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H Nikaido

Publications and source records attributed to H Nikaido.

At least 109 records · Page 6Linked to original sources
PubMed1993

Molecular cloning and characterization of acrA and acrE genes of Escherichia coli.

The DNA fragment containing the acrA locus of the Escherichia coli chromosome has been cloned by using a complementation test. The nucleotide sequence indicates the presence of two open reading frames (ORFs). Sequence analysis suggests that the first ORF encodes a 397-residue lipoprotein with a 24-amino-acid signal peptide at its N terminus. One inactive allele of acrA from strain N43 was shown to contain an IS2 element inserted into this ORF. Therefore, this ORF was designated acrA. The second downstream ORF is predicted to encode a transmembrane protein of 1,049 amino acids and is named acrE. Genes acrA and acrE are probably located on the same operon, and both of their products are likely to affect drug susceptibilities observed in wild-type cells. The cellular localizations of these polypeptides have been analyzed by making acrA::TnphoA and acrE::TnphoA fusion proteins. Interestingly, AcrA and AcrE share 65 and 77% amino acid identity with two other E. coli polypeptides, EnvC and EnvD, respectively. Drug susceptibilities in one acrA mutant (N43) and one envCD mutant (PM61) have been determined and compared. Finally, the possible functions of these proteins are discussed.

Acriflavine↗
PubMed1993

15-Ketoprostaglandin delta 13-reductase activity in bovine ocular tissues.

The present study provides the first evidence for delta 13-reduction of 15-ketoprostaglandins (15-keto-PGs) by bovine ocular tissues. The 9,000xg supernatants of cornea, iris, ciliary body, retina, and RPE-choroid except lens exhibited delta 13-reductase activity toward 15-keto-PG E2 and F2 alpha in the presence of NADPH or NADH as an electron donor. Among the tissues tested, the highest activity was observed in ciliary body and iris, followed by RPE-choroid, retina, and cornea. The NADPH- and NADH-linked double bond reductase activities were inhibited by dicumarol, quercitrin, indomethacin and disulfiram, but not by potassium cyanide. NADPH-linked 15-keto-PG F2 alpha delta 13-reductase was purified from bovine iris-ciliary body cytosol by fractionation with ammonium sulfate and high performance liquid chromatography (HPLC) with TSK gel DEAE-5PW, and TSK gel Blue-5PW. The purified enzyme was homogenous by the criterion of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its molecular weight was estimated to be about 57,000 by electrophoresis, and about 55,000 by gel filtration HPLC with Superose 12.

15-Oxoprostaglandin 13-Reductase↗
PubMed1992

Transport proteins in bacteria: common themes in their design.

Bacterial transport proteins mediate passive and active transport of small solutes across membranes. Comparison of amino acid sequences shows strong conservation not only among bacterial transporters, but also between them and many transporters of animal cells; thus the study of bacterial transporters is expected to contribute to our understanding of transporters in more complex cells. During the last few years, structures of three bacterial outer membrane transporters were solved by x-ray crystallography. Much progress has also occurred in the biochemical and molecular genetic studies of transporters in the cytoplasmic membranes of bacteria, and a unifying design among membrane transporters is gradually emerging. Common structural motives and evolutionary origins among transporters with diverse energy-coupling mechanisms suggest that many transporters contain a central module forming a transmembrane channel through which the solute may pass. Energy-coupling mechanisms can be viewed as secondary features added on to these fundamental translocation units.

Bacteria↗
PubMed1992

UV difference spectroscopy of ligand binding to maltose-binding protein.

We have used UV absorbance spectroscopy to study the binding of linear and circular maltodextrins to maltose-binding protein (MBP). Titrations with maltose yield three isosbestic points in the difference spectrum of MBP, consistent with two protein conformations: ligand-free and ligand-bound. In contrast, titrations with maltotetraose reveal three conformations: ligand-free, a low-affinity liganded state, and a high affinity liganded state. These results confirm and extend the results from tritium NMR spectroscopy, namely, that MBP can bind maltodextrin either by the sugar's anomeric end (high affinity) or by the middle of the maltodextrin chain (low affinity).

