BaTiO3 doped Na0.5K0.5NbO3 thin films deposited by using eclipse shutter enhanced pulsed laser deposition method.

We have investigated structural, electrical, and electro-mechanical properties of lead-free piezoelectric BaTiO3 doped Na0.5K0.5NbO3 (BTO-NKN) thin films deposited by pulsed laser deposition (PLD) methods. BTO-NKN thin films have been deposited on La0.5Sr0.5CoO3 (LSCO) bottom electrodes with LaAlO3 (LAO) substrates. X-ray diffraction data have shown that all the BTO-NKN and bottom electrodes are highly oriented with their c-axes normal to the substrates. In order to improve the morphology of BTO-NKN thin films, we have located an eclipse shutter between a target and a substrate. Root-mean-square roughness was changed from 91 nm to 21 nm with eclipse shutter enhanced PLD (E-PLD) method. Furthermore, the enhanced surface morphology leads to the improvement in electrical or electro-mechanical properties mainly due to increased density. Typical capacitance and d33 values of a BTO-NKN film deposited by E-PLD method are 1000 pF and 30 pmN, respectively.

Pseudonocardia kongjuensis sp. nov., isolated from a gold mine cave.

The taxonomic position of an isolate that was recovered from a gold mine cave near Kongju, Republic of Korea, was determined by 16S rDNA sequence studies and chemotaxonomic characterization. Comparative studies of 16S rDNA sequences indicated that this organism was phylogenetically related to members of the genus Pseudonocardia, branching outside a cluster encompassing Pseudonocardia autotrophica and Pseudonocardia compacta. The affiliation to the genus was also supported by the cell chemistry, which was represented by a type IV cell wall, MK-8(H4) as the major menaquinone, a phospholipid type PIII pattern (phosphatidylcholine as a diagnostic phospholipid) and a DNA G+C content of 71 mol%. The fatty acid profile contained saturated, unsaturated and 10-methyl branched fatty acids, but tuberculostearic acid and hydroxy fatty acids were not present. The isolate differed from its phylogenetic neighbours in the presence of phosphatidylethanolamine, dodecanoate, 16-methylheptadecenoate and 16-methylheptadecanoate and the absence of phosphatidylinositol mannoside and phosphatidylmethylethanolamine. The unique combination of physiological properties, the cellular fatty acid profile and DNA-DNA hybridization data indicates that this organism is readily differentiated from the type strains of all of the validly published species of the genus Pseudonocardia. The name Pseudonocardia kongjuensis sp. nov. is proposed for the type strain, LM 157T (= IMSNU 50583T = KCTC 9990T = DSM 44525T).

Syntheses and structural and electrochemical characterizations of vanadatricarbadecaboranyl analogues of vanadocene and the structural characterization of the [Li(CH(3)CN)2+](6-CH(3)-nido-5,6,9-C(3)B(7)H(9-) tricarbadecaboranyl anion.

A single-crystal X-ray determination of the [Li(CH(3)CN)(2)(+)](6-CH(3)-nido-5,6,9-C(3)B(7)H(9)(-)) salt has shown that the 6-CH(3)-nido-5,6,9-C(3)B(7)H(9)(-) tricarbadecaboranyl anion has a nido-cage geometry based on an octadecahedron missing the unique six-coordinate vertex. The resulting six-membered open face is puckered, with two of the cage carbons (C6 and C9) occupying the low-coordinate cage positions above the plane of the four remaining atoms (C5, B7, B8, and B10). The Li(+) ion is centered over the open face and is solvated by two acetonitrile molecules. The reactions of the 6-CH(3)-nido-5,6,9-C(3)B(7)H(9)(-) anion with various vanadium halide salts, including VCl(4), VCl(3), and VBr(2), each resulted in the isolation of the same five paramagnetic products (2-6) of composition V(CH(3)-C(3)B(7)H(9))(2). X-ray crystallographic determinations of 2-5 showed that the complexes consist of two octadecahedral VC(3)B(7) fragments sharing a common vanadium vertex and established their structures as commo-V-(1-V-4'-CH(3)-2',3',4'-C(3)B(7)H(9))(1-V-2-CH(3)-2,3,4-C(3)B(7)H(9)) (2), commo-V-(1-V-5'-CH(3)-2',3',5'-C(3)B(7)H(9))(1-V-4-CH(3)-2,3,4-C(3)B(7)H(9)) (3), commo-V-(1-V-5'-CH(3)-2',3',5'-C(3)B(7)H(9))(1-V-2-CH(3)-2,3,4-C(3)B(7)H(9)) (4), and commo-V-(1-V-2-CH(3)-2,3,4-C(3)B(7)H(9))(2) (5). These complexes can be considered as tricarbadecaboranyl analogues of vanadocene, (eta(5)-C(5)H(5))(2)V. However, unlike vanadocene, these complexes are air- and moisture-stable and have only one unpaired electron. The five complexes differ with respect to one another in that they either (1) contain different enantiomeric forms of the CH(3)-C(3)B(7)H(9) cages, (2) have a different twist orientation of the two cages, or (3) have the methyl group of the CH(3)-C(3)B(7)H(9) cage located in either the 2 or 4 position of the cage. Subsequent attempts to oxidize the compounds with reagents such as Br(2) and Ag(+) were unsuccessful, illustrating the ability of the tricarbadecaboranyl anion to stabilize metals in low oxidation states. Consistent with this, both the electrochemical oxidation and the reduction of 2 were much more positive than those of the same oxidation state changes in vanadocene. The one-electron reduction of 2 is a remarkable 2.9 V positive of that of Cp(2)V.

