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T Nakae

Publications and source records attributed to T Nakae.

At least 37 records · Page 2Linked to original sources
PubMed2000

Assignment of the substrate-selective subunits of the MexEF-OprN multidrug efflux pump of Pseudomonas aeruginosa.

Pseudomonas aeruginosa expresses a low level of the MexAB-OprM efflux pump and shows natural resistance to many structurally and functionally diverse antibiotics. The mutation that has been referred to previously as nfxC expresses an additional efflux pump, MexEF-OprN, exhibiting resistance to fluoroquinolones, imipenem, and chloramphenicol and hypersusceptibility to beta-lactam antibiotics. To address the antibiotic specificity of the MexEF-OprN efflux pump, we introduced a plasmid carrying the mexEF-oprN operon into P. aeruginosa lacking the mexAB-oprM operon. The transformants exhibited resistance to fluoroquinolones, trimethoprim, and chloramphenicol but, unlike most nfxC-type mutants, did not show beta-lactam hypersusceptibility. The transformants exhibited additional resistance to tetracycline. In the next experiment, we analyzed the MexEF-OprN pump subunit(s) responsible for substrate selectivity by expressing MexE, MexF, OprN, and MexEF in strains lacking MexA, MexB, OprM, and MexAB, respectively. The MexEF-OprM/DeltaMexAB transformants exhibited MexEF-OprN-type pump function that rendered the strains resistant to fluoroquinolones and chloramphenicol but did not change susceptibility to beta-lactam antibiotics compared with the host strain. The MexAB-OprN/DeltaOprM, MexAF-OprM/DeltaMexB, and MexEB-OprM/DeltaMexA mutants exhibited antibiotic susceptibility indistinguishable from that in the mutant lacking both types of efflux pumps. The results imply that the MexEF-OprM pump selects substrates by a MexEF functional unit. Interestingly, OprN did not link functionally with the MexAB complex, despite the fact that OprM interacted functionally with MexEF.

Anti-Bacterial Agents↗
PubMed2000

Atrial double potentials associated with the elimination of the electrical connection between the coronary sinus (CS) and the left atrium in two cases of Wolff-Parkinson-White syndrome with a CS-connected accessory pathway.

A curious retrograde conduction in connection with the coronary sinus (CS) musculature was observed in 2 patients. After the failed ablation procedure, the atrial electrogram during ventricular pacing presented double potentials, the first component of which was sharp and with an activation sequence that was the same before ablation (CS distal to proximal). The second component of the double potentials was dull and had a decremental property; its activation sequence was in reverse (proximal to distal). In both cases, the first component disappeared after successful ablation. These findings suggest that the first component was the CS electrogram conducted over the accessory pathway and the second component was the left atrial electrogram conducted through the inter-atrial septum. The separation of each electrogram is probably the result of a block between the accessory pathway connected to the CS musculature and the left atrium. These are unusual cases of an accessory pathway connected to the CS musculature, which separates the left atrial myocardium at the distal portion from the ostium.

Adult↗
PubMed1999

nalB-type mutations causing the overexpression of the MexAB-OprM efflux pump are located in the mexR gene of the Pseudomonas aeruginosa chromosome.

Two mutations, one at the nalB and the other at the mexR locus, in the Pseudomonas aeruginosa chromosome are known to cause overexpression of the MexAB-OprM efflux pump. Based on the following results, we concluded that nalB is the mutation that has occurred within the mexR gene of the P. aeruginosa chromosome. (i) Nucleotide sequencing of the mex operon upstream region of 21 independent nalB-type mutants including the original nalB9 revealed that all the mutations were located within the mexR gene. The mutations were classified into three different groups and nine types including single base substitutions, single base deletions and base insertions. (ii) Substitution of the mutant mexR with the wild-type mexR and replacement of the wild-type mexR with a defined mexR mutation resulted in the expression of wild-type and nalB-type MexAB-OprM respectively, which was confirmed by testing the antibiotic susceptibility and beta-galactosidase activity of the mexA-lacZ translational fusion.

Bacterial Outer Membrane Proteins↗
PubMed1999

Interplay between the efflux pump and the outer membrane permeability barrier in fluorescent dye accumulation in Pseudomonas aeruginosa.