Bacterial Proteins↗
PubMed1992

Mechanism of maltose transport in Escherichia coli: transmembrane signaling by periplasmic binding proteins.

Maltose transport across the cytoplasmic membrane of Escherichia coli is dependent on the presence of a periplasmic maltose-binding protein (MBP), the product of the malE gene. The products of the malF, malG, and malK genes form a membrane-associated complex that catalyzes the hydrolysis of ATP to provide energy for the transport event. Previously, mutants were isolated that had gained the ability to grow on maltose in the absence of MBP. After reconstitution of the transport complex into proteoliposomes, measurement of the ATPase activity of wild-type and mutant complexes in the presence and absence of MBP revealed that the wild-type complex hydrolyzed ATP rapidly only when MBP and maltose were both present. In contrast, the mutant complexes have gained the ability to hydrolyze ATP in the absence of maltose and MBP. The basal rate of hydrolysis by the different mutant complexes was directly proportional to the growth rate of that strain on maltose, a result indicating that the constitutive ATP hydrolysis and presumably the resultant cyclic conformational changes of the complex produce maltose transport in the absence of MBP. These results also suggest that ATP hydrolysis is not directly coupled to ligand transport even in wild-type cells and that one important function of MBP is to transmit a transmembrane signal, through the membrane-spanning MalF and MalG proteins, to the MalK protein on the other side of the membrane, so that ATP hydrolysis can occur.

ATP-Binding Cassette Transporters↗
PubMed1992

Carnitine administration to juvenile visceral steatosis mice corrects the suppressed expression of urea cycle enzymes by normalizing their transcription.

Previous studies in our laboratories have revealed that juvenile visceral steatosis mice show suppressed transcription of urea cycle enzyme genes during development and are systemically deficient in carnitine. It has not yet been explained, however, how this carnitine deficiency relates to the abnormal gene expression. We investigated the effect of carnitine on abnormal gene expression, growth retardation, and fatty liver. Carnitine administration relieved the suppression of the developmental induction of two urea cycle enzymes examined, carbamoyl-phosphate synthetase and argininosuccinate synthase, and kept the activities of enzymes normal. However, carnitine did not reduce accumulated lipid in the liver to the normal level. These results suggest that carnitine deficiency plays an important role in the abnormal expression of urea cycle enzyme genes and that the abnormal expression of the genes is not directly caused by lipid accumulation in the liver.

Aging↗
PubMed1992

Abnormal expression of urea cycle enzyme genes in juvenile visceral steatosis (jvs) mice.

Juvenile visceral steatosis (jvs) mice from the C3H-H-2 degrees strain have markedly low levels of all the hepatic urea cycle enzymes (Imamura et al. (1990) FEBS Lett. 260, 119-121). The steady state levels of messenger RNA for the four urea cycle enzymes examined and also for albumin and serine dehydratase were severely reduced in the liver. The levels of mRNA for other liver-specific enzymes including aldolase B and phospho enol pyruvate carboxykinase did not vary significantly from normal littermates. As for extrahepatic expression of the urea cycle enzymes, only argininosuccinate synthetase in the kidney was decreased. Nuclear run-on experiments showed reduced transcription of the corresponding genes, which mostly accounts for the low mRNA levels. Furthermore, the time-course of mRNA accumulation from 5 days of age showed that the developmental induction of hepatic carbamyl phosphate synthetase and argininosuccinate synthetase mRNAs was strongly suppressed. These results suggest that jvs affects not only the regulation of the tissue-specific expression of the urea cycle enzymes but also the regulation of their developmental induction.

Animals↗
PubMed1992

Pore-forming activity of OmpA protein of Escherichia coli.