Mitochondrial NADH-cytochrome b(5) reductase plays a crucial role in the reduction of D-erythroascorbyl free radical in Saccharomyces cerevisiae.

The relevance of NADH-cytochrome b(5) reductase to the NADH-dependent reduction of D-erythroascorbyl free radical was investigated in Saccharomyces cerevisiae. MCR1, which is known to encode NADH-cytochrome b(5) reductase in S. cerevisiae, was disrupted by the insertion of URA3 gene into the gene of MCR1. In the mcr1 disruptant cells, the activity of NADH-D-erythroascorbyl free radical reductase almost disappeared and the intracellular level of D-erythroascorbic acid was about 11% of that of the congenic wild-type strain. In the transformant cells carrying MCR1 in multicopy plasmid, the intracellular level of D-erythroascorbic acid and the activity of NADH-D-erythroascorbyl free radical reductase increased up to 1.7-fold and 2.1-fold, respectively. Therefore, it indicated that the MCR1 product, mitochondrial NADH-cytochrome b(5) reductase, plays a key role in the NADH-dependent reduction of D-erythroascorbyl free radical in S. cerevisiae. On the other hand, the mcr1 disruptant cells were hypersensitive to hydrogen peroxide and menadione, and overexpression of MCR1 made the cells more resistant against oxidative stress. These results suggested that the mitochondrial NADH-cytochrome b(5) reductase functions as NADH-D-erythroascorbyl free radical reductase and plays an important role in the response to oxidative damage in S. cerevisiae.

Deficiency of D-erythroascorbic acid attenuates hyphal growth and virulence of Candida albicans.

In some lower eukaryotes, D-erythroascorbic acid, a five-carbon analog of L-ascorbic acid, is present instead of L-ascorbic acid. We have cloned ALO1, the gene encoding D-arabinono-1,4-lactone oxidase, which catalyzes the final step of D-erythroascorbic acid biosynthesis in Candida albicans. The ALO1 gene contained a continuous open reading frame of 1,671 bp that encodes a polypeptide consisting of 557 amino acids with a calculated molecular mass of 63,428 Da. To investigate the functional roles of D-erythroascorbic acid in C. albicans, we disrupted or overexpressed the ALO1 gene. In the alo1/alo1 null mutants, the activity of D-arabinono-1,4-lactone oxidase was completely lost and D-erythroascorbic acid could not be detected. When ALO1 on a multicopy plasmid was transformed in C. albicans, the enzyme activity and the intracellular D-erythroascorbic acid level were increased up to 3.4-fold and 4.0-fold, respectively. The alo1/alo1 null mutants of C. albicans showed increased sensitivity towards oxidative stress. Overexpression of ALO1 made the cells more resistant to the same stress. The alo1/alo1 mutants showed defective hyphal growth and attenuated virulence. Taken together, our results suggest that D-erythroascorbic acid functions as an important antioxidant and can be considered one of the virulence factors enhancing the pathogenicity of C. albicans.

Characterization of the gene family encoding alternative oxidase from Candida albicans.

Candida albicans possesses a cyanide-resistant respiratory pathway mediated by alternative oxidase (AOX), which seems to be encoded by a gene family with two members. Cloning and expression of AOX1a, one of the genes encoding alternative oxidase from C. albicans, has previously been reported [Huh and Kang (1999) J. Bacteriol. 181, 4098-4102]. In the present study we report the isolation of another gene coding for alternative oxidase, designated AOX1b. AOX1b contains a continuous open reading frame that encodes a polypeptide consisting of 365 amino acids. Interestingly, AOX1a and AOX1b were found to be located in tandem on one of the chromosomes of C. albicans. The presence of cyanide in the culture medium remarkably retarded the growth of the aox1a/aox1a mutants. The growth of the aox1b/aox1b mutants and the aox1a/aox1a aox1b/aox1b double mutants was almost completely inhibited in the same medium. beta-Galactosidase reporter assays indicated that, whereas AOX1a was expressed constitutively, the expression of AOX1b was dependent on growth phase and was induced by treatment with cyanide, antimycin A, H(2)O(2), menadione and paraquat. Growth of the cells in media with non-fermentable carbon sources also enhanced the expression of AOX1b. CaSLN1, which encodes a histidine kinase, seems to be involved in the regulation of AOX expression in C. albicans on the basis of the observation that the activity of cyanide-resistant respiration and the expression level of AOX in the casln1/casln1 mutants were found to be significantly low under normal conditions and slightly increased in the presence of respiratory inhibitors compared with the wild-type strain. Like AOX1a, AOX1b could also be functionally expressed in AOX-deficient Saccharomyces cerevisiae and confer cyanide-resistant respiration on the organism.

The Escherichia coli glucose transporter enzyme IICB(Glc) recruits the global repressor Mlc.

In addition to effecting the catalysis of sugar uptake, the bacterial phosphoenolpyruvate:sugar phosphotransferase system regulates a variety of physiological processes. Exposure of cells to glucose can result in repression or induction of gene expression. While the mechanism for carbon catabolite repression by glucose was well documented, that for glucose induction was not clearly understood in Escherichia coli. Recently, glucose induction of several E.coli genes has been shown to be mediated by the global repressor Mlc. Here, we elucidate a general mechanism for glucose induction of gene expression in E.coli, revealing a novel type of regulatory circuit for gene expression mediated by the phosphorylation state-dependent interaction of a membrane-bound protein with a repressor. The dephospho-form of enzyme IICB(Glc), but not its phospho-form, interacts directly with Mlc and induces transcription of Mlc-regulated genes by displacing Mlc from its target sequences. Therefore, the glucose induction of Mlc-regulated genes is caused by dephosphorylation of the membrane-bound transporter enzyme IICB(Glc), which directly recruits Mlc to derepress its regulon.

Synthesis and reactivity of an efficient 1,2-dehydrocarborane precursor, phenyl[o-(trimethylsilyl)carboranyl]iodonium acetate.

The reaction of [o-(trimethylsilyl)carboranyl]lithium with IPh(OAc)2 in diethyl ether affords an efficient 1,2-dehydro-o-carborane precursor 4: the facile 2 + 4 cycloaddition of 4 with dienes in the presence of the desilylating agent is reported.

The first 1,3-digermyla-2-nickela-carboranylene and the Ni-catalyzed double germylation of unsaturated organic substrates.

The reaction of o-bis(dimethylgermyl)carborane with Ni(PEt3)4 in pentane affords the reactive intermediate, [o-(GeMe2)2C2B10H10]Ni(PEt3)2 2: the facile double germylation of unsaturated organic substrates catalyzed by 2 is reported.

Self-assembly of one-dimensional coordination polymers from AgX (X = CF(3)SO(3)(-), ClO(4)(-), and NO(3)(-)) and 2-aminomethylpyridinedipropionitrile (2-AMPDPN).

Three novel supramolecules whose topologies depend on the counteranion, [Ag(2-AMPDPN)X] (X = CF(3)SO(3)(-) (1), ClO(4)(-) (2), and NO(3)(-) (3)), have been prepared by the self-assembly of AgX (X = CF(3)SO(3)(-), ClO(4)(-), and NO(3)(-)) with 2-aminomethylpyridinedipropionitrile (2-AMPDPN). The crystal structures reveal different packing arrangements of the one-dimensional infinite coordination polymers. Compound 1 is made up of ladder chains that are interlocked by cyano groups, while the polymeric chain of 2 is isomorphous to 1 except for the Ag-Ag interaction. Compound 3 consists of helical chains that are surrounded by nitrate. The adjacent helical chains are racemic. The structures of 1, 2, and 3 suggest the role that the counterions may play in the network construction.

Protein glycation: creation of catalytic sites for free radical generation.

In a glycation reaction, alpha-dicarbonyl compounds such as deoxyglucosone, methylglyoxal, and glyoxal are more reactive than the parent sugars with respect to their ability to react with amino groups of proteins to form inter- and intramolecular cross-links of proteins, stable end products called advanced Maillard products or advanced end products (AGEs). The AGEs, which are irreversibly formed, accumulate with aging, atherosclerosis, and diabetes mellitus, and are especially associated with long-lived proteins such as collagens, lens crystallins, and nerve proteins. It was suggested that the formation of AGEs not only modifies protein properites but also induces biological damage in vivo. In this report, we summerize results obtained from our studies for (1) identifying the structure of the cross-linked radical species formed in the model system-the reaction between alpha-dicarbonyl methylglyoxal with amino acids, and (2) the reactivity of the radical center of the protein created by the similar reaction. These results indicate that glycation of protein generates active centers for catalyzing one-electron oxidation-reduction reactions. This active center, which exhibits enzyme-like character, is suggested to be the cross-linked Schiff-based radical cation of the protein. It mimics the characteristics of the metal-catalyzed oxidation system. These results together indicate that glycated proteins accumulated in vivo provide stable active sites for catalyzing the formation of free redicals.

Identification of cis site involved in nickel-responsive transcriptional repression of sodF gene coding for Fe- and Zn-containing superoxide dismutase of Streptomyces griseus.

A sodF gene coding for iron- and zinc-containing superoxide dismutase (FeZnSOD) of Streptomyces griseus was cloned and sequenced. A 5' end of 0.8-kb sodF transcript was mapped at the 57 nucleotides upstream from an ATG initiation codon. Employing expressions of sodF::xylE fusions in trans in Streptomyces lividans, nickel-responsive transcriptional repression was found to be relieved if mutations were introduced into an operator sequence of inverted-repeat, TTGCAN(7)TGCAA, which traverses the 5' end (+1, G) of the sodF mRNA. Nickel-dependent interaction between cell extracts and sodF regulatory DNA, monitored through gel-mobility shift assay, was abolished when the operator was mutated. Recombinant sodF operon having operator mutations showed protein level and enzyme activity, which were no longer repressed by nickel, suggesting that nickel-responsive repression of FeZnSOD is regulated mainly at the level of transcription through the operator.

Enzyme-like activity of glycated cross-linked proteins in free radical generation.

The structure and property of cross-linked amino acids and proteins produced by a three- carbon alpha-dicarbonyl methylglyoxal in glycation reaction were investigated. Our results showed that these reactions generated yellow fluorescent products and several free radical species. From the reaction with alanine, three types of free radicals were identified by EPR spectroscopy: 1) the cross-linked radical cation, methylglyoxal diaklylimine cation radical; 2) the methylglyoxal radical anion as the counterion; 3) the superoxide radical anion produced only in the presence of oxygen. Glycation of bovine serum albumin by methylglyoxal also generated the protein-bound, cross-linked free radical, probably the cation radical of the cross-linked Schiff base as observed with alanine. The glycated protein reduced ferricytochrome c to ferrocytochrome c in the absence of oxygen or added metal ions. This reduction of cytochrome c was accompanied by a large increase in the amplitude of the electron paramagnetic resonance signal originated from the protein-bound free radical. In addition, the glycated protein catalyzed the oxidation of ascorbate in the presence of oxygen while the protein-free radical signal disappeared. These results indicate that glycation of protein generates active centers for catalyzing one-electron oxidation-reduction reactions. This active center, which exhibits enzyme-like character, was suggested to be the cross-linked Schiff base/the cross-linked Schiff base radical cation of the protein. It mimics the characteristics of metal-catalyzed oxidation system. These results together indicate that glycated proteins accumulated in vivo provide stable active-sites for catalyzing the formation of free radicals.

Catellatospora koreensis sp. nov., a novel actinomycete isolated from a gold-mine cave.

A new actinomycete strain, LM 042T, which was isolated from a gold-mine cave in Kongju, Republic of Korea, is described by phenotypic and genotypic characters. The organism formed short chains of non-motile spores and globose bodies from substrate mycelium. An aerial mycelium was absent. This organism was chemotaxonomically characterized by the presence of meso-diaminopimelic acid, rhamnose, xylose, glucose, mannose and ribose in whole-cell hydrolysates (a type II cell wall and a variant of sugar pattern D), a glycolyl type of muramic acid, DNA G+C content of 70.4 mol%, a type PII phospholipid pattern (phosphatidylethanolamine as a diagnostic nitrogenous phospholipid), a tetrahydrogenated menaquinone with 10 isoprene units as a major menaquinone, and fatty acid profiles predominated by iso-branched hexadecanoic acid, iso-branched pentadecanoic acid and heptadcenoic acid. A comparative analysis of 16S rDNA sequences indicated that this organism formed a distinct clade within the evolutionary radiation of the family Micromonosporaceae and clustered with members of the genus Catellatospora. The 16S rDNA similarity values between the isolate and its phylogenetic neighbours, the two subspecies of Catellatospora citrea and Catellatospora tsunoense, were 95.0-95.2% and 94.9%, respectively. An equidistant relationship was observed among the isolate, Catellatospora ferruginea and all other members of the Micromonosporaceae genera (levels of similarity 93.0-94.0%). The combination of physiological, chemotaxonomic and DNA-DNA hybridization data supported that this organism is a novel species of the genus Catellatospora, for which the name Catellatospora koreensis sp. nov. is proposed. The type strain is LM 042T (= IMSNU 50729T).

Saccharothrix violacea sp. nov., isolated from a gold mine cave, and Saccharothrix albidocapillata comb. nov.

The generic position of two isolates from soils inside a gold mine cave in Kongju, Korea, was determined by 16S rDNA sequencing and chemotaxonomic characteristics. Phylogenetic analysis indicated that both of the isolates formed a clade with Lentzea albidocapillata and members of the genus Saccharothrix of the family Pseudonocardiaceae. The chemical composition of the isolates and of Lentzea albidocapillata was consistent with that of the genus Saccharothrix, which is characterized by a type III cell wall (the meso-isomer of diaminopimelic acid, and galactose and rhamnose as characteristic whole-cell sugars), MK-9(H4) as the major menaquinone, and a phospholipid type PII pattern (phosphatidylethanolamine as a diagnostic phospholipid). The combination of morphological features, chemotaxonomic characters and phylogenetic data supported the proposal that Lentzea albidocapillata, the only and type strain of the genus Lentzea, should be transferred to the genus Saccharothrix. On the basis of physiological properties, cellular fatty acid composition and DNA-DNA hybridization data, two new species within the genus Saccharothrix are proposed: Saccharothrix violacea sp. nov., type strain LM 036T (= IMSNU 50388T), and Saccharothrix albidocapillata comb. nov., type strain DSM 44073T (=IMSNU 21253T).

Hongia gen. nov., a new genus of the order Actinomycetales.

An aerobic, nocardioform actinomycete, named LM 161T, was isolated from a soil sample obtained from a gold mine in Kongiu, Republic of Korea. This organism formed well-differentiated aerial and substrate mycelia and produced branched hyphae that fragmented into short or elongated rods. The cell wall contains major amounts of LL-diaminopimelic acid, alanine, glycine, glutamic acid, mannose, glucose, galactose, ribose and acetyl muramic acid. The major phospholipids of this isolate are phosphatidylcholine, diphosphatidylglycerol, phosphatidylglycerol and phosphatidylinositol, and the major isoprenologue is a tetrahydrogenated menaquinone with nine isoprene units. The whole-cell hydrolysate of strain LM 161T contains 12-methyltetradecanoic and 14-methylpentadecanoic acids as the predominant fatty acids, but does not contain mycolic acids. The G+C content of the DNA is 71.3 mol%. The phylogenetic position of the test strain was investigated using an almost complete 16S rDNA sequence. The isolate formed the deepest branch in the clade encompassing the members of the suborder Propionibacterineae Rainey et al. 1997. On the basis of chemical, phenotypic and genealogical data, it is proposed that this isolate be classified within a new genus as Hongia koreensis gen. nov., sp. nov. in the order Actinomycetales. The type strain is LM 161T (= IMSNU 50530T).

Enhanced sensitivity of Streptomyces seoulensis to menadione by superfluous lipoamide dehydrogenase.

Lipoamide dehydrogenase from Streptomyces seoulensis could facilitate menadione-mediated cytochrome c reduction, which was mostly inhibited by superoxide dismutase, indicating the obvious involvement of superoxide radical anion. In this reaction, the production of superoxide radical anion occurred via a menadione semiquinone radical anion. When exposed to menadione, lipoamide dehydrogenase-overexpressing cells showed a much lower survival rate with a concomitant decrease of intracellular protein thiol than the wild-type strain. These results suggest that lipoamide dehydrogenase is a facilitating agent in the redox cycling of quinone compounds in vivo as well as in vitro and could inevitably increase the potential toxicity of the compounds.

Amycolatopsis thermoflava sp. nov., a novel soil actinomycete from Hainan Island, China.

A soil isolate, which had been assigned to the genus Nocardia, was shown to have properties consistent with its classification in the genus Amycolatopsis. An almost complete nucleotide sequence of the 16S rDNA of the strain was determined following cloning and sequencing of the amplified gene. The sequence was aligned with those available for members of the family Pseudonocardiaceae and related taxa and phylogenetic trees were inferred using three tree-making algorithms. The organism consistently formed a distinct monophyletic clade with the type strain of Amycolatopsis methanolica, but DNA-DNA relatedness data showed that the two strains belonged to distinct genomic species. The organism was also distinguished from the type strains of all validly described species of Amycolatopsis using a battery of phenotypic properties. The genotypic and phenotypic data show that the strain merits recognition as a new species of the genus Amycolatopsis. The name proposed for the new species is Amycolatopsis thermoflava sp. nov. The type strain is IFO 14333T.

Bacterial production of D-erythroascorbic acid and L-ascorbic acid through functional expression of Saccharomyces cerevisiae D-arabinono-1,4-lactone oxidase in Escherichia coli.

D-Arabinono-1,4-lactone oxidase, which catalyzes the terminal step in the biosynthesis of D-erythroascorbic acid in Saccharomyces cerevisiae, was functionally expressed in Escherichia coli inherently lacking the enzyme. The recombinant E. coli strain expressing the enzyme could overproduce D-erythroascorbic acid and L-ascorbic acid when supplied with D-arabinono-1,4-lactone and L-galactono-1,4-lactone, respectively.

Spectral characterization and chemical modification of FMN-containing ascorbyl free-radical reductase from Pleurotus ostreatus.

Ascorbyl free-radical reductase was purified 1143-fold with an overall yield of 9.9% from the cytosolic fraction of Pleurotus ostreatus. The native enzyme had a molecular mass of 127 kDa and SDS/PAGE revealed that the enzyme consists of two subunits, each with a molecular mass of 62 kDa. The enzyme utilized only NADH as an electron donor. The enzyme was highly specific for ascorbyl free radical as an electron acceptor and capable of catalysing the reduction of ferricyanide and 2,6-dichloroindophenol as artificial electron acceptors. The apparent K(m) values of the enzyme towards NADH and ascorbyl free radical were 35+/-0.22 and 2.1+/-0.03 microM, respectively. The catalytic mechanism of this enzyme is of Ping Pong type. The enzyme contained FMN as a prosthetic group and showed the characteristic absorption spectrum ascribed to the charge-transfer interaction of thiolate anion with FMN. The enzyme contained eight cysteine residues per monomer and was inactivated more rapidly by mercurials than by thiol-alkylating reagents. Kinetic analysis of the inactivation process revealed that the enzyme had 1 mol of thiol group/mol of subunit in the active site with a pK(a) of 6.9. The modification of the thiol group of the enzyme caused the loss of charge-transfer absorbance centred at 540 nm and blocked the electron-transfer process from NADH to FMN. The modification of lysine, arginine and histidine residues led to the loss of its activity. Unlike the active enzyme, the fluorescence quenching of NADH was not observed in the lysine-modified enzyme, which implies that lysine residues can participate in the interaction with NADH.