Biomedical subjects

T Nakae

Publications and source records attributed to T Nakae.

At least 73 records · Page 4Linked to original sources

PubMed1995

[Protective effect of human macrophage colony-stimulating factor (hM-CSF) on fungal infection (1). In vivo effect of hM-CSF on systemic candidiasis and in vitro effect of hM-CSF on macrophages activities].

We studied the protective effect of human macrophage colony-stimulating factor (hM-CSF) on fungal infection due to systemic candidiasis in vivo and the activities of macrophages in vitro, in order to demonstrate the usefulness of M-CSF on fungal infection. The effect of hM-CSF on systemic candidiasis was examined by using normal and immunosuppressed mice. In addition, the effects of hM-CSF on the activity of reticuloendothelial system (RES) organ and on the phagocytic activity and NBT reduction activity of mouse macrophage were also examined in vitro. HM-CSF improved the survival rate of systemic candidiasis in both normal and immunosuppressed mice. Combination therapy with hM-CSF and fluconazole showed higher survival rate more than in the therapy with either hM-CSF or fluconazole alone. Furthermore, hM-CSF enhanced the activity of RES organ, phagocytosis by macrophages and NBT reduction by macrophages, significantly. These results indicate that hM-CSF enhances the phagocytic cactivity and candicidal activity by macrophages in vivo, thereby preventing dissemination of fungal infection.

Animals↗

PubMed1994

[Protective effect and antibody titer of intravenous immunoglobulin (IVIG) against clinical isolates of opportunistic bacteria].

Neonates and leukopenic, immunosuppressed patients are at high risk for severe infection of opportunistic pathogens despite of the availability of potent antimicrobial agents. In this study, antibody titers of immunoglobulin preparations (IVIG) were contrasted with the protective effect in mice against each of bacterial infection. Antibody titers were determined by ELISA. The antigens were 70-80 clinical isolates of Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae and Staphylococcus aureus, respectively. Antibody titers of IVIG against these three gram-negative bacteria ranged 3200 to 102,400. ICR mice were inoculated intraperitoneally with each of several strains against which IVIG showed various titers. IVIG showed rather high protective activities against well-reactive strains, while it showed little protective activities against poor-reactive strains. In the case of P. aeruginosa, statistical analysis of the results obtained with the antibody titer and efficacy showed a good correlation (p < 0.01). On the other hand, IVIG showed a high and complicate antibody titer against S. aureus IVIG ranging 400,000 to 12,800,000, since apparent titers contained non-specific binding of Fc portion of IgG with protein A on the cell wall. IVIG was active in mice, where protein A was less and specific binding was stronger. Bacterial cells have various components; lipopolysaccharide, lipid A, capsule, flagella, pill, etc. that are responsive to specific antibodies. This study indicates that IVIG have such antibodies and that is associated with protective activity against bacterial infection in proportion to antibody titer.

Animals↗

PubMed1993

Calcium ion-mediated opening of the channel gate in the Pseudomonas aeruginosa porin.

The gate-forming domain of protein D2 (OprD2) in the outer membrane of Pseudomonas aeruginosa contains an amino acid sequence homologous to the calcium-binding site of the myosin light chain. This observation lets us to test the effect of Ca2+ on the channel function of OprD2. The diffusion rate of p-nitrophenyl phosphate (PNPP) through OprD2 was 2.3 times higher in the presence of mM order of Ca2+, but not Mg2+ or Mn2+. Concomitantly, the intrinsic fluorescence emission of OprD2 became about 10% lower in the presence of Ca2+. As the proteolytic cleavage of the gate-forming domain of OprD2 results the channel activity about 7 times higher, we tested the effect of Ca2+ on the solute permeability through the trypsin-treated OprD2. The diffusion rate of PNPP in the trypsin-treated OprD2 appeared to be nearly identical in the presence and absence of Ca2+. The result suggests that Ca2+ activates the OprD2 channel exerting its effect on the gate-forming domain.

Bacterial Outer Membrane Proteins↗

PubMed1993

Lipopolysaccharide promoted opening of the porin channel.