Escherichia coli outer membrane protein OmpA was purified to homogeneity, as a monomer, from a K12 derivative deficient in both OmpF and OmpC porins. When proteoliposomes reconstituted from the purified OmpA, phospholipids, and lithium dodecyl sulfate were tested for permeability to small molecules by osmotic swelling, it was found that OmpA produced apparently nonspecific diffusion channels that allowed the penetration of various solutes. The pore-forming activity was destroyed by the heat denaturation of the OmpA protein, and the use of an OmpA-deficient mutant showed that the activity was not caused by copurifying contaminants. The size of the OmpA channel, estimated by comparison of diffusion rates of solutes of different sizes, was rather similar to that of E. coli OmpF and OmpC porins, i.e. about 1 nm in diameter. The rate of penetration of L-arabinose caused by a given amount of OmpA protein, however, was about a hundredfold lower than the rate produced by the same amount of E. coli OmpF porin. The addition of large amounts of lithium dodecyl sulfate to the reconstitution mixture increased the permeability through the OmpA channel, apparently by facilitating the correct insertion of OmpA into the bilayer.

Arabinose↗
PubMed1992

Outer membranes of gram-negative bacteria are permeable to steroid probes.

The permeability of bacterial outer membranes was assayed by coupling the influx of highly hydrophobic probes, 3-oxosteroids, with their subsequent oxidation catalysed by 3-oxosteroid delta 1-dehydrogenase, expressed from a gene cloned from Pseudomonas testosteroni. In Salmonella typhimurium producing wild-type lipopolysaccharide, the permeability coefficients for uncharged steroids were 0.45 to 1 x 10(-5) cm s-1, and the diffusion appeared to occur mainly through the lipid bilayer domains of the outer membrane. These rates are one or two magnitudes lower than that expected for their diffusion through the usual biological membranes. The permeation rates were markedly increased (up to 100 times) when the lipopolysaccharide leaflet was perturbed either by adding deacylpolymyxin or by introducing mutations leading to the production of deep rough lipopolysaccharides. An amphiphilic, negatively charged probe, testosterone hemisuccinate, penetrated much more slowly than the uncharged steroids. Study of various Gram-negative species revealed that P. testosteroni, Pseudomonas acidovorans, and Acinetobacter calcoaceticus showed higher outer membrane permeability to steroid probes and higher susceptibility to hydrophobic agents such as fusidic acid, novobiocin and crystal violet relative to S. typhimurium and Escherichia coli.

Bacterial Outer Membrane Proteins↗
PubMed1992

Interaction between maltose-binding protein and the membrane-associated maltose transporter complex in Escherichia coli.

Active transport of maltose in Escherichia coli requires the presence of both maltose-binding protein (MBP) in the periplasm and a complex of MalF, MalG, and MalK proteins (FGK2) located in the cytoplasmic membrane. Earlier, mutants in malF or malG were isolated that are able to grow on maltose in the complete absence of MBP. When the wild-type malE+ allele, coding for MBP, was introduced into these MBP-independent mutants, they frequently lost their ability to grow on maltose. Furthermore, starting from these Mal- strains, Mal+ secondary mutants that contained suppressor mutations in malE were isolated. In this study, we examined the interaction of wild-type and mutant MBPs with wild-type and mutant FGK2 complexes by using right-side-out membrane vesicles. The vesicles from a MBP-independent mutant (malG511) transported maltose in the absence of MBP, with Km and Vmax values similar to those found in intact cells. However, addition of wild-type MBP to these mutant vesicles produced unexpected responses. Although malE+ malG511 cells could not utilize maltose, wild-type MBP at low concentrations stimulated the maltose uptake by malG511 vesicles. At higher concentrations of the wild-type MBP and maltose, however, maltose transport into malG511 vesicles became severely inhibited. This behaviour of the vesicles was also reflected in the phenotype of malE+ malG511 cells, which were found to be capable of transporting maltose from a low external concentration (1 microM), but apparently not from millimolar concentrations present in maltose minimal medium. We found that the mutant FGK2 complex, containing MalG511, had a much higher apparent affinity towards the wild-type MBP than did the wild-type FGK2 complex.(ABSTRACT TRUNCATED AT 250 WORDS)

ATP-Binding Cassette Transporters↗
PubMed1992

Porins and specific channels of bacterial outer membranes.

