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In this study, a novel electrochemical biosensor is reported for carboxin detection based on the fabrication of the deoxyribonucleic acid/poly l-methionine-silver nanoparticles/pencil graphite electrode (DNA/PMT-AgNPs/PGE). The electrochemical response of carboxin on the DNA/PMT-AgNPs/PGE was measured using adsorptive differential pulse voltammetry (AdDPV). The electrochemical impedance spectroscopy studies showed lower Rct values for PMT-AgNPs/PGE and DNA/PMT-AgNPs/PGE compared to PGE, demonstrating their good conductivity and faster electron transfer rate. Also, the interaction of DNA with carboxin led to the better accumulation of carboxin on the electrode surface. The proposed biosensor afforded excellent selectivity for carboxin against other pesticides. According to the findings, the DNA/PMT-AgNPs/PGE presents a linear response over the carboxin concentration ranges from 8.0 pM to 1.0 µM. The detection limit was obtained as 5.0 pM under the optimized conditions. The applied DNA/PMT-AgNPs/PGE could successfully detect carboxin in the tap and river water. [ABSTRACT FROM AUTHOR]

The aggregation behavior of mixed micellar solutions of cationic hexadecylpiperidinium surfactants and nonionic surfactant Tween 80 is studied. The critical micelle concentration is determined by varying the component ratios, and a negative deviation from the ideal mixing model can be observed (synergistic effect). The adsorption parameters and surface potential of mixed micelles are estimated. The solubilizing effect individual and mixed compositions have on such hydrophobic biologically active compounds as bioflavonoid quercetin and systemic fungicide carboxin is characterized quantitatively via spectrophotometry. [ABSTRACT FROM AUTHOR]

The accurate manipulation of genomic integration of mutant alleles or fluorescent fusion-protein constructs is necessary for understanding of pathogenic mechanism of Magnaporthe oryzae. Recently, this can be achieved by integrating of exogenous DNA randomly into genome of this pathogen, but ectopic integration may result in alteration of gene expression or gene disruption due to unpredictable position effects and/or disruption of protein-coding regions. In this study, we establish a novel strategy for locus-specific integration of exogenous DNA via carboxin-resistance reconstitution by a point-mutation (H245L) on succinate dehydrogenase subunit Mosdi1. Independent transformants derived from the same reconstitution construct showed consistent fluorescent signal and undiversified phenotypes, including hyphae growth, conidiation and pathogenicity, in M. oryzae. Meanwhile, 96 % of all transformants integrate correctly into the Mosdi1 locus as a single copy. Furthermore, we provide a vector carrying yeast recombination cassette and thus allow assembly of multiple overlapping DNA fragments by yeast in vivo recombination for gene complementation and protein localization assay. [ABSTRACT FROM AUTHOR]

Kluyveromyces marxianus converts whey-borne sugar into ethyl acetate, an environmentally friendly solvent with many applications. K. marxianus DSM 5422 presumably synthesizes ethyl acetate from acetyl-SCoA. Iron limitation as a trigger for this synthesis is explained by a diminished aconitase and succinate dehydrogenase activity (both enzymes depend on iron) causing diversion of acetyl-SCoA from the tricarboxic acid cycle to ester synthesis. Copper limitation as another trigger for ester synthesis in this yeast refers to involvement of the electron transport chain (all ETC complexes depend on iron and complex IV requires copper). This hypothesis was checked by using several ETC inhibitors. Malonate was ineffective but carboxin partially inhibited complex II and initiated ester synthesis. Antimycin A and cyanide as complexes III and IV inhibitors initiated ester synthesis only at moderate levels while higher concentrations disrupted all respiration and caused ethanol formation. A restricted supply of oxygen (the terminal electron acceptor) also initiated some ester synthesis but primarily forced ethanol production. A switch from aerobic to anaerobic conditions nearly stopped ester synthesis and induced ethanol formation. Iron-limited ester formation was compared with anaerobic ethanol production; the ester yield was lower than the ethanol yield but a higher market price, a reduced number of process stages, a faster process, and decreased expenses for product recovery by stripping favor biotechnological ester production. [ABSTRACT FROM AUTHOR]

An outbreak of typhoid fever in rural Malawi triggered an investigation by the Malawi Ministry of Health and the Centers for Disease Control and Prevention in July 2009. During the investigation, villagers were directly consuming washed, donated, pesticide-treated wheat seed meant for planting. The objective of this study was to evaluate the potential for pesticide exposure and health risk in the outbreak community. A sample of unwashed (1430 g) and washed (759 g) wheat seed donated for planting, but which would have been directly consumed, was tested for 365 pesticides. Results were compared with each other (percentage change), the US Environmental Protection Agency's (EPA) health guidance values and estimated daily exposures were compared with their Reference dose (RfD). Unwashed and washed seed samples contained, respectively: carboxin, 244 and 57p.p.m.; pirimiphos methyl, 8.18 and 8.56p.p.m.; total permethrin, 3.62 and 3.27p.p.m.; and carbaryl, 0.057 and 0.025 p.p.m. Percentage change calculations (unwashed to washed) were as follows: carboxin, -76.6%; pirimiphos methyl, + 4.6%; total permethrin, -9.7%; and carbaryl -56.1%. Only carboxin and total permethrin concentration among washed seed samples exceeded US EPA health guidance values (285 x and seven times, respectively). Adult estimated exposure scenarios (1 kg seed) exceeded the RfD for carboxin (8 x ) and pirimiphos methyl (12 x ). Adult villagers weighing 70 kg would have to consume 0.123, 0.082, 1.06, and 280 kg of washed seed daily to exceed the RfD for carboxin, pirimiphos methyl, permethrins, and carbaryl, respectively. Carboxin, pirimiphos methyl, permethrins, and carbaryl were detected in both unwashed and washed samples of seed. Carboxin, total permethrin, and carbaryl concentration were partially reduced by washing. Health risks from chronic exposure to carboxin and pirimiphos methyl in these amounts are unclear. The extent of this practice among food insecure communities receiving relief seeds and resultant health impact needs further study. [ABSTRACT FROM AUTHOR]

The sdhB gene encoding an iron–sulfur (Ip) subunit of succinate dehydrogenase (SDH, EC 1.3.99.1) complex was cloned from Mortierella alpina 1S-4. The deduced amino acid sequence of SdhB from M. alpina 1S-4 showed high similarity to those of SdhB from other organisms. The mutated sdhB ( CBXB) gene encodes a modified SdhB with an amino-acid substitution (a highly conserved histidine residue within the third cysteine-rich cluster of SdhB replaced by a leucine residue) and is known to confer carboxin resistance. We succeeded in transforming M. alpina 1S-4 by using the CBXB gene as a selectable marker gene and expressing the heterologous uidA gene encoding β-glucuronidase of Escherichia coli. Moreover, transformation efficiency was up to 40–50 transformants per 4.0 × 108 spores. This carboxin-transformation system, characterized by marginal background growth and mitotic stability in M. alpina 1S-4, is considered to be widely useful for the wild strain, M. alpina 1S-4, and various derivative mutants without laborious preparation of auxotrophic mutants as a host strain. [ABSTRACT FROM AUTHOR]

In an attempt to get a marker gene suitable for genetical transformation of the ectomycorrhizal fungus Hebeloma cylindrosporum, the gene Hc.SdhR that confers carboxin-resistance was isolated from a UV mutant of this fungus. It encodes a mutant allele of the Fe–S subunit of the succinate dehydrogenase gene that carries a single amino acid substitution known to confer carboxin-resistance. This gene was successfully used as the selective marker to transform, via Agrobacterium tumefaciens, monokaryotic and dikaryotic strains of H. cylindrosporum. We also successfully transformed hygromycin-resistant insertional mutants. Transformation yielded mitotically stable carboxin-resistant mycelia. This procedure produced transformants, the growth of which was not affected by 2 μg l−1 carboxin, whereas wild-type strains were unable to grow in the presence of 0.1 μg l−1 of this fungicide. This makes the carboxin-resistance cassette much more discriminating than the hygromycin-resistance one. PCR amplification and Southern blot hybridisation indicated that more than 90% of the tested carboxin-resistant mