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T Nakae

Publications and source records attributed to T Nakae.

At least 109 records · Page 6Linked to original sources
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↗
PubMed1987

Interaction of the alpha-toxin of Staphylococcus aureus with the liposome membrane.

When the liposome membrane is exposed to the alpha-toxin of Staphylococcus aureus, fluorescence of the tryptophan residue(s) of the toxin molecule increases concomitantly with the degree of toxin-hexamer formation (Ikigai, H., and Nakae, T. (1985) Biochem. Biophys. Res. Commun. 130, 175-181). In the present study, the toxin-membrane interaction was distinguished from the hexamer formation by the fluorescence energy transfer from the tryptophan residue(s) of the toxin molecule to the dansylated phosphatidylethanolamine in phosphatidylcholine liposome. Measurement of these two parameters yielded the following results. The effect of the toxin concentration and phospholipid concentration on these two parameters showed first order kinetics. The effect of liposome size on the energy transfer and the fluorescence increment of the tryptophan residue(s) was only detectable in small liposomes. Under moderately acidic or basic conditions, the fluorescence energy transfer always preceded the fluorescence increment of the tryptophan residue(s). The fluorescence increment at 336 nm at temperatures below 20 degrees C showed a latent period, whereas the fluorescence energy transfer did not. These results were thought to indicate that when alpha-toxin damages the target membrane, the molecule interacts with the membrane first, and then undergoes oligomerization within the membrane.

Energy Transfer↗
PubMed1987

Assembly of the alpha-toxin-hexamer of Staphylococcus aureus in the liposome membrane.

It has been shown that the access of the alpha-toxin of Staphylococcus aureus to the target membrane and assembly of the hexamer can be monitored independently by respectively measuring the fluorescence energy transfer from the tryptophan residue(s) of the toxin to the dansylated phosphatidylethanolamine in the liposome membrane and the fluorescence increment of the toxin at 336 nm (Ikigai, H., and Nakae, T., (1987) J. Biol. Chem. 262, 2150-2155). Measurement of these parameters under various conditions showed the following results: when phosphatidylcholine (PC) liposomes composed of saturated fatty acids were mixed with the toxin, the fluorescence energy transfer occurred below, at, and above the transition temperature of the lipid, but the change of fluorescence at 336 nm was never detectable; when PC-liposomes containing unsaturated fatty acids were used, both the fluorescence energy transfer and the fluorescence increment of 336 nm were observed. These results suggested that the toxin-membrane interaction occurs in PC-membranes containing saturated and/or unsaturated fatty acids and that the oligomerization occurs only in the presence of PC containing unsaturated fatty acid(s). This conclusion was supported by the results of quantitative determination of the toxin-hexamer assembly and leakage of carboxyfluorescein from PC-liposomes under conditions similar to the above.

Energy Transfer↗
PubMed1987

Liposome mediated dissipation of valinomycin-imposed potassium potential across erythrocytes membrane.

Influence of liposomes made of phosphatidylcholine (PC) on the valinomycin-imposed potassium potential across erythrocyte membrane was examined by measuring the fluorescence change of the potential-sensitive cyanine dye. We concluded that the liposomes modulate ion selectivity of the membrane embedded valinomycin, on the basis of the following lines of evidence. (i) The valinomycin-imposed potassium potential across erythrocyte membrane (interior negative) was dissipated in the presence of PC-liposomes. (ii) When PC-liposomes were added to the cell suspension before the valinomycin, a membrane potential could not be imposed. (iii) Liposomes containing only the PC of saturated fatty acids were inactive in the potential dissipation, whereas the liposomes containing PC of unsaturated fatty acids were fully active. (iv) Liposome-mediated dissipation of the imposed-membrane potential was similarly observed in the resealed erythrocyte ghosts. (v) The liposomes did not show a detectable effect on the gramicidin-mediated proton potential. (vi) The effect of liposome was somewhat analogous to the nigericin-mediated dissipation of the valinomycin-imposed potassium potential.

