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Background: Vasodilators, such as nitroglycerin, have long been first-line treatments for acute heart failure syndromes (AHFS). Nicorandil is a vasodilator with dual potassium channel opening and nitrate properties. However, there are no randomized controlled studies of intravenous nicorandil safety and efficacy in the urgent phase AHFS. We examined the symptomatic, hemodynamic, and echocardiographic effects and safety, and 60-day clinical outcomes of intravenous nicorandil, in addition to standard therapy, in patients with AHFS in the urgent phase., Methods: In this prospective, randomized controlled trial, 106 AHFS patients were randomized within one hour of arrival to receive either standard therapy (control group, n=56) or standard therapy plus simultaneous intravenous nicorandil (0.2 mg/kg bolus followed by 0.2 mg/kg/h for 24 h; nicorandil group, n=50). Outcomes were assessed at 60 days., Results: Patients in the nicorandil group exhibited greater improvement of dyspnea as measured by change in a five-point Likert scale compared to those in the control group (after 1 h infusion: p=0.006, 6 h; p<0.001). The nicorandil group also showed significantly improved E/e', an estimate of left ventricular filling pressure, at 1 and 24 h ( p=0.001 and p=0.004, respectively). In addition, intravenous nicorandil therapy was safe and did not cause side effects such as excessive hypotension or reflex tachycardia. However, it did not reduce all-cause mortality and readmission rates at 60 days., Conclusions: Addition of intravenous nicorandil to standard therapy for urgent phase AHFS improved dyspnea and left ventricular diastolic function but not 60-day outcome.

The csnR gene, localized at the beginning of an operon, csnR-K, which organization is conserved through many actinomycete genomes, was previously shown to repress the transcription of the chitosanase gene csnA in Streptomyces lividans. However, knowledge on the function of the whole csnR-K operon in the metabolism of chitosan (an N-deacetylated derivative of chitin) remained limited. Mutants of S. coelicolor A3(2) harboring partial or total deletions of the csnR-K operon were analyzed for their capacity to uptake glucosamine oligosaccharides (GlcN)n. The csnR-K operon was autoregulated by CsnR repressor and its transcription was inducible by GlcN oligosaccharides. The operon controlled the uptake of GlcN oligosaccharides in S. coelicolor A3(2), with a minor contribution to the consumption of monomeric GlcN but not chitin-related N-acetylated derivatives. The deletion of the whole operon abolished the uptake of GlcN oligosaccharides. The CsnEFG transporter encoded by this operon is the front door for the assimilation of chitosan-derived hydrolysis products in S. coelicolor A3(2). The ATP-binding component MsiK was essential for CsnEFG transport function. Also, deletion of msiK abolished the induction of csnA transcription by GlcN oligosaccharides., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Arabidopsis thaliana BOR1 was the first boron (B) transporter identified in living systems. There are four AtBOR1-like genes, OsBOR1, 2, 3 and 4, present in the rice genome. We characterized the activity, expression and physiological function of OsBOR4. OsBOR4 is an active efflux transporter of B. Quantitative PCR analysis and OsBOR4 promoter-green fluorescent protein (GFP) fusion revealed that OsBOR4 was both highly and specifically expressed in pollen. We obtained five Tos17 insertion mutants of osbor4. The pollen grains were viable and development of floral organs was normal in the homozygous osbor4 mutants. We observed that in all Tos17 insertion lines tested, the frequency of osbor4 homozygous plants was lower than expected in the progeny of self-fertilized heterozygous plants. These results establish that OsBOR4 is essential for normal reproductive processes. Pollen from osbor4 homozygous plants elongated fewer tubes on wild-type stigmas, and tube elongation of mutant pollen was less efficient compared with the wild-type pollen, suggesting reduced competence of osbor4 mutant pollen. The reduced competence of mutant pollen was further supported by the crosses of independent Tos17-inserted alleles of OsBOR4. Our results suggest that OsBOR4, a boron efflux transporter, is required for normal pollen germination and/or tube elongation.