Porins and specific channels both produce water-filled pores that allow the transmembrane diffusion of small solutes, but the latter contain specific ligand-binding sites within the channels. Recent structural studies show that many or most of these proteins exist as beta-barrels with the beta-strands traversing the thickness of the outer membrane. The channels often have diameters in the range of 1 nm, and thus the penetration rates of solutes through porin channels are likely to be affected strongly by what appear to be minor differences in the size, shape, hydrophobicity or charge of the solute molecule. With the specific channels, the presence of binding sites can accelerate very significantly the diffusion of some ligands when they are present at low concentrations. Thus these simple channels can sometimes achieve a surprising degree of real or apparent specificity. Recent data tend to favour the idea that these proteins are first exported into the periplasm, and then inserted into the outer membrane. Although lipopolysaccharides seem to play a significant role in the final assembly of the trimeric porins, the details of the targeting process still remain to be elucidated.

Amino Acid Sequence↗
PubMed1992

Oxidation of 1,1-diphenylhydrazine to N-nitrosodiphenylamine by superoxide radical in the eye.

By microsomes obtained from bovine ciliary body, 1,1-diphenylhydrazine was oxidized to N-nitrosodiphenylamine in the presence of NADPH. This reaction was stimulated by riboflavin which was recognized to be an electron carrier. The oxidizing activity by microsomes was markedly inhibited by superoxide dismutase, but not by SKF 525-A or carbon monoxide. Similarly, the oxidation of 1,1-diphenylhydrazine to its corresponding nitrosamine occurred in varying degrees when the hydrazine derivative was exposed to visible light in the presence of photosensitizers such as riboflavin, flavin adenine dinucleotide, flavin mononucleotide, lumiflavin, lumichrome, NAD+, NADH, NADP+, or NADPH. The photochemical oxidation was inhibited by active oxygen-scavengers such as superoxide dismutase, L-ascorbic acid or alpha-tocopherol. The superoxide radical involved in the photochemical reaction was determined by measuring the oxidation of epinephrine to adrenochrome. The oxidation of epinephrine was well correlated to that of 1,1-diphenylhydrazine. Thus, the present study provided evidence that the superoxide radical is responsible for the oxidation of a hydrazine derivative to a corresponding nitrosamine by ocular tissue microsomes and by photosensitizers.

Adrenochrome↗
PubMed1991

Trimerization of an in vitro synthesized OmpF porin of Escherichia coli outer membrane.

The assembly of outer membrane proteins of Escherichia coli was examined using the OmpF porin as a model. Since this protein is made as a precursor, which is processed to a protein of Mr 37,000 before being assembled into trimers in the outer membrane, we synthesized a modified OmpF, which lacked 16 out of 22 amino acid residues from its signal sequence, in a coupled transcription-translation system. This modified protein resembled the unfolded, monomeric OmpF in its electrophoretic behavior, but much of the protein apparently existed in a more tightly folded conformation as it was recognized by a monoclonal antibody specific to a surface epitope of the native, trimeric OmpF porin. At least some conformers of this protein could be further incorporated into outer membrane or lipopolysaccharide bilayers, and assembled into trimers. The trimers formed were trypsin-resistant and heat-stable in sodium dodecyl sulfate up to 70 degrees C, thus showing the characteristics of the native trimeric protein. These results extend our earlier observation that OmpF monomer secreted by spheroplasts of E. coli can be trimerized in vitro (Sen, K., and Nikaido, H (1990) Proc. Natl. Acad. Sci. U.S. A 87, 743-747) and show that the trimerization can occur, albeit at a low efficiency, with porin monomers synthesized in vitro, presumably not contaminated by membrane fragments or other components of the cell envelope. However, comparison of trimerization efficiency of the nascent in vitro product with that of the same product already exposed to aqueous medium, as well as with that of the spheroplast-secreted product, leads us to the working hypothesis that the trimerization process in intact cells is accelerated either by accessory components or by the conformational changes accompanying the secretion through the cytoplasmic membrane and that the reactions observed in this study represent only part of the physiological process.

Amino Acid Sequence↗
PubMed1991

Purification and characterization of the membrane-associated components 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. The three membrane-associated components of the transport system, the MalK, MalF, and MalG proteins, have been solubilized from the membrane and maltose transport activity has been reconstituted in proteoliposome vesicles (Davidson, A. L., and Nikaido, H. (1990) J. Biol. Chem. 265, 4254-4260). A modification of the reconstitution technique is presented which permits reconstitution from the detergent dodecyl maltoside. Utilizing reconstitution of maltose transport as an assay, we have purified these proteins in the presence of n-dodecyl-beta-D-maltoside. The purified proteins catalyze both maltose transport activity and ATP hydrolysis. In all experiments, the MalF, MalG, and MalK proteins behaved as a multiprotein complex; all three proteins were immunoprecipitated using antibody prepared against MalF, and they copurified, eluting from a gel filtration column between markers of Mr 160,000 and 200,000. Each complex contains two MalK, one MalF, and one MalG proteins, providing two putative sites for ATP hydrolysis. Chemical cross-linking detected specific interactions between MalF and MalG and between MalF and MalK.

ATP-Binding Cassette Transporters↗
PubMed1991

Identification and characterization of porins in Pseudomonas aeruginosa.

Earlier studies have shown that the major porin species in Pseudomonas aeruginosa outer membrane is protein F (OprF), which produces channels wider than those produced by Escherichia coli porins. In contrast, Yoshihara and Nakae ((1989) J. Biol. Chem. 264, 6297-6301) reported that protein F has no pore-forming activity as measured by the flux of L-arabinose, and that the channels in P. aeruginosa outer membrane, being produced by proteins C, "D," and "E," are much narrower than E. coli porin channels. In this study, we followed the protein purification scheme of Yoshihara and Nakae as closely as possible, and found that protein F had a specific activity for pore formation similar to that of proteins D1, D2, and E2. Furthermore, proteoliposome reconstitution assays showed conclusively that the channels formed by protein F, as well as by unfractionated outer membranes, allowed the diffusion of a tetrasaccharide, stachyose, at a significant rate, indicating that these channels are much larger than E. coli porin channels. It appears likely that in the study of Yoshihara and Nakae protein F was inadvertently inactivated during purification. We further suggest a hypothesis that resolves the apparent conflict between the presence of large diameter channels and the low permeability of the outer membrane in P. aeruginosa.

Arabinose↗
PubMed1991

Primary orbital malignant lymphoma: a clinicopathology study of 17 cases.

We investigated the clinicopathologic characteristics of 17 patients (13 men and 4 women) with primary orbital malignant lymphoma using the Working Formulation. Most of the cases belonged to the low-grade malignancy group, and more women than men were in the histologically high-grade malignancy group. The phenotype of the tumor cells was investigated immunohistochemically. All cases showed the monoclonal feature of a B-cell lineage. All patients received chemotherapy with or without radiotherapy. Of 16 subjects, 15 achieved a complete remission; none of these patients has had a recurrence since the completion of the initial therapy (range of follow-up from 16 months to 10 years). One patient died.

Adult↗
PubMed1991

Specificity of the sex-influenced esterase (ES-SI) isozyme in rat liver.

A carboxylesterase which shares common antigenicity with sex-influenced esterase (ES-SI) was found in both the male and the female liver of the rat but not in the following tissues: erythrocyte, heart, kidney, lung, spleen, small intestine, testis, thymus, and lymph node. Subcellular fractionation showed the esterase localizes in the microsome-rich fraction. The strain distribution of the presence or absence of the esterase in inbred rats was identical to that of ES-SI, although in adult males a considerable amount of the esterase exists, unlike ES-SI. The esterase had a higher isoelectric point than ES-SI but after neuraminidase treatment the difference disappeared, suggesting that the esterase has a sialic acid moiety. Because this esterase has different properties from those previously reported, it is proposed that it is designated liver-ES-SI. The common antigenicity and similar strain distribution between ES-SI and liver-ES-SI suggest that liver-ES-SI is a precursor molecule of ES-SI and therefore the two esterases are products of a single gene.

Animals↗