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T Nakae

Publications and source records attributed to T Nakae.

At least 55 records · Page 3Linked to original sources
PubMed1997

The role of mex-gene products in antibiotic extrusion in Pseudomonas aeruginosa.

The antibiotic extrusion machinery in Pseudomonas aeruginosa is assembled from the mex-operon encoded proteins, OprM and MexA-MexB, connecting the outer and inner membranes. To envisage the role of these proteins in antibiotic extrusion and resistance, we employed the gene replacement technique to construct mutants deficient in mexA, mexB, or oprM, and all possible combinations of these genes. Using the Southern and the Western blotting methods, we confirmed that only the target genes were disrupted. All the mutants deficient in OprM exhibited a 4 to 16 times higher susceptibility against quinolone antibiotics, chloramphenicol, and gentamicin than the parent strain. The mutants deficient in MexA or MexB or both MexA and MexB were only 2 to 4 times more susceptible to these antibiotics than the parent strain. All the mutants lacking MexA, MexB, or OprM showed stereospecific hypersusceptibility to beta-lactam antibiotics than the parent strain. However, the extent of susceptibility to each beta-lactam was comparable among the mutants. Strains lacking OprM accumulated the highest level of ciprofloxacin among all these isogenic strains. The strains lacking either MexA or MexB accumulated lower levels of ciprofloxacin than the mutant lacking OprM, but the levels were still higher than in the parent strain. The results are consistent with the antibiotic susceptibility of these strains. These results suggest that the extrusion of antibiotics occurs most efficiently with a whole assembly of MexA/B-OprM, but it remains a possibility that OprM interacts with a putative inner membrane pump(s).

4-Quinolones↗
PubMed1997

Trisubstituted benzene leukotriene B4 receptor antagonists: synthesis and structure-activity relationships.

A series of trisubstituted benzenes which demonstrate leukotriene B4 (LTB4, 1) receptor affinity was prepared. Previous trisubstituted benzenes from our laboratory showed high affinity to the LTB4 receptor but demonstrated agonist activity in functional assays. Compound 3a, the initial lead compound of this new series, showed only modest affinity (IC50 = 0.20 microM). However, 3a was a receptor antagonist with no demonstrable agonist activity up to 30 microM. Further modification of the lipid tail and aryl head groups region led to the discovery of 3b (ONO-4057). This compound, free of agonist activity, possesses high affinity to the LTB4 receptor (Ki = 3.7 +/- 0.9 nM).

Benzene Derivatives↗
PubMed1997

Synthesis of structural analogues of leukotriene B4 and their receptor binding activity.

Structural analogues of leukotriene B4 (LTB4) were designed based on the plausible conformation of LTB4 (1). Joining C-7-C-9 of the conformer A or B into an aromatic ring system led to the discovery of benzene analogues 2, 4 and 6a. Joining C-4-C-9 of the conformer C or D into an aromatic ring system led to the discovery of analogues 3, 5 and 7. The compounds examined in this study were evaluated as to their inhibition of [3H] LTB4 binding to human neutrophils, and by a secondary intact human neutrophil functional assay for agonist/antagonist activity. The first analogues prepared, compounds 2-7, demonstrated moderate potency in the LTB4 receptor binding assay. The modification of these compounds by the introduction of another substituent into the aromatic ring produced a marked increase in receptor binding (28c, IC50 = 0.020 microM; 38c, IC50 = 0.020 microM; 52a, IC50 = 0.020 microM; 52b, IC50 = 0.018 microM). Most of these structural analogues of LTB4 demonstrated agonist activity. Of the analogues prepared in this study, only compound 57 demonstrated weak LTB4 receptor antagonist activity, at 10 microM.

Cell Aggregation↗
PubMed1997

A novel cryohemagglutinin associated with adherence of enteroaggregative Escherichia coli.

