Biomedical subjects

H Nikaido

Publications and source records attributed to H Nikaido.

At least 127 records · Page 7Linked to original sources

PubMed1991

Contribution of the cell-surface-associated enzyme in the Zimmermann-Rosselet assay of outer membrane permeability of beta-lactam antibiotics.

Analysis of beta-lactam hydrolysis by intact cells of several strains of enteric bacteria suggests that a significant fraction of beta-lactamase molecules is present at a location directly accessible for the substrates in the medium. We propose a method to correct for the error introduced by these "surface-exposed" enzymes in the Zimmermann-Rosselet assay of outer membrane permeability.

Anti-Bacterial Agents↗

PubMed1991

Imipenem- and meropenem-resistant mutants of Enterobacter cloacae and Proteus rettgeri lack porins.

Carbapenems such as imipenem and meropenem are not rapidly hydrolyzed by commonly occurring beta-lactamases. Nevertheless, it was possible, by mutagenesis and selection, to isolate mutant strains of Enterobacter cloacae and Proteus rettgeri that are highly resistant to meropenem and imipenem. Two alterations were noted in the E. cloacae mutants. First, the mutant strains appeared to be strongly derepressed in the production of beta-lactamases, which reached a very high level when the strains were grown in the presence of imipenem. Second, these mutants were deficient in the production of nonspecific porins, as judged by the pattern of outer membrane proteins as well as by reconstitution assays of permeability. As with most porin-deficient mutants, their cultures were unstable, and their cultivation in the absence of carbapenems rapidly led to an overgrowth of porin-producing revertants. Analysis of the data suggests that the synergism between the lowered outer membrane permeability and the slow but significant hydrolysis of carbapenems by the overproduced enzymes can explain the resistance phenotypes quantitatively, although the possibility of alteration of the target cannot be excluded at present. With P. rettgeri mutants, there was no indication of further derepression of beta-lactamase, but the enzyme hydrolyzed imipenem much more efficiently than the E. cloacae enzyme did. In addition, the major porin was absent in one mutant strain. These results suggest that a major factor for the carbapenem resistance of these enteric bacteria is the porin deficiency, and this conclusion forms a contrast to the situation in Pseudomonas aeruginosa, in which the most prevalent class of imipenem-resistant mutants appears to lack the specific channel protein D2 yet retains the major nonspecific porin F.

Bacterial Outer Membrane Proteins↗

PubMed1991

Interplay of cell wall barrier and beta-lactamase activity determines high resistance to beta-lactam antibiotics in Mycobacterium chelonae.

We calculated the target access index, which reflects the probability that a drug will reach its target at an effective concentration, and the predictive MICs of cephaloridine, cephalothin, and cefazolin for Mycobacterium chelonae from the drug concentrations inhibiting the binding of benzylpenicillin to penicillin-binding proteins, the permeability coefficients, and the beta-lactamase properties. Despite a low level of beta-lactamase activity and because of a very effective permeability barrier, the target access indices were very low, e.g., 100 times lower than for penicillinase-producing Escherichia coli. The predicted MICs were within one to three twofold dilutions of the experimentally determined values (which ranged between 512 and greater than or equal to 2,048 micrograms/ml). These results demonstrate that the interplay of the cell wall barrier and beta-lactamase activity determines high resistance of M. chelonae to beta-lactam antibiotics.

Cefazolin↗

PubMed1991

Lipopolysaccharide structure required for in vitro trimerization of Escherichia coli OmpF porin.

Deep rought mutants, which produce very defective lipopolysaccharides, are unable to export normal levels of porins into the outer membrane. In this study, we showed that lipopolysaccharides from such mutants were also unable to facilitate the trimerization, in vitro, of monomeric OmpF porin secreted by spheroplasts of Escherichia coli B/r. In contrast, lipopolysaccharides containing most or all of the core oligosaccharides were able to facilitate trimerization.

Bacterial Outer Membrane Proteins↗

PubMed1991

Stoichiometry of maltodextrin-binding sites in LamB, an outer membrane protein from Escherichia coli.