Animals↗
PubMed1986

The permeability property of the outer membrane of Bacteroides fragilis, a strictly anaerobic opportunistic pathogen.

It was concluded that the exclusion limit of the outer membrane of Bacteroides fragilis is close to the size of uncharged saccharides of Mr, 340-400 and of anionic solutes of Mr, 500 on the basis of the following lines of evidence. Penetration of methylhexoses and salicin into the NaCl-expanded periplasm appeared to be 85 and 50%, respectively, of that of pentoses, whereas penetration of the saccharides of Mr larger than 340-400 was negligibly small. The cells treated with 500 mosM di-through pentasaccharides were about 36% lighter than the isotonic NaCl-treated cells, whereas the 500 mosM pentoses- or hexoses-treated cells appeared to be 16 or 21%, respectively, lighter. The result indicates that the saccharides of Mr more than 340 exerted osmolarity at the outer membrane and hence these saccharides are assumed merely permeable. The phosphorylated solutes and beta-lactam antibiotics of Mr a little larger than the above threshold exclusion limit are partially diffusible.

Anti-Bacterial Agents↗
PubMed1986

A small diffusion pore in the outer membrane of Pseudomonas aeruginosa.

The permeability properties of the outer membrane of Pseudomonas aeruginosa were re-examined, since the reported conclusions are conflicting [Decad, M. G. and Nikaido, H. (1976) J. Bacteriol. 128, 325-336; Caulcott, C. A., Brown, M. R. W. and Gonda, I. (1984) FEMS Microbiol. Lett. 21, 119-123]. On the basis of the experimental evidence to be described below we conclude that the exclusion limit of the outer membrane of P. aeruginosa is smaller than the size of uncharged disaccharides but larger than the size of hexose. This conclusion is based on the following evidence. Penetration of monosaccharides into the expanded periplasm was large and that of disaccharides was small, after the cells were plasmolyzed with 600 mosM NaCl. A significant amount of protein was released after osmotic down-shock of cells treated with the hypertonic monosaccharides but not of cells treated with the hypertonic saccharides larger than disaccharides. Centrifuged pellets of cells treated with hypertonic di, tri and tetrasaccharides weighed about 15-20% less than that of cells treated with the isotonic monosaccharide, suggesting that the osmotic pressure was exerted on the outer membrane causing dehydration and shrinking of the cells. By contrast, cells treated with the hypertonic pentose and hexoses weighed about 0.1% and 6% less, respectively, than cells treated with the isotonic saccharide, suggesting that pentose diffused through the outer membrane freely.

Cell Membrane Permeability↗
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↗
PubMed1986

The role of the maltodextrin-binding site in determining the transport properties of the LamB protein.

We have examined by the liposome swelling technique the permeability properties of the modified LamB proteins isolated from mutants of Escherichia coli K12 with altered affinities toward starch and/or maltose (Ferenci, T., and Lee, K-S. (1982) J. Mol. Biol. 160, 431-444). The results revealed the following. A mutant strain exhibiting a markedly lowered affinity toward starch produced a LamB protein that has lost the ability to permeate longer maltodextrins. This protein retained a nonspecific pore for a wide variety of small sugars. A mutant strain with partially reduced affinity for starch produced a LamB protein which still permeated maltodextrins, maltose, and non-maltose sugars but had also gained an ability to permit the diffusion of sucrose and raffinose; in this strain sucrose and raffinose could now compete for the starch-binding site. A mutant with enhanced affinity for both maltose and starch produced a protein which exhibited elevated rates of diffusion for longer maltodextrins but still permeated other small sugars. Two other mutants with altered affinities showed relatively minor changes in the diffusion of maltose and non-maltose sugars. It could be concluded from these studies that the LamB proteins form pores allowing the diffusion of a wide variety of monosaccharides irrespective of the presence or the absence of affinity of a binding site for maltodextrins. However, the presence of a sugar-binding site is crucial in determining the rate of the diffusion of maltodextrins or other oligosaccharides.