Strain O42 (serotype O44:H18) of enteroaggregative Escherichia coli (EAggEC) has been shown to be pathogenic in volunteer experiments. This strain exhibited plasmid (pO42)-encoded D-mannose-resistant hemagglutinating activity (MRHA) that was detected only at low temperatures (e.g., 0 degrees C) and only with human erythrocytes. The production of this cryogenic MRHA (cryo-MRHA) was observed when the bacteria were grown in liquid media and was strictly regulated by bacterial growth temperatures. Transposon-insertion mutagenesis revealed that this MRHA is associated with (i) bacterial clump formation in liquid cultures, (ii) bacterial adherence to HEp-2 cells as well as (Formalin-fixed) human colonic mucosa, and (iii) production of a 16-kDa outer membrane protein. The PCR designed on the basis of the determined cryo-MRHA-associated DNA sequence sharply distinguished strain O42 from eight other EAggEC strains whose MRHAs were detected at both cold and room temperatures to the same (or similar) extent. Strain O42 possessed a surface layer that may enhance the pO42-mediated adherence. The data suggest that a plasmid-encoded cryo-MRHA is a candidate for a major adhesin of EAggEC strain O42.

Animals↗
PubMed1997

[Antibiotic extrusion and multidrug resistance].

Many bacteria evolved to have machineries that extrude noxious compounds across the cell membranes. Extrusion of such compounds through membranes of gram-negative bacteria is a complex, since the compounds need to cross two membranes. Typically, Pseudomonas aeruginosa and Escherichia coli produce power-operated antibiotic extrusion pumps and the exit membrane channel located in the inner and the outer membranes, respectively. The membrane fusion protein anchoring in the inner membrane and largely protruding the periplasmic space connects these two membrane proteins to facilitate extrusion of antibiotics.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Multiantibiotic resistance caused by active drug extrusion in Pseudomonas aeruginosa and other gram-negative bacteria.

All living organisms have been exposed to noxious compounds throughout their long evolutionary history and those surviving have evolved to fabricate devices that detoxicate and extrude these life threatening substances. It is likely, therefore, that all viable organisms, from bacteria to mammals, are equipped with active extrusion machinery. When bacteria are attacked by antibiotics, they use these tactics to combat the drugs and to develop resistance. Drugs extrusion machinery in Gram-negative bacteria is complex, consisting of the inner membrane transporter which acts as an energy-dependent extrusion pump; a binding protein which presumably connect both membranes; and the outer membrane exit channel. The extrusion pump assemblies are often encoded by chromosomal genes and might be expressed by mutation(s) or induced in the presence of drug(s).

Anti-Bacterial Agents↗
PubMed1996

Protein D2 porin of the Pseudomonas aeruginosa outer membrane bears the protease activity.

We report here our discovery that protein D2 of the outer membrane of Pseudomonas aeruginosa is a novel porin bearing protease activity. Homogeneously purified protein D2 hydrolyzed several synthetic peptides according to the Michaelis-Menten kinetics. A specific serine protease inhibitor, diisopropyl fluorophosphate (DFP), inactivated the protease activity and [3H]DFP covalently labeled protein D2. We tested the effect of two monoclonal antibodies raised against protein D2 on the protease activity. One antibody lowered the protease activity to about 20%, while the other enhanced it to about 300% of that without antibody. In addition, the fractions derived from the outer membrane of the protein D2-deficient mutants showed negligible protease activity, whereas similarly fractionated outer membrane proteins of the protein D2-positive parent strain showed strong protease activity.

Amino Acid Sequence↗
PubMed1996

Specific interaction of the protein-D2 porin of Pseudomonas aeruginosa with antibiotics.

The protein-D2 porin of Pseudomonas aeruginosa is lacking in carbapenem or fluoroquinolone-resistant strains and hence was thought to facilitate the diffusion of these antibiotics. We examined the effect of several antibiotics on the single channel conductivity of protein-D2 in planar lipid bilayers and found that fluoroquinolones and carbapenems at concentrations of around 1 mM caused closure of the protein-D2 channel. Tetracycline, ampicillin, piperacillin, and latamoxef did not exert any detectable effect on the protein-D2 channel activity.

Anti-Bacterial Agents↗
PubMed1996

Protein C (OprC) of the outer membrane of Pseudomonas aeruginosa is a copper-regulated channel protein.