We have directly measured the stoichiometry of maltodextrin-binding sites in LamB. Scatchard plots and computer fitting of flow dialysis (rate-of-dialysis) experiments clearly establish three independent binding sites per LamB trimer, with a dissociation constant of approximately 60 microM for maltoheptaose. The current model for LamB's function as a specific pore is discussed with respect to the symmetry in LamB's kinetic properties and the implications of our results.

Bacterial Outer Membrane Proteins↗

PubMed1990

Regulation of the maltose transport system of Escherichia coli by the glucose-specific enzyme III of the phosphoenolpyruvate-sugar phosphotransferase system. Characterization of inducer exclusion-resistant mutants and reconstitution of inducer exclusion in proteoliposomes.

Maltose transport in Escherichia coli is regulated at the protein level by the glucose-specific enzyme III (IIIglc) of the phosphoenolpyruvate-sugar phosphotransferase system, by a mechanism known as inducer exclusion. We have isolated and characterized four mutants in the maltose transport system, all of which are in malK, which are resistant to inducer exclusion. The mutations in three of these mutants fall within the COOH-terminal domain of MalK and suggest the first reported function for this domain. Two of these are in a region which shows sequence similarity to lacY and melB, both of which are also regulated by IIIglc, and thus may define a IIIglc-binding domain. We have also reconstituted inducer exclusion in proteoliposomes made from membranes overexpressing the maltose permease. Maltose transport is inhibited by 50-60% when IIIglc is included in the intravesicular space. The inhibition is due to a decrease in the Vmax of transport by a factor of 2. IIIglc does not affect the coupling of ATP hydrolysis to maltose transport, since the ratio of ATP hydrolyzed/maltose transported remained constant in the presence and absence of IIIglc. Finally, the Ki for IIIglc was 40 microM, roughly the same as the in vivo concentration of IIIglc.

Amino Acid Sequence↗

PubMed1990

Protein D2 channel of the Pseudomonas aeruginosa outer membrane has a binding site for basic amino acids and peptides.

Protein D2 of Pseudomonas aeruginosa outer membrane is known to facilitate the specific permeation of imipenem (N-formimdoylthienamycin) across this membrane barrier. We have characterized the binding site in the protein D2 channel by studying the competitive inhibition, by various solutes, of imipenem diffusion into the periplasm. We found that basic amino acids, lysine, arginine, histidine, and ornithine, were effective inhibitors. L- and D-lysine were found to be competitive inhibitors with approximate Ki values of 0.6 and 0.3 mM, respectively. Peptides containing L-lysine at the carboxyl terminus, as well as dipeptides containing L-lysine at the amino terminus, were also able to inhibit the transport. Wild type cells transported tripeptide Thr-Ser-Lys into the periplasm three to four times as rapidly as the mutant cells lacking the D2 protein. These results suggest that protein D2 plays a physiologically significant role in the uptake of basic amino acids and peptides containing these amino acids across the outer membrane of P. aeruginosa.

Amino Acid Sequence↗

PubMed1990

Partitioning of hydrophobic probes into lipopolysaccharide bilayers.

Lipophilic solutes permeate rapidly through lipid bilayer membranes. However, the outer membrane of enteric bacteria, which is composed of a lipopolysaccharide monolayer outer leaflet and the glycerophospholipid inner leaflet, shows extremely low permeability to hydrophobic solutes. In order to examine the cause of this exceptionally low permeability, the lipid/water partition behavior of various lipophilic probes was determined by using lipopolysaccharides of various chemotypes and glycerophospholipids. With all probes, under many different conditions, the lipopolysaccharide/water partition coefficients were generally about an order of magnitude smaller than the phospholipid/water partition coefficients, and this result is consistent with the low permeability of the lipopolysaccharide monolayer, and hence the asymmetric bilayer found in the outer membrane. Furthermore, organic polycations significantly increased the partition of N-phenylnaphthylamine into lipopolysaccharides, a result again consistent with the permeability-increasing effect of such cations on intact outer membrane. Very defective, 'deep rough' lipopolysaccharides of chemotypes Rd2, Rd1 and Re, had only slightly (20-75%) higher partition coefficients in comparison with the more complete lipopolysaccharides, and this difference is probably not enough to explain the approximately 100-fold increase in lipophile permeability seen in deep rough strains.

