the responsiveness of animals to the inducer were observed even with use of the liver homogenate obtained from untreated and phenobarbital- or beta-naphthoflavone-pretreated animals, similar to what was observed with the microsomal system.

7-Alkoxycoumarin O-Dealkylase↗
PubMed1982

Biotransformation of coumarin derivatives (1). 7-alkoxycoumarin O-dealkylase in liver microsomes.

The in vitro metabolic fate of 7-alkoxycoumarin was studied using liver microsomes. Microsomal enzyme catalyzed dealkylation of 7-alkoxycoumarin to 7-hydroxycoumarin in the presence of NADPH and molecular oxygen as cofactors was found to be one of the metabolic pathways. The metabolite 7-hydroxycoumarin was further metabolized to unidentified metabolite(s) in the presence of NADPH and O2 at a very slow rate, while the formation of the conjugate of 7-hydroxycoumarin with glucuronic acid was observed in the presence of UDPGA. Microsomal 7-alkoxycoumarin O-dealkylase activity was altered by the substitution of the alkyl group of the substrate, and the substitutions to either an O-propyl or an O-butyl group resulted in a decrease of the enzyme activity. Species differences were observed in the substrate specificity of microsomal O-dealkylation. The O-dealkylase activities in rat liver microsomes were stimulated by pretreatment of the animals with phenobarbital, regardless of the O-alkyl substituent at the 7 position of the coumarin ring. On the other hand, pretreatment with 3-methylcholanthrene or beta-naphthoflavone resulted in marked increase of O-deethylation. O-depropylation and O-debutylation activities, but not of O-demethylation activity. Pretreatment of animals with beta-naphthoflavone also resulted in remarkable stimulation of 7-hydroxycoumarin-glucuronide formation by the microsomal enzyme, while the conversion of 7-hydroxycoumarin to unidentified metabolite(s) was activated by the pretreatment of rats with only phenobarbital. The O-dealkylation activities in liver microsomes from intact and phenobarbital pretreated rats were inhibited markedly by the addition of hexobarbital to the incubation mixture, but no inhibition was observed with alpha-naphthoflavone. On the other hand, the O-dealkylation activities in microsomes from beta-naphthoflavone-pretreated rats were inhibited remarkably by alpha-naphthoflavone. These results confirmed that several microsomal enzymes, including the cytochrome P-450's and UDP-glucuronyltransferase, participate in the biotransformation of 7-alkoxycoumarin, and these enzymes are regulated differently by inducers.

7-Alkoxycoumarin O-Dealkylase↗