We show here that the imipenem (a carbapenem, beta-lactam antibiotic)-permeable porin channels (protein D2 or OprD2) of Pseudomonas aeruginosa were closed mostly in the lipopolysaccharide (LPS)-free membrane and were openable by adding LPS to the membrane as assayed by ion conductivity measurements using planar lipid bilayers. Open and closed states of the OprD2 channels exhibited conductivities of about 400 and 30 pS, respectively, in 1 M NaCl. The OprD2 channel in the LPS-containing membrane showed very rapid opening and closing events in a second order and the duration of closure became longer at low membrane potentials.

Bacterial Outer Membrane Proteins↗

PubMed1993

Bactericidal catechins damage the lipid bilayer.

The mode of antibacterial action of, the green tea (Camellia sinensis) extracts, (-)-epigallocatechin gallate (EGCg) and (-)-epicatechin (EC) was investigated. Strong bactericidal EGCg caused leakage of 5,6-carboxyfluorescein from phosphatidylcholine liposomes (PC), but EC with very weak bactericidal activity caused little damage to the membrane. Phosphatidylserine and dicetyl phosphate partially protected the membrane from EGCg-mediated damage when reconstituted into the liposome membrane with PC. EGCg, but not EC, caused strong aggregation and NPN-fluorescence quenching of PC-liposomes and these actions were markedly lowered in the presence of negatively charged lipids. These results show that bactericidal catechins primarily act on and damage bacterial membranes. The observation that Gram-negative bacteria are more resistant to bactericidal catechins than Gram-positive bacteria can be explained to some extent by the presence of negatively charged lipopolysaccharide.

Anti-Bacterial Agents↗

PubMed1993

Mechanism of efficient elimination of protein D2 in outer membrane of imipenem-resistant Pseudomonas aeruginosa.

Most imipenem-resistant Pseudomonas aeruginosa isolates produce an immunologically undetectable level of protein D2 (OprD2). To study the efficient elimination of the protein, we selected 23 independent imipenem-resistant mutants from a strain harboring the plasmid carrying cloned oprD and having a mutation in chromosomal oprD. All these oprD/oprD (plasmid/chromosomal) mutants expressed undetectable levels of OprD2, as shown from an assay by the immunoblotting method. Restriction maps of the DNAs from all 23 mutant plasmids could be divided into two groups. Restriction mapping and sequencing analysis of DNA from one representative plasmid from each group showed that both mutant oprD genes had a deletion. One had an 11-bp deletion in the coding region generating a frameshift mutation and a premature termination codon. Another had a large deletion encompassing the upstream site of its putative promoter region through the coding region. Northern blotting analysis showed that the gene with the 11-bp deletion was transcribed to about 1.5 kb of mRNA, but the gene with the large deletion produced undetectable RNA complementary to the oprD DNA probe. Since we analyzed only plasmid-borne oprD, we cannot exclude the possibility that the imipenem resistance caused by the chromosomal mutation is by a different mechanism(s). It is suggested, yet, that clear elimination of OprD2 from most imipenem-resistant P. aeruginosa isolates is due to efficient selection of the oprD deletion mutants.

Bacterial Proteins↗

PubMed1993

[Bullectomy utilizing EndoGIA under thoracoscopic guidance].

We report 5 cases of pneumothorax successfully treated with an endoscopic GIA stapling device (EndoGIA) under thoracoscopic guidance. There were no complications such as air leak or bleeding, and patients were discharged 2 to 7 days after operation. Surgical procedure utilizing EndoGIA has been proved to have advantages such as less pain, early return to normal activity and more cosmetic incision.

Adolescent↗

PubMed1992

Porin of Pseudomonas aeruginosa forms low conductance ion channel in planar lipid bilayers.

Protein E1, a porin of the outer membrane of Pseudomonas aeruginosa, was reconstituted into planar lipid bilayers. Single channel conductance of the protein appeared to be 230 pS (pico siemens) in 1 M KCl-10 mM Hepes, pH7.2. This value is approximately 5 times lower than the conductance of the OmpF channel of Escherichia coli. Conductance increased linearly as the membrane potential was raised from -200 mV to +200 mV, and was nearly proportional to the KCl concentration. These results show that protein E1 is probably a genuine porin in the P. aeruginosa outer membrane supporting the earlier conclusion that protein E1 forms a small channel.