The dasD gene is located just downstream of the dasABC gene cluster, encoding components of an ABC transporter for uptake of a chitin-degradation product N,N'-diacetylchitobiose [(GlcNAc)(2) ] in Streptomyces coelicolor A3(2). To clarify the roles of the DasD protein in the degradation and assimilation of chitin, we obtained and characterized a recombinant DasD protein and a dasD-null mutant of S. coelicolor A3(2). The recombinant DasD protein produced in Escherichia coli showed N-acetyl-β-d-glucosaminidase (GlcNAcase) activity and its optimum temperature and pH were 40 °C and 7, respectively. dasD transcription was strongly induced in the presence of chitin, weakly by chitosan, but not by cellulose or xylan in S. coelicolor A3(2). Immuno-blot analysis demonstrated that DasD is a cytoplasmic protein. The dasD-null mutant exhibited cellular GlcNAcase activity which was comparable with that of the parent strain M145. DasD, thus, did not seem to be a major GlcNAcase. Induced extracellular chitinase activity in the dasD-null mutant was, interestingly, higher than M145, in the presence of colloidal chitin or (GlcNAc)(2) . In contrast to M145, (GlcNAc)(2) temporally accumulated in the culture supernatant of the dasD-null mutant in the presence of colloidal chitin., (© 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

The hydrolytic specificities of chitosanases were determined using N¹,N⁴-diacetylchitohexaose [(GlcN)₂-GlcNAc-(GlcN)₂-GlcNAc]. The results for the hydrolytic specificities of chitosanases belonging to subclasses I, II, and III toward chitohexaose and N¹,N⁴-diacetylchitohexaose agreed with previous results obtained by analysis of the hydrolysis products of partially N-acetylated chitosan. N¹,N⁴-Diacetylchitohexaose is a useful substrate to determine the hydrolytic specificity of chitosanase. On the other hand, chitosanases from Amycolatopsis sp. CsO-2 and Pseudomonas sp. A-01 showed broad cleavage specificity. They cleaved both the GlcNAc-GlcN and the GlcN-GlcNAc bonds in addition to the GlcN-GlcN bond in the substrate. Thus, both enzymes were new chitosanases. The chitosanases were divided into four subclasses according to their specificity for hydrolysis of the β-glycosidic linkages in partially N-acetylated chitosan.

Aleuria aurantia lectin (AAL) is an L-fucose-specific lectin produced in the mycelia and fruit-bodies of the widespread ascomycete fungus Aleuria aurantia. It is extensively used in the detection of fucose, but its physiological role remains unknown. To investigate this, we analyzed the interaction between AAL and, a zygomycete fungus Mucor racemosus, which is assumed to contain fucose in its cell wall. AAL specifically bound to the hyphae of M. racemosus, because binding was inhibited by L-fucose but not by D-fucose. It inhibited the growth of the fungus at 1 µM, and the M. racemosus cells were remarkably disrupted at 7.5 µM. In contrast, two other fucose-specific lectins, Anguilla anguilla agglutinin and Ulex europaeus agglutinin, did not inhibit the growth of M. racemosus. These results suggest that the growth inhibition activity is unique to AAL, and that AAL could act as an antifungal protein in natural ecosystems.

Although Streptomyces species are major chitin-degraders in soil ecosystems, the expression of the diverse chitinase genes within Streptomyces coelicolor grown in soil has not been assessed. As a first step, the induction pattern of nine chitinase genes in S. coelicolor growing in autoclaved soil was compared with those in liquid cultures. The relative expression levels of nine chitinase genes were measured using real-time reverse transcription PCR. The expression of all chitinase genes was induced by chitin in both autoclaved soil and liquid cultures, but to different levels. The expression levels of five chitinase genes in autoclaved soil were significantly higher than those in the liquid cultures. In particular, a putative chitinase gene, chitinase H, showed the highest induction in autoclaved soil. The same induction pattern was confirmed in nonautoclaved soil, indicating that soil contains some factors affecting the expression of chitinase genes. The chiH gene product, ChiH, cloned in Streptomycetes lividans was secreted and exhibited chitin degradation activity that was stable within a wide range of acidic pHs. The disruption of dasR, a transcriptional regulator for the uptake of N-acetylglucosamine, abolished the expression of chiH, demonstrating that DasR is required for the regulation of ChiH expression., (© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Because of the high demand for iron of the photosynthetic apparatus in thylakoid membranes, iron deficiency primarily affects the electron transfer between the two photosystems (PSI and PSII), resulting in photooxidative damage in plants. However, in barley, PSII is protected against photoinhibition, and the plant survives even with a low iron content in its chlorotic leaves. In this study, we report an adaptation mechanism of the photosynthetic apparatus in barley to iron deficiency, which is concomitant with the remodeling of a PSII antenna system. Transcriptome analysis revealed that long-term iron deficiency induced the expression of two genes of the major light-harvesting Chl a/b-binding protein of PSII (LHCII), namely HvLhcb1.11 and HvLhcb1.12. Chl fluorescence analysis of the wild type and Lhcb1-less chlorina mutants clearly showed that non-photochemical quenching (NPQ) of the wild type was increased by approximately 200% by iron deficiency, whereas NPQ of chlorina mutants did not change significantly under iron deficiency. The mutant showed severe photodamage in young leaves under prolonged iron deficiency, suggesting that the HvLhcb1 protein is essential for both thermal dissipation and photoprotection in iron-deficient barley. Analysis of thylakoid protein complexes revealed that the proportion of the monomeric form of Lhcb1 significantly increased in barley grown under iron-deficient conditions. We hypothesize that alteration of the HvLhcb1 subpopulations modifies the organization of LHCII in the thylakoid membranes, which is a key step for thermal dissipation to compensate for excess excitation energy and thereby protect the photosystems from serious damage in iron-deficient barley leaves.