1-Naphthylamine↗

PubMed1990

Selective purification of sex-influenced esterase from rat serum by immunoaffinity chromatographies.

Sex-influenced esterase (ES-SI) is a female-specific serum esterase of rats. There are two alleles at the Es-Si locus, Es-Sia coding for ES-SI and Es-Sib, a silent allele. ES-SI was purified to electrophoretic homogeneity from the serum of adult female SI3 rats by a four-step purification. First, albumin was removed from the serum by Blue-Sepharose CL-6B affinity chromatography. Sera of rats carrying Es-Sia and Es-Sib were utilized to make two novel immunoaffinity columns. Rat anti-(ES-SI) alloantibody was obtained after the immunization of rats carrying Es-Sib with rat ES-SI-positive serum. ES-SI bound to the gel in this antibody-conjugated affinity chromatography. Simultaneously, rabbit anti-(rat serum protein lacking ES-SI) antibody was raised against ES-SI-negative rat serum and used to exclude other serum proteins. ES-SI was recovered in the flow-through fractions in this antibody-conjugated immunoaffinity chromatography, whereas the other serum proteins bound to the gel. ES-SI was selectively purified about 240-fold by combining these immunoaffinity chromatographies. This combination is a very powerful technique for purification of the protein. Finally, ES-SI was purified by preparative polyacrylamide gel electrophoresis after which it appeared as a single protein band (using silver staining) on SDS/polyacrylamide gel electrophoresis with a molecular mass of 72 kDa. In the ordinary estimation of enzyme purification, the purification of ES-SI was 30-fold at most, because the specific activity of the enzyme was calculated from the bulky esterase activity. However, ES-SI was selectively purified 2280-fold comparing its recovery to that of total protein. ES-SI exhibited a pI of 4.6 and optimum pH of 6.7. The activity of ES-SI was completely inhibited by diisopropyl fluorophosphate at 10 nM but not at all by eserine.

Animals↗

PubMed1990

Determinants of OmpF porin antigenicity and structure.

Sixty-six murine hybridomas raised to Escherichia coli B/r porin were used to identify and differentiate the epitopes of this outer membrane protein. Anti-porin monoclonal antibodies (mAb) were raised against outer membrane fragments, purified native trimeric porin (trimer), and purified sodium dodecyl sulfate-denatured monomeric porin (monomer). Immunochemical and flow cytometric methods identified five distinct cell surface-exposed determinants on OmpF. The peptide composition of porin epitopes was determined by analysis of mAb reactivity with cyanogen bromide-generated peptide fragments. Four of 43 anti-monomer mAb reacted with surface exposed sites on OmpF, defining epitopes that consist of residues within CNBr peptides d2, d3, and B. The anti-porin mAb panel was also used to evaluate changes in porin antigenic structure in strains with short ompF deletions. Flow cytometric experiments indicated that despite changes in porin permeability, little if any alteration of surface epitopes occurred in these strains. Western immunoblot analysis of the mutant porins showed loss of reactivity with numerous mAb, which was caused by changes in three spatially distinct epitopes at residues 108-111, 118-123, and 124-129. Our findings indicate that in these ompF mutants the residues responsible for altering porin permeability are not exposed on the cell surface, but are buried within the tertiary structure of the protein. One of these regions, which is apparently involved in the determination of channel permeability characteristics, is conserved among 15 of 16 different porin molecules which were screened with the anti-OmpF mAb panel.

Amino Acid Sequence↗

PubMed1990

Overproduction, solubilization, and reconstitution of the maltose transport system from Escherichia coli.

Maltose is transported across the cytoplasmic membrane of Escherichia coli by a binding protein-dependent transport system. We observed a 10-fold increase in the level of transport activity in assays with membrane vesicles when the three membrane-associated components of the transport system (the MalF, MalG, and MalK proteins) were overproduced. In addition, we have successfully reconstituted maltose transport activity in proteoliposome vesicles from solubilized proteins using a detergent dilution procedure. The addition of ATP as an energy source was sufficient to obtain transport, and this activity was dependent on the presence of maltose binding protein and was not seen in proteoliposomes prepared from a strain with a deletion of the maltose genes. We determined that hydrolysis of ATP was directly coupled to maltose uptake. In the majority of these experiments, an average of 1.4 mol of ATP was hydrolyzed for each mole of maltose accumulated. However, in the remaining experiments, ATP hydrolysis was observed to be much higher and averaged 17 mol of ATP hydrolyzed per mol of maltose transported. Possible explanations for a variable stoichiometry are discussed. These results provide strong evidence that it is the hydrolysis of ATP by a component of the transport complex that provides the energy required for active maltose transport.