Bacterial Outer Membrane Proteins↗

PubMed1992

Separation of gate- and channel-forming domains in the pore-forming protein of the outer membrane of Pseudomonas aeruginosa.

The domains of the pore-forming protein responsible for the gate and channel formations were separated and identified in the outer membrane of Pseudomonas aeruginosa. The proteolytic cleavage of the 46K channel protein, protein D2, yielded two major domains with apparent M(r) of 27K and 19K. We identified the 27K polypeptide to be the channel-forming domain by an in vitro permeability assay. The channel size of purified 27K domain was indistinguishable from that of native protein D2. Degradation of the 19K domain into small subfragments increases the channel activity about ten times suggesting that the 19K polypeptide forms the gate or cap.

Bacterial Outer Membrane Proteins↗

PubMed1992

Calcium ion-mediated regulation of the alpha-toxin pore of Staphylococcus aureus.

The water-soluble alpha-toxin monomers of Staphylococcus aureus become hexamers forming the transmembrane pore when exposed to the membranes. This pore is freely permeable to small hydrophilic molecules, e.g. carboxyfluorescein, and becomes less permeable in the presence of calcium ions. Calcium ion-mediated decrease of the carboxyfluorescein leakage could not be eliminated by EDTA added in the medium, but the carboxyfluorescein could be freed by EDTA added in the intraliposomal space. This result suggests that the alpha-toxin pore changes its conformation as the calcium ion is bound and that the binding site is exposed to the intraliposomal side of the membrane. The interaction between the alpha-toxin hexamer and 8-anilino-1-naphthalene-sulfonic acid (ANS) was monitored by determining the fluorescence in the presence and absence of calcium chloride. The mean distances between the tryptophan residues of the alpha-toxin hexamer and the bound ANS were calculated to be 1.90 and 1.80 nm in the absence and presence, respectively, of calcium ions. The results showed the calcium ion mediated conformational change of the membrane-embedded alpha-toxin hexamer.

Anilino Naphthalenesulfonates↗

PubMed1992

Investigation of the selectivity of maltoporin channels using mutant LamB proteins: mutations changing the maltodextrin binding site.

Wild-type and seven mutant maltoporins were purified and their channel-forming activities studied after reconstitution into black lipid membranes. The proteins were assayed for alterations at the maltodextrin binding site by measuring the sugar-dependent blockage of ion flux through these channels. Some substitutions (R8H, W74R) caused reduced channel affinity for all maltodextrins without changing single channel conductivities. The channel with a GlySer insertion after residue 9 was also poorly blocked by sugars but unique to this protein, the channel showed a striking, almost exponential increase of affinity with increasing maltodextrin chain length. In mutants with AspPro insertions after residues 79 and 183, there was an increase in affinity for glucose and maltose but not longer maltodextrins. The additional negative charge in the AspPro insertion mutants increased the cation selectivity of maltoporin channels, as did the decrease in positive charge resulting from the R8H substitution. A mutant with a W120C substitution also showed an increased affinity for glucose and maltose but reduced affinity for longer maltosaccharides. In contrast, a Y118F substitution resulted in an 8-fold increase in maltotriose affinity, but lesser improvements for other sugars. These results are interpreted to reflect changes in subsites contributing to an extended binding site within the channel, which in turn determines the overall sugar affinity of maltoporin.

Amino Acid Sequence↗

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↗

PubMed1992

Interactions of lung cancer cells with the human mesothelial cell monolayer: an in vitro model for cancer invasion.

An in vitro monolayer model for cancer invasion was developed, comprising cultured layers of human pleura-derived mesothelial cells (PM cells). The cells were isolated from normal human pleura at autopsy and cultured in RPMI-1640 supplemented with 10% fetal calf serum; they were polygonal in shape, forming a pavement-like structure, and preserved the morphologic characteristics of mesothelial cells. When small cell lung cancer cells (OC-10 cells) were seeded on to the monolayer of PM cells, they attached themselves to the monolayer, invaded it and formed flattened cancer cell nests beneath it. By counting the number of cancer cells penetrated, the in vitro invasiveness (invasive capacity) of the cancer cells was assayed. The invasive capacity of poorly invasive cells (10N), which were cloned from OC-10 cells, was five times less than that of the parental OC-10 cells. These results indicate our system to provide a useful model for studying human cancer cell invasion.