Protein C (OprC) of the outer membrane of Pseudomonas aeruginosa forms small channels, as assayed by the liposome swelling method. We report here that OprC functions as a channel-forming and copper-binding protein. OprC purified to homogeneity formed a channel in planar lipid bilayers with an ion conductance of about 200 pS in 1 M NaCl. Cloning and sequencing of the gene encoding OprC revealed that it specified a polypeptide comprising 723 and 668 amino acid residues for the precursor and mature polypeptides (M(r) 73,372), respectively. The amino acid sequence of OprC showed the highest degree of similarity with that of NosA of Pseudomonas stutzeri (65% sequence identity) which conveys Cu2+ to intracellular acceptor(s). OprC showed high copper-binding activity (Kd = 2.6 microM) in aqueous solution containing surfactant. The expression of OprC appeared to be repressed by exogenous Cu2+ and derepressed by anaerobiosis in the presence of nitrate. These results suggest that OprC might be involved in copper utilization.

Amino Acid Sequence↗
PubMed1996

Mechanism of membrane damage by El Tor hemolysin of Vibrio cholerae O1.

El Tor hemolysin (ETH; molecular mass, 65 kDa) derived from Vibrio cholerae O1 spontaneously assembled oligomeric aggregates on the membranes of rabbit erythrocyte ghosts and liposomes. Membrane-associated oligomers were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting into two to nine bands with apparent molecular masses of 170 to 350 kDa. ETH assembled oligomers on a liposomal membrane consisting of phosphatidylcholine and cholesterol, but not on a membrane of phosphatidylcholine alone. Cholesterol could be replaced with diosgenin or ergosterol but not with 5alpha-cholestane-3-one, suggesting that sterol is essential for the oligomerization. The treatment of carboxyfluorescein-encapsulated liposomes with ETH caused a rapid release of carboxyfluorescein into the medium. Because dextrin 20 (molecular mass, 900 Da) osmotically protected ETH-mediated hemolysis, this hemolysis is likely to be caused by pore formation on the membrane. The pore size(s) estimated from osmotic protection assays was in the range of 1.2 to 1.6 nm. The pore formed on a rabbit erythrocyte membrane was confirmed morphologically by electron microscopy. Thus, we provide evidence that ETH damages the target by the assembly of hemolysin oligomers and pore formation on the membrane.

Animals↗
PubMed1996

Characterization of a novel hemagglutinin of diarrhea-associated Escherichia coli that has characteristics of diffusely adhering E. coli and enteroaggregative E. coli.

Escherichia coli 73-1 (serotype O73:H33) and 5-2 (serotype O89:H-) isolated from patients with diarrhea adhered to tissue culture cells (HeLa and HEp-2) as well as coverslips (plastic and glass) in a diffuse pattern. Adherence of strain 73-1 was mediated by a 110-kbp plasmid designated pEDA1 and correlated with D-mannose-resistant hemagglutinin (MRHA) detected with bovine, sheep, or human erythrocytes. The MRHA region was duplicated on pEDA1 and mediated the production of the 57-kDa outer membrane protein whose N-terminal amino acid sequence was hydrophobic. In accordance with MRHA and adherence, the 57-kDa outer membrane protein was observed best at 37 degrees C and to a lesser extent at 25 degrees C. In human intestine, adherence to mucus and colonic epithelium was obvious. No detectable pili were observed. The enteroaggregative E. coli heat-stable enterotoxin 1 (EAST1) gene, whose nucleotide sequence was 99.1% homologous to that of enteroaggregative E. coli, was present adjacent to the MRHA region on pEDA1. Strain 5-2 also exhibited MRHA activities and adherence and had sequences corresponding to those of the MRHA region and EAST1 gene. The data suggest that strain 73-1 (and strain 5-2), which has characteristics of both diffusely adhering E. coli and enteroaggregative E. coli, possesses a novel hemagglutinin associated with diffuse adherence.

Amino Acid Sequence↗
PubMed1996

Acceptor specificity of cyclodextrin glucanotransferase from an alkalophilic Bacillus species and synthesis of glucosyl rhamnose.

Cyclodextrin glucanotransferase [1,4-alpha-D-glucan 4-alpha-D-(1,4-alpha-D-glucano)-transferase (cyclizing), EC 2. 4. 1. 19] from an alkalophilic Bacillus species A2-5a had a wider acceptor specificity than that from B. macerans, which was similar to those from B. stearothermophilus and B. circulans. Glucosyl rhamnose produced by the CGTase was identified as glucopyranosyl-alpha-1,4-rhamnopyranose by alpha- and beta-glucosidase treatments, and 1H- and 13C-NMR analyses.