Bacterial Outer Membrane Proteins↗
PubMed1986

The outer membrane of Pseudomonas aeruginosa is a barrier against the penetration of disaccharides.

The outer membrane of Pseudomonas aeruginosa acted as a barrier against the penetration of di- (Mr, 342), tri- (Mr, 504) and tetrasaccharides (Mr, 666), whereas the membrane allowed the penetration of pentose (Mr, 150) and methylhexoses (Mr, 194) into the periplasm. When the intact cells of P. aeruginosa were treated with 600 mosM saccharides of various sizes and observed under an electron microscope, saccharides of Mr larger than 342 caused the extensive shrinking of the outer membrane. Whereas the cells treated with the saccharides of Mr less than 194 or with sucrose in the presence of EDTA showed plasmolysis. Determination of the extent of saccharide penetration into the periplasm of the cells treated with 600 mosM sodium chloride or with 600 mosM saccharides of various sizes showed that only pentose and hexoses, so far examined, were penetrable but di-, tri- and tetrasaccharides were impenetrable.

Disaccharides↗
PubMed1986

Leishmania braziliensis: localization of glycoproteins in promastigotes.

Two species of glycoproteins from Leishmania braziliensis promastigotes of apparent molecular weights of 53,000 (glycoprotein 53) and 47,000 (glycoprotein 47) were localized. Four lectins with different sugar specificities bound to the blotting sheet to which the electrophoretically separated materials were transferred. Concanavalin A and Ricinus communis agglutinin bound to the band of glycoprotein 53 and the lectin from Dolichos biflorus bound to the band of glycoprotein 47. Wheat germ agglutinin bound to the bands of both glycoproteins. Histochemical examinations using fluorescence labeled lectins demonstrated that the glycoproteins 53 and 47 were located on the cell surface and in the cytoplasm of promastigotes, respectively. The results are consistent with the result of agglutination test.

Agglutination Tests↗
PubMed1986

Pore formation by LamB of Escherichia coli in lipid bilayer membranes.

Lipid bilayer experiments were performed in the presence of different Escherichia coli LamB preparations. These LamB preparations formed two types of pores in the membranes. Large pores, which had a single-channel conductance of 2.7 nS and comprised about 1 to 6% of the total pores, were presumably contaminants which might have been induced together with LamB. LamB itself formed small pores with a single-channel conductance of 160 pS in 1 M KCl. These pores could be completely blocked by the addition of maltose and maltodextrins. Titration of the pore conductance with maltotriose suggested that there was a binding site inside the pores with a Ks of 2.5 X 10(-4) M for maltotriose. On the basis of our data we concluded that the structure of the LamB channels is quite different from the structures of the channels of general diffusion porins, such as OmpF and OmpC.

Bacterial Outer Membrane Proteins↗
PubMed1986

Outer-membrane permeability of bacteria.

Gram-negative bacteria evolved to survive under the conditions in which a number of hazardous compounds are abundant. The outer membrane which protects the cell interior acts as a barrier against such hazardous agents, yet the cells must incorporate the chemicals that are essential for the cellular activity. The devices that Gram-negative bacteria developed to incorporate such essence are the transmembrane pores. These pores could be subdivided into three categories: (1) pore made of porins has a weak solute selectivity; (2) pore made of lamB protein and tsx proteins hold intermediate solute specificity. and (3) pores for the diffusion of vitamin B12 and ferric ion-chelator complexes have a tight solute specificity. Porins are identified from a number of Gram-negatives and from the outer membrane of mitochondria of various sources. Studies on the diffusion properties of these outer-membrane proteins provided essential information to understand membrane transports.

Anti-Bacterial Agents↗
PubMed1985

The mechanism of ion selectivity of OmpF-porin pores of Escherichia coli.