ATP-Binding Cassette Transporters↗

PubMed1990

Biogenesis of prokaryotic pores.

The prokaryotic pore-forming proteins are synthesized in the cytoplasm, and are assembled in their functional form in the outer membrane. They begin to traverse the cytoplasmic membrane via the SecY/SecA export pathway, which is shared also by periplasmic proteins. The sorting signals that direct these proteins to the outer membrane could be present in the three-dimensional conformations of the proteins, but some results suggest that they may be present in short, contiguous sequences. Outer membrane proteins share a rather hydrophilic amino acid composition, and appear to be rich in beta-sheets (with the exception of lipoproteins). This observation as well as the demonstration of periplasmic export intermediates favor the secretion pathway through the periplasm, as opposed to export through fusion sites between the inner and the outer membrane, but such intermediates have not yet been observed with the wild type proteins under physiological conditions.

Amino Acid Sequence↗

PubMed1990

Urea cycle disorder in C3H-H-2 degree mice with juvenile steatosis of viscera.

We determined the activities of urea cycle enzymes in the liver of C3H-H-2 degree-jsv mice. The activities of all urea cycle enzymes decreased in the latter period of lactation. The activities of carbamylphosphate synthetase and ornithine transcarbamylase in some affected mice were undetectable. On the other hand, the activities of enzymes other than urea cycle enzymes were normal. We consider that the decrease in the urea cycle enzymes is caused by an abnormality in the mechanism of gene expression.

Ammonia↗

PubMed1990

In vitro trimerization of OmpF porin secreted by spheroplasts of Escherichia coli.

It is not yet clear how bacterial outer membrane proteins reach their correct destination after they are secreted across the cytoplasmic membrane. We show here that porin OmpF is secreted into the medium as a water-soluble monomeric protein by spheroplasts of Escherichia coli. Furthermore, this monomeric porin is taken up by cell envelope preparations or purified lipopolysaccharides in the presence of 0.03% Triton X-100 and is converted correctly into the mature trimeric conformation. These results appear to reproduce a part of the physiological export and targeting steps of this protein.

Bacterial Outer Membrane Proteins↗

PubMed1990

Outer membrane protein D2 catalyzes facilitated diffusion of carbapenems and penems through the outer membrane of Pseudomonas aeruginosa.

The outer membrane of imipenem-resistant mutants of Pseudomonas aeruginosa with decreased permeability to imipenem was shown by Western (immuno-) blotting to contain protein D1 and to lack protein D2. Protein D2 was purified and was shown to allow the permeation of imipenem at a rate higher than expected from its molecular weight. Spontaneous imipenem-resistant mutants of P. aeruginosa PAO1 appeared at a frequency of 10(-8) in the laboratory and did not synthesize protein D2. Experiments performed with intact cells carrying plasmid pHN4 containing the gene for L-1 beta-lactamase from Pseudomonas maltophilia showed that this channel could also be used by SM-7338, Sch 33755, and Sch 33440 but apparently not by Sch 34343 or Sch 29482.

Anti-Bacterial Agents↗

PubMed1990

Outer membrane permeability and beta-lactamase stability of dipolar ionic cephalosporins containing methoxyimino substituents.