Bacillus↗
PubMed1996

Synthesis of neohesperidin glycosides and naringin glycosides by cyclodextrin glucanotransferase from an alkalophilic Bacillus species.

Cyclodextrin glucanotransferase from an alkalophilic Bacillus species produced neohesperidin monoglucoside and a series of its maltooligoglucosides by transglycosylation with neohesperidin as an acceptor and soluble starch as a donor. As the reaction using beta-CD as a donor at an alkaline pH was very effective for solubilizing neohesperidin, the amount of glycosides formed was increased. As a result, its amount with beta-CD at pH 10 was about 7 times greater than that with soluble starch at pH 5. Neohesperidin monoglucoside was purified from the reaction mixture by glucoamylase and naringinase treatments, an Amberlite XAD-16 column, a Sephadex LH20 column, and HPLC on an ODS column. The structure of the purified monoglucoside was identified as 3G-alpha-D-glucopyranosyl neohesperidin by FAB-MS, methylation analysis, and 1H- and 13C-NMR. The solubility of neohesperidin monoglucoside in water was approximately 1500 times higher than that of neohesperidin, and the bitterness of the monoglucoside was about 10 times less than that of neophesperidin. In addition, naringin was also glycosylated by the same method as neohesperidin, and its monoglucoside was identified as 3G-alpha-D-glucopyranosyl naringin. The solubility of naringin monoglucoside in water was also at least 1000 times higher than that of naringin without altering its bitterness.

Alkalies↗
PubMed1995

Role of porins in the antibiotic susceptibility of Pseudomonas aeruginosa: construction of mutants with deletions in the multiple porin genes.

We inserted deletions in the chromosomal genes of Pseudomonas aeruginosa coded for the outer membrane porins, proteins C, D2, or E1, and all possible combinations of these proteins by the gene replacement technique and selecting for imipenem-resistance. Determination of the minimum inhibitory concentrations of beta-lactams, fluoroquinolones, chloramphenicol and gentamicin in these mutants revealed that most mutants showed equal susceptibility to the porin-sufficient strain. The only exception was that imipenem and meropenem showed increased minimum inhibitory concentrations in all of the mutants lacking protein D2. These results firmly established that the P. aeruginosa porins identified so far form the pores do not accommodate the passage of most antipseudomonal antibiotics, with the exception of carbapenems.

Bacterial Outer Membrane Proteins↗
PubMed1995

Outer membrane permeability of beta-lactamase inhibitors in Pseudomonas aeruginosa.

Evaluation of four beta-lactamase inhibitors in terms of their outer membrane permeability in Pseudomonas aeruginosa revealed that sulbactam and tazobactam diffused most efficiently and equally well. That of BRL42715 appeared to be a factor of ten lower than that of the above two, but it showed the strongest beta-lactamase inhibitory activity. This is most likely due to its better beta-lactamase inactivating activity. BRL42715 at 1.56 micrograms ml-1 lowered the minimum inhibitory concentrations of ceftazidime and imipenem in a strain producing fully derepressed beta-lactamase and an undetectable level of the outer membrane protein OprD2.

Cell Membrane↗
PubMed1995

Expression of genes associated with antibiotic extrusion in Pseudomonas aeruginosa.

We cloned the gene(s) associated with multiantibiotic resistance in Pseudomonas aeruginosa from a mutant with elevated drug extrusion. The strain harboring cloned gene accumulated less amount of ofloxacin than the strain without the clone. By using Southern and northern blot analyses, we investigated whether multiantibiotic resistance is caused by gene amplification or overexpression. We found that all our multiantibiotic resistance is caused by gene amplification or overexpression. We found that all our multiantibiotic resistant mutants isolated earlier overexpressed mRNA homologous to the cloned gene(s). Overexpression of the 41 KDa and 110 KDa inner and the 50 KDa outer membrane proteins were detected. Other multiantibiotic resistant mutants including the nalB mutant overexpressed the mRNA and the membrane proteins, but the nfxB and nfxC mutants did little. We concluded that low antibiotic accumulation in the multiantibiotic resistant mutants is attributable to overexpression of the antibiotic extrusion machinery.

Cloning, Molecular↗