The OmpF porin from the outer membrane of Escherichia coli acts as a lightly cation-selective pore, allowing the diffusion of small cations and cationic molecules, whose Mr are a little larger than the threshold exclusion limit. To ascertain the mechanism of this cation selectivity, we have examined a possible influence of cationic solutes on the fluorescence emission and the circular dichroic spectrum of tryptophan residues of the porin trimer, searching for conformational change(s). The diffusion of cationic solutes was determined with the native and the amidated porins in the presence or the absence of the effector cations. The following results were obtained. (a) Cations, e.g. spermidine, caused fluorescence quenching in the native trimer, with a half-maximum fluorescence quenching at 11-18 microM. A change in the circular dichroic spectrum was also recorded at around 280 nm. (b) The dissociation constant of spermidine to the native trimer was calculated to be 16 microM as determined by the method of equilibrium dialysis. (c) The cation-caused fluorescence quenching was reversed when the carboxyl groups of the trimer were modified by the amidation reaction, though amidation of the trimer resulted in no significant change in the fluorescence intensity. (d) The diffusion rate of N-benzyloxycarbonyl-glycyl-L-prolyl-L-arginine p-nitroanilide through the native and the amidated porins was lowered in the presence and the absence, respectively, of cations. Both the extent of fluorescence quenching in the presence of cation and the rate of cation diffusion were inversely proportional to the number of amidated carboxyl residues. The relative fluorescence quenching of the porin trimer (the amidated versus the native) in the presence of cations was linearly related to the relative solute diffusion via the porin (the amidated versus the native). These results suggested that cations caused a conformational change in the trimer, resulting in an easier diffusion of the solutes. The results suggested further that a limited number of carboxyl groups in the pore interior are involved in the cation selectivity of OmpF-porin pores.

Bacterial Outer Membrane Proteins↗
PubMed1985

Conformational alteration in alpha-toxin from Staphylococcus aureus concomitant with the transformation of the water-soluble monomer to the membrane oligomer.

The membrane-damaging alpha-toxin aggregate of Staphylococcus aureus was characterized physicochemically. The aggregate weight of the toxin formed by various methods appeared to be 6 times higher than the molecular weight of the monomer as determined by the laser light scattering technique, suggesting the presence of a hexamer in the membrane. The aggregates fluoresced 20 to 50% more than the monomer at 336 nm. Circular dichroism measurements revealed that both the monomer and the oligomer showed essentially beta-sheet structure with the maximum ellipticity about -8,400 deg.cm2.dmol-1 at 215 nm. Circular dichroism spectrum of the oligomers showed ellipticity difference of -6,600, -44 and +84 deg.cm2.dmol-1, at 200, 250 and 280 nm, respectively, compared with the monomer. All these results suggest that the conformational change in the toxin molecule occurs concomitant with the transformation of the water-soluble monomer to the membrane-embedded hexamer.

Bacterial Toxins↗
PubMed1984

Properties of chemically modified porin from Escherichia coli in lipid bilayer membranes.

Purified porin OmpF from Escherichia coli outer membrane was chemically modified by acetylation and succinylation of amino groups and by amidation of the carboxyl groups. Native and chemically modified porins were incorporated into lipid bilayer membranes and the permeability properties of the pores were studied. Acetylation and succinylation of the porin trimers had almost no influence on the single channel conductance in the presence of small cations and anions and the cation selectivity remained essentially unchanged as compared with the native porin. Amidation had also only little influence on the single channel conductance and changed the pore conductance at maximum by less than 50%, whereas the cation selectivity of the porin is completely lost after amidation. The results suggest that the structure of the porin pore remains essentially unchanged after chemical modification of the pores and that their cation selectivity is caused by an excess of negatively charged groups inside the pore and/or on the surface of the protein. Furthermore, it seems very unlikely that the pore contains any positively charged group at neutral pH.

Acetylation↗