Pseudomonas aeruginosa encodes three types of xenobiotic efflux pumps, MexAB-OprM, MexCD-OprJ, and MexEF-OprN, which are regulated by the nalB, nfxB, and nfxC genes, respectively, and their high expression renders the cells resistant to multiple species of antibiotics. We evaluated the role of the outer membrane permeability barrier and the efflux pump in lowering the intracellular concentration of fluorescent probes. The wild-type, nalB, nfxB, and nfxC strains with an intact outer membrane showed equally high capability in draining out intracellular fluorescent dye, 2-(4-dimethylaminostyryl)-1-ethylpyridinium and ethidium bromide. When the outer membrane barrier was dismantled by the EDTA treatment, wild-type, nfxC, nfxB, and nalB strains showed significantly different levels of dye accumulation. The polymyxin B-treated cells showed an even more pronounced difference in dye accumulation among the nfxC, nfxB, and nalB mutants. We concluded from these results that the xenobiotic extrusion pumps interplay with the outer membrane permeability barrier in lowering the intracellular substrate concentration. Among three extrusion pumps in P. aeruginosa, MexAB-OprM was the most efficient, followed by MexCD-OprJ and MexEF-OprN pumps for the fluorescent dye extrusion.

Bacterial Outer Membrane Proteins↗
PubMed1999

Membrane topology of the xenobiotic-exporting subunit, MexB, of the MexA,B-OprM extrusion pump in Pseudomonas aeruginosa.

The MexA,B-OprM efflux pump assembly of Pseudomonas aeruginosa consists of two inner membrane proteins and one outer membrane protein. The cytoplasmic membrane protein, MexB, appears to function as the xenobiotic-exporting subunit, whereas the MexA and OprM proteins are supposed to function as the membrane fusion protein and the outer membrane channel protein, respectively. Computer-aided hydropathy analyses of MexB predicted the presence of up to 17 potential transmembrane segments. To verify the prediction, we analyzed the membrane topology of MexB using the alkaline phosphatase gene fusion method. We obtained the following unique characteristics. MexB bears 12 membrane spanning segments leaving both the amino and carboxyl termini in the cytoplasmic side of the inner membrane. Both the first and fourth periplasmic loops had very long hydrophilic domains containing 311 and 314 amino acid residues, respectively. This fact suggests that these loops may interact with other pump subunits, such as the membrane fusion protein MexA and the outer membrane protein OprM. Alignment of the amino- and the carboxyl-terminal halves of MexB showed a 30% homology and transmembrane segments 1, 2, 3, 4, 5, and 6 could be overlaid with the segments 7, 8, 9, 10, 11, and 12, respectively. This result suggested that the MexB has a 2-fold repeat that strengthen the experimentally determined topology model. This paper reports the structure of the pump subunit, MexB, of the MexA,B-OprM efflux pump assembly. This is the first time to verify the topology of the resistant-nodulation-division efflux pump protein.

Amino Acid Sequence↗
PubMed1999

Two forms of Vibrio cholerae O1 El Tor hemolysin derived from identical precursor protein.

Vibrio cholerae O1 grown in heart infusion broth produces two forms of El Tor hemolysin (ETH) monomers of 65 and 50 kDa. These monomers form several different sizes of mixed oligomers ranging from 180 to 280 kDa in the liposomal membranes. We found that the N-terminal amino acid sequences, NH2-Trp-Pro-Ala-Pro-Ala-Asn-Ser-Glu, of both the 65- and 50-kDa toxins were identical. We assumed, therefore, that the 65- and 50-kDa toxins were derivatives of the identical precursor protein and the 50-kDa protein was a truncated derivative of 65-kDa ETH. To substantiate this assumption, we treated the 260-kDa oligomer with trypsin and obtained a 190-kDa oligomer. This 190-kDa oligomer consisted of only the 50-kDa subunits. Both 260- and 190-kDa oligomers formed ion channels indistinguishable from each other in planar lipid bilayers. These results suggest that the essential part of the ETH in forming the membrane-damaging aggregate is a 50-kDa protein.

Bacterial Proteins↗
PubMed1999

Plummer's disease with spontaneous progression to hypothyroidism.