Carcinoma, Small Cell↗

PubMed1991

Ofloxacin-resistant Pseudomonas aeruginosa mutants with elevated drug extrusion across the inner membrane.

We selected the quinolone-resistant mutants from the protein F deficient Pseudomonas aeruginosa. The mutants showed cross-resistance to tetracycline, minocycline and chloramphenicol, but not to the beta-lactam antibiotics. The MIC of ofloxacin (OFLX) against the mutants, but not in the parent, became 2 to 4 times lower as the medium pH was raised from 6.5 to 8.5. The mutants accumulated about half as much OFLX as the parent. The OFLX accumulation in the mutants increased 6.5- to 8-fold in the presence of 100 mM carbonyl cyanide m-chlorophenylhydrazone, while that in the parent was 4-fold. The OFLX sensitivity of the mutant DNA-gyrase was comparable with that of the parent's enzyme. These results suggest that the resistance of these mutants to OFLX is associated with the membrane potential dependent drug efflux.

Anti-Bacterial Agents↗

PubMed1991

Cloning of the protein D2 gene of Pseudomonas aeruginosa and its functional expression in the imipenem-resistant host.

Protein D2 forms the water-filled pore across the outer membrane of Pseudomonas aeruginosa and allows the penetration of imipenem. We cloned the protein D2 gene by the antibody screening technique. When the imipenem-resistant mutant lacking protein D2 harbored the plasmid with the cloned D2 gene, the mutant overproduced protein D2 in the outer membrane. These transformants exhibited fully-restored imipenem susceptibility. The results prove unequivocally that protein D2 forms the imipenem-permeable pore in the P. aeruginosa outer membrane.

Bacterial Proteins↗

PubMed1991

Factors that influence the permeability assay of the outer membrane of Pseudomonas aeruginosa.

Brief exposure of Pseudomonas aeruginosa to a temperature of 10 degrees C or lower caused a significant leakage of the periplasmic beta-lactamase into the medium. The extent of leakage increased as the incubation temperature was lowered to 4 degrees C and reached a maximum at 0 degrees C. Cells grown in the presence of beta-lactamase inducers were unsuitable for the permeability assay. It was found that the diffusion rates of beta-lactams through the outer membrane of P. aeruginosa were much lower than those previously reported, as assayed under refined conditions. The diffusion rates of beta-lactams in one of the mutants tested were an order of magnitude lower than those of the other strains, despite the fact that the outer membrane protein profile of the strain appeared to be indistinguishable from those of the others. These results suggest that beta-lactam antibiotics diffuse through the outer membrane of P. aeruginosa, at least partly, through a non-porin pathway.

Cell Membrane Permeability↗

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↗

PubMed1991

Outer membrane permeability of Acinetobacter calcoaceticus and its implication in antibiotic resistance.

In order to understand high and broad antibiotic resistance of Acinetobacter calcoaceticus, the outer membrane permeability was studied. The permeability coefficients of zwitterionic cephalosporins in the intact cell outer membrane were 0.14-1.12 x 10(-4) cm3/min/mg protein. These values were two to seven times lower than the permeability coefficients of the same beta-lactams in the outer membrane of Pseudomonas aeruginosa. The diffusion rates of carbapenems and zwitterionic cephalosporins into liposomes containing purified outer membrane appeared to be about 1-3%, that of the Escherichia coli B outer membrane. These results indicate that the outer membrane of A. calcoaceticus acts as a substantial barrier against the penetration of these antibiotics. About 80 times of the A. calcoaceticus outer membrane was needed in the liposome compared with E. coli B to show the same extent of saccharide permeability. Two minor outer membrane proteins (less than 5% of total outer membrane protein) were identified to be the porins. These results suggest that one of the causes for the high antibiotic resistance of A. calcoaceticus is attributable to the presence of a small number of small-sized porins.

Acinetobacter calcoaceticus↗