We have purified and characterized an oligoxylosyl transfer enzyme (OxtA) from Bacillus sp. strain KT12. In the present study, a N-terminally His-tagged recombinant form of the enzyme, OxtA(H)(E), was overproduced in Escherichia coli and applied to the reaction with xylan and hydroquinone to produce 4-hydroxyphenyl beta-D-oligoxylosides, beta-(Xyl)(n)-HQ (n=1-4), by one step reaction. The obtained beta-(Xyl)(n)-HQ inhibited mushroom tyrosinase, which catalyzes the oxidation of L-DOPA to L-DOPA quinine, and the IC(50) values of beta-Xyl-HQ, beta-(Xyl)(2)-HQ, beta-(Xyl)(3)-HQ, and beta-(Xyl)(4)-HQ were 3.0, 0.74, 0.48, and 0.18 mM respectively. beta-(Xyl)(4)-HQ showed 35-fold more potent inhibitory activity than beta-arbutin (4-hydroxyphenyl beta-D-glucopyranoside), of which the IC(50) value was measured to be 6.3 mM. Kinetic analysis revealed that beta-(Xyl)(2)-HQ, beta-(Xyl)(3)-HQ, and beta-(Xyl)(4)-HQ competitively inhibited the enzyme, and the corresponding K(i) values were calculated to be 0.20, 0.29, and 0.057 mM respectively.

An actinomycete strain, Amycolatopsis sp. CsO-2, produces a 27-kDa chitosanase. To reveal the molecular characteristics of the enzyme, its corresponding gene ctoA was cloned by a reverse genetic technique, based on the N-terminal amino acid sequence of the protein. The encoded CtoA protein was deduced to be composed of 286 amino acids, including a putative signal peptide (1-48), and exhibited 83% identity in the amino acid sequence with the family 46 chitosanases from Streptomyces sp. N174 or Nocardioides sp. N106. The active recombinant CtoA protein was successfully overproduced in Escherichia coli. The mutant protein E22Q, in which the glutamic acid residue 22 was replaced with glutamine, abolished the chitosanase activity, showing that the Glu22 residue is required for the enzymatic activity. CtoA exhibited antifungal activity against Rhizopus oryzae, which is known to produce chitosan probably as a cell wall component. In contrast, E22Q did not inhibit the growth of the fungus, suggesting that chitosan-hydrolyzing activity is essential for the antifungal activity. It is noteworthy that the antifungal effect of CtoA against R. oryzae was drastically enhanced by the simultaneous addition of the family 19 chitinase ChiC from Streptomyces griseus.

Pseudomonas sp. A-01, isolated as a strain with chitosan-degrading activity, produced a 28 kDa chitosanase. Following purification of the chitosanase (Cto1) and determination of its N-terminal amino acid sequence, the corresponding gene (cto1) was cloned by a reverse-genetic technique. The gene encoded a protein, composed of 266 amino acids, including a putative signal sequence (1-28), that showed an amino acid sequence similar to known family-46 chitosanases. Cto1 was successfully overproduced and was secreted by a Brevibacillus choshinensis transformant carrying the cto1 gene on expression plasmid vector pNCMO2. The purified recombinant Cto1 protein was stable at pH 5-8 and showed the best chitosan-hydrolyzing activity at pH 5. Replacement of two acidic amino acid residues, Glu23 and Asp41, which correspond to previously identified active centers in Streptomyces sp. N174 chitosanase, with Gln and Asn respectively caused a defect in the hydrolyzing activity of the enzyme.