A case of Plummer's disease that spontaneously progressed to hypothyroidism is presented. A 49-year-old female visited our hospital because of a 3 kg decrease in body weight during the previous month and a painless nodule in the right anterior area of her neck. A diagnosis of Plummer's disease was made based on the results of thyroid function tests, thyroid scintigrams, and an ultrasonogram, but the patient's disease followed an usual clinical course. About two months later, she gradually developed manifestations of permanent hypothyroidism, and anti-thyroid autoantibodies became positive. In spite of continuous administration of levothyroxine sodium, uptake of 99mTcO4- to the nodule was unchanged or rather increased according to the consecutive thyroid scintigraphies. These results suggested that this case represented an autonomously functioning nodule with underlying silent thyroiditis and Hashimoto's disease.

Autoantibodies↗
PubMed1999

Molecular cloning and characterization of the oprQ gene coding for outer membrane protein OprE3 of Pseudomonas aeruginosa.

We cloned and characterized the oprQ gene coding for outer membrane protein OprE3 of Pseudomonas aeruginosa PAO1. The oprQ gene was composed of 1,275 base pairs including a sequence encoding for the signal sequence and a mature protein with a Mr of 44,602. Computer-aided alignment and hydropathy analyses of the predicted amino acid sequences suggested that OprE3 is a transmembrane protein homologous to outer membrane proteins of P. aeruginosa such as OprD2 (OprD) porin and OprE1 (OprE) porin. Susceptibility to several antibiotics of the strains lacking or overproducing OprE3 was indistinguishable from that of the wild-type strain, suggesting that OprE3 is unlikely involved in the diffusion of carbapenems and other beta-lactam antibiotics.

Amino Acid Sequence↗
PubMed1999

Resistance to beta-lactam antibiotics in Pseudomonas aeruginosa due to interplay between the MexAB-OprM efflux pump and beta-lactamase.

We evaluated the roles of the MexAB-OprM efflux pump and beta-lactamase in beta-lactam resistance in Pseudomonas aeruginosa by constructing OprM-deficient, OprM basal level, and OprM fully expressed mutants from beta-lactamase-negative, -inducible, and -overexpressed strains. We conclude that, with the notable exception of imipenem, the MexAB-OprM pump contributes significantly to beta-lactam resistance in both beta-lactamase-negative and beta-lactamase-inducible strains, while the contribution of the MexAB-OprM efflux system is negligible in strains with overexpressed beta-lactamase. Overexpression of the efflux pump alone contributes to the high level of beta-lactam resistance in the absence of beta-lactamase.

Bacterial Outer Membrane Proteins↗
PubMed1999

A simple technique for anatomical slow pathway ablation in atrioventricular nodal reentrant tachycardia.

The slow pathway potential or the slow potential serves as a useful marker in catheter ablation of the slow pathway. However, an anatomical approach without recording of these potentials is also an effective way to cure atrioventricular nodal reentrant tachycardia (AVNRT). Moreover, the origin of these potentials is a matter of controversy. We compared 2 approaches to ascertain whether or not recording of these potentials is necessary in eliminating the slow pathway and to estimate the usefulness of the simple anatomical approach. The study population consisted of 24 patients with a conventional approach (Group P) and 19 patients with an anatomical approach (Group A). In group A, the ablation site was determined by fluoroscopy, which was the lowest one-third of the area between the His bundle electrogram recorded position and the coronary sinus orifice at the right anterior oblique view, and just in front of and above the coronary sinus orifice also posterior to the His catheter at the left anterior oblique view where the His catheter was seen tangentially. The slow pathway was successfully ablated in all patients without any complications, including more than first-degree AV block. Although there were no significant differences in total energy or number of applications between the 2 groups, the procedure time was significantly shorter in group A (p < 0.01). In conclusion, recording of the slow pathway potential or the slow potential is not always necessary for slow pathway ablation in the treatment of AVNRT. Because our anatomical approach was performed simply, effectively and safely, it is recommended for the slow pathway ablation of AVNRT.

Adolescent↗
PubMed1998

Antibiotic stress induces a large amount of outer membrane protein in Pseudomonas aeruginosa.

To investigate the bacterial response to antibiotic stress, we analyzed the outer membrane proteins of Pseudomonas aeruginosa grown in the presence of a sub-minimum inhibitory concentration of antibiotics. Among the antibiotics tested, fluoroquinolones and streptonigrin induced a large amount of outer membrane protein with a molecular mass of 43 kDa. This protein is most likely the stress-responsive protein, since the quinolone-resistant mutants with a higher minimum inhibitory concentration of antibiotic than the wild-type strain produced a large amount of 43-kDa protein only in the presence of sub-minimum inhibitory concentration of the mutants itself, but not that of the antibiotic-susceptible wild-type strain. The sequence of N-terminal 15 amino acids of the 43-kDa protein was identical to that of pyocin R1. However, purified pyocin R1 failed to accumulate in the outer membrane. Thus, we concluded that the 43-kDa protein (pyocin R1) is the antibiotic-stress-induced outer membrane protein.

Amino Acid Sequence↗
PubMed1998

Identification of the catalytic triad of the protein D2 protease in Pseudomonas aeruginosa.

We reported recently that protein D2 (OprD) porin of Pseudomonas aeruginosa bears protease activity (FEBS Letters 394, 179-182, 1996). To identify the catalytic residues of OprD, we introduced the site-directed mutations replacing the putative catalytic triad His156, Asp208, and Ser296 with glutamine, asparagine, and alanine, respectively. The OprD proteins purified from the chromosomal oprD-deficient mutants harboring the plasmids encoding the site-directed mutations showed protease activity less than 0.1% of that of the wild-type OprD. These site-directed mutageneses caused undetectable changes in the pore-forming activity of OprD as measured by single-channel conductance by the planar lipid bilayer. The minimum inhibitory concentration of imipenem in mutants having the replaced catalytic triads was identical with that in the wild-type strain. On the other hand, introduction of the mutation at His367 replacing with glutamine, the site that is supposed to be unrelated to the catalytic sites, showed the unchanged protease activity. These results unequivocally demonstrate that OprD is the protease bearing porin and catalyzes the reaction at His156, Asp208, and Ser296 residues.

Alanine↗
PubMed1998

Subunit swapping in the Mex-extrusion pumps in Pseudomonas aeruginosa.

Pseudomonas aeruginosa encodes three sets of antibiotic extrusion proteins designated as MexA,B.OprM, MexC,D-OprJ and MexE,F-OprN regulated by the nalB, nfxB and nfxC genes, respectively. MexB,D,F, OprM, J,N and MexA,C,E function as the inner membrane pumps, the outer membrane channels and the membrane fusion proteins, respectively. To investigate the possibility of subunit interchangeability, we constructed the following combinations of chimeric pumps: MexA,D-OprM/delta MexB, MexC,B-OprM/delta MexA, and MexA,B-OprJ/delta OprM. The strains producing MexA,D-OprM/delta MexB and MexC,B-OprM/delta MexA failed to restore the antibiotic resistance shown in the strains producing the natural combinations of the subunit proteins. These results suggested that the inner membrane components cannot be interchanged. In contrast, the stains producing MexA,B-OprJ/delta OprM exhibited higher resistance to several antibiotics than the mutant lacking OprM and lower resistance than the strain overexpressing OprM. This result suggests that OprJ may complement the OprM function partially. A spectrum of antibiotics, of which the minimum inhibitory concentrations were restored partially by the complementation, was the same as the spectrum to which the nalB type mutant shows resistance. We surmised from these results that the MexA/MexB unit sustains the substrate specificity of the MexA,B-OprM machinery.

Anti-Bacterial Agents↗
PubMed1998

Expression of cathepsin B in small cell lung carcinoma cells in relation to in vitro invasiveness.

Cathepsin B expression and its relation to in vitro invasiveness were investigated in small cell lung carcinoma cells (OC-10 cells). A subclone derived from OC-10 cells (10N) was similar to the parental cells both in growth pattern and morphology. However, the in vitro invasive capacity of OC-10 cells was 4 times higher than that of 10N cells. Cathepsin B activities in the plasma membrane fraction and spent medium of OC-10 cells were 2-fold higher than those of 10N cells. The invasiveness of OC-10 cells was markedly blocked by the addition of cysteine proteinase inhibitors, E-64C or leupeptin, while treatment of 10N cells with 2% (v/v) DMSO resulted in a 2-fold increment both in invasive capacity and cathepsin B activity. Immunoblot analysis demonstrated that the intensity of the cathepsin B band from OC-10 or 10N cells was not remarkably different regardless of DMSO treatment. Although no significant correlation was observed between the biochemical activity and protein of cathepsin B, in vitro invasive capacity of OC-10 cells strongly correlated with cathepsin B activity. These results suggest that cathepsin B plays an important role in the invasiveness of human small cell lung carcinoma cells.

Carcinoma, Small Cell↗
PubMed1997

Antibiotic diffusion pathways in the outer membrane of Pseudomonas aeruginosa.

We investigated the effect of a temperature shift from 37 degrees C to 17 degrees C on the steady-state diffusion rate of imipenem and cephalothin by evaluating periplasmic drug concentrations in intact cells of Pseudomonas aeruginosa, which overexpresses the extended spectrum beta-lactamase. We found that the ratio of periplasmic imipenem concentration at 17 degrees C relative to that of 37 degrees C was 1.03+/-0.1, whereas that of cephalothin was 0.43+/- 0.09. Accumulation rates of cell-associated tetracycline and fluoroquinolone at 17 degrees C were roughly 1/16 and 1/8, respectively, compared with that at 37 degrees C. We concluded from these data that cephalothin and possibly most other antibiotics excepting carbapenems cross the outer membrane of P. aeruginosa mainly by dissolving in the lipid phase but probably not passing through the porin channel. This may explain why the outer membrane of P. aeruginosa is a tight barrier against the penetration of antibiotics.

Anti-Bacterial Agents↗
PubMed1997

Use of fluorescence probes to monitor function of the subunit proteins of the MexA-MexB-oprM drug extrusion machinery in Pseudomonas aeruginosa.

The MexA-MexB-OprM efflux pump of Pseudomonas aeruginosa consists of two inner membrane proteins, MexA and MexB, and one outer membrane protein, OprM. We investigated the role of the components of this drug extrusion system by evaluating the repercussions of deleting these subunit components on the accumulation of several fluorescent probes. Fluorescence intensities of positively charged 2-(4-dimethylaminostyryl)-1ethylpyridinium and uncharged N-phenyl-1-naphtylamine were 7 and 4 times higher, respectively, in the mutant lacking OprM and 4 and 1.7 times higher, respectively, in the mutants lacking MexA or MexB than in the wild type strain. This order of fluorescence intensity was fully consistent with a previously reported minimum inhibitory concentration of antibiotics such as tetracycline, chloramphenicol, and fluoroquinolones. Ethidium bromide accumulation in all the Mex mutants proceeded at about 5 times faster than the rate in the wild type cells. This result is in accord with the minimum inhibitory concentration of beta-lactam antibiotics. These results suggest that the fluorescence probes could be successfully used in real time monitoring of the function of the drug extrusion machinery in Gram-negative bacteria. The downhill extrusion kinetics of 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene, which orients perpendicular to the inner leaflet of the cytoplasmic membrane, from preloaded cells lacking the extrusion pump was preceded by a slow increase in fluorescence intensity, whereas the wild type cell immediately released the dye. This observation was explained by a slow trans-cytoplasmic membrane crossing of intracellular dye in the mutants. These results reflected higher accumulation of the probe in the cytoplasmic membrane in the mutants and strengthened the hypothesis that extrusion of hydrophobic substrate mediated by MexA-MexB-OprM mainly takes place from the interior of the cytoplasmic membrane.

1-Naphthylamine↗
PubMed1997

El Tor hemolysin of Vibrio cholerae O1 forms channels in planar lipid bilayer membranes.

We investigated the channel formation by El Tor hemolysin (molecular mass, 65 kDa) of Vibrio cholerae O1 biotype El Tor in planar lipid bilayers. The El Tor hemolysin channel exhibited asymmetric and hyperbolic membrane current with increasing membrane potential, meaning that the channel is voltage dependent. The zero-current membrane potential measured in KCI solution showed that permeability ratio PK+/PCl- was 0.16, indicating that the channel is 6-fold more anion selective over cation. The hemolysin channel frequently flickered in the presence of divalent cations, suggesting that the channel spontaneously opens and closes. These data imply that the El Tor hemolysin damages target cells by the formation of transmembrane channels and, consequently, is the cause of osmotic cytolysis.

Bacterial Toxins↗