Some enteric bacteria, such as Enterobacter cloacae, can develop high-level resistance to broad-spectrum cephalosporins by overproducing their chromosomally encoded type I beta-lactamases. This is because these agents are hydrolyzed rapidly at pharmacologically relevant, low (0.1 to 1 microM), concentrations, owing to their high affinity for type I enzymes. In contrast, the more recently developed cephalosporins, with quaternary-nitrogen-containing substituents at the 3 position, show increased efficacy against beta-lactamase-overproducing strains and, indeed, have a much lower affinity for type I enzymes. However, the possible contribution of an improved outer membrane permeability in their increased efficacy has not been studied. We found by proteoliposome swelling assays that cefepime, cefpirome, and E-1040 all penetrated the porin channels of Escherichia coli and E. cloacae much more rapidly than did ceftazidime and at least as rapidly as did cefotaxime. Considering that the influx of anionic compounds such as cefotaxime and ceftazidime will be further retarded in intact cells, owing to the Donnan potential, we expect that the newer compounds will penetrate intact cells 2 to 10 times more rapidly than will cefotaxime and ceftazidime. The kinetic parameters of hydrolysis of these agents by E. cloacae beta-lactamase showed that at 0.1 microM, they were hydrolyzed much more slowly than was cefotaxime and at about the same rate as or a lower rate than was ceftazidime. The combination of these two effects explains nearly quantitatively why these newer agents are more effective against some of the beta-lactamase-overproducing gram-negative bacteria.

Bacteria↗

PubMed1990

Cir and Fiu proteins in the outer membrane of Escherichia coli catalyze transport of monomeric catechols: study with beta-lactam antibiotics containing catechol and analogous groups.

Recently, beta-lactam agents containing iron-chelating moieties, such as E0702, which contains catechol, and pirazmonam and U-78,608, which contain 3-hydroxypyridone, have been developed. By determining the susceptibility to these agents of Escherichia coli mutants lacking various iron-repressible outer membrane proteins, we showed that two of these proteins with hitherto unknown functions, Fiu and Cir, were apparently involved in the transport of monomeric catechol and its analogs. These results confirm the conclusion of Curtis and co-workers, which was obtained by using a different set of catechol-containing antibiotics (N. A. C. Curtis, R. L. Eisenstadt, S. J. East, R. J. Cornford, L. A. Walker, and A. J. White, Antimicrob. Agents Chemother. 32:1879-1886, 1988). E0702 was shown to enhance the uptake of radioactive ferric iron into wild-type cells but not into cir fiu double mutants. By combining the influx of E0702 with its hydrolysis by a periplasmic beta-lactamase, we showed that the wild-type cells transported unliganded E0702 at a rate comparable to or even higher than the rate of transport of the E0702-Fe3+ complex. We postulate that the main function of Cir and Fiu may be to recapture the hydrolytic products of enterobactin, such as 2,3-dihydroxybenzoic acid and 2,3-dihydroxybenzoylserine.

Anti-Bacterial Agents↗

PubMed1990

Permeability barrier to hydrophilic solutes in Mycobacterium chelonei.

In order to define the permeability barrier to hydrophilic molecules in mycobacteria, we used as a model a smooth, beta-lactamase-producing strain of Mycobacterium chelonei. The rates of hydrolysis of eight cephalosporins by intact and sonicated cells were measured, and the permeability coefficient (P) was calculated from these rates by the method of Zimmermann and Rosselet (W. Zimmermann and A. Rosselet, Antimicrob. Agents Chemother. 12:368-372, 1977). P ranged from (0.9 +/- 0.3) x 10(-8) (benzothienylcephalosporin) to (10 +/- 3.3) x 10(-8) cm/s (cephaloridine); i.e., the P values were lower than those reported for Pseudomonas aeruginosa and Escherichia coli by 1 and 3 orders of magnitude, respectively. The permeability barrier was shown to reduce drastically the stream of drug molecules entering the cell, allowing the rather low level of beta-lactamase (0.1 U/mg of protein with penicillin G) to decrease radically the concentration of the drug at the target; this explains the poor in vitro activities of the beta-lactams against M. chelonei. We also estimated P for small, hydrophilic molecules (glucose, glycerol, glycine, leucine), by studying their uptake kinetics. The values found, ranging from 15 x 10(-8) to 490 x 10(-8) cm/s, were consistent again with a very low permeability of M. chelonei cell wall. The permeation of cephalosporins was not very dependent on the hydrophobicity of the molecules or on the temperature, suggesting a hydrophilic pathway of penetration for these molecules.

Biological Transport↗