Streptomyces coelicolor A3(2) has 13 chitinase genes encoding 11 family 18 and two family 19 chitinases. To compare enzymatic properties of family 19 chitinase and family 18 chitinases produced by the same organism, the four chitinases (Chi18bA, Chi18aC, Chi18aD, and Chi19F), whose genes are expressed at high levels in the presence of chitin, were produced in Escherichia coli and purified. The effect of pH on the hydrolytic activity was very different not only among the four chitinases but also among the substrates. The hydrolytic activity of Chi19F, family 19 chitinase, against soluble substrates was remarkably high as compared with three family 18 chitinases, but was the lowest against crystalline substrates among the four chitinases. On the contrary, Chi18aC, a family 18-subfamily A chitinase, showed highest activity against crystalline substrates. Only Chi19F exhibited significant antifungal activity. Based on these observations, the roles of family 19 chitinases are discussed.

The ATP-binding cassette (ABC) transporter Ngc for N-acetylglucosamine (GlcNAc) of the chitin-degrader Streptomyces olivaceoviridis comprises the solute-binding protein NgcE, which has highest affinity for GlcNAc and N,N'-diacetylchitobiose {(GlcNAc)2} and reduced affinity for longer chitooligomers. NgcE was used to develop a generally applicable enzyme-linked immunosorbent assay (ELISA) system. As a prerequisite, the reducing end of (GlcNAc)2 was coupled with the ethylamino group of 2-(4-aminophenyl)ethylamine. The resulting conjugate was linked with amino groups of bovine serum albumin (BSA) to gain the neoglycoprotein BSA-APEA-(GlcNAc)2, which was fixed to wells in microtitre-plates. The NgcE protein was shown to bind efficiently to the immobilized BSA-APEA-(GlcNAc)2. In competition assays, the affinity of NgcE was 1,000-fold higher for GlcNAc and (GlcNAc)2 than for (GlcNAc)3 and (GlcNAc)4. These results are consistent with those previously obtained by surface plasmon resonance. Since the ELISA method can be performed very rapidly at low cost, it should be an efficient general tool to determine the affinity of a ligand to its cognate solute-binding protein.

A lectin named GFL was isolated from the fruiting body of the basidiomycete mushroom Grifola frondosa, which belongs to Aphyllophorales. The lectin had a molecular mass of 24 kDa on SDS-PAGE. The hemagglutinating activity of GFL was not inhibited by any monosaccharide, and inhibited only by porcine stomach mucin so far as tested. The occurrence of GFL was studied at three stages during fruiting body formation. The largest quantity of hemagglutinating activity was found in the fruiting body, and lesser amounts in the mycelial mat and the primordium. The 24-kDa band of GFL was found at all three stages, and the band-intensity corresponded to the level of activity in each sample. By cloning and sequencing the GFL-cDNA, the primary structure of this lectin was determined. GFL is composed of 181 amino acids, having no signal peptide. The amino acid sequence was found to be homologous to those of so-called jacalin-related plant lectins, suggesting that GFL is the first example of a jacalin-related lectin of fungal origin.

A protein that binds specifically to the promoter region of chiA was purified from the cell lysate of Streptomyces lividans by using an affinity purification method. Determining the amino-terminal amino acid sequence of the purified protein led to cloning of a gene (cpb1) encoding the chitinase promoter-binding protein, Cpb1. The deduced amino acid sequence of Cpb1 showed significant similarity to the sequences of a group of hypothetical proteins of S. coelicolor that have been revealed by the genome project, and the amino-terminal region of Cpb1 showed similarity to the DNA-binding domains of several transcription factors. The Cpb1 proteins expressed in S. lividans or Escherichia coli showed specific binding activity to the chiA promoter. The disruption of cpb1 resulted in partial relief of the glucose repression of chitinase production, indicating that cpb1 took part in the regulation of chitinase expression in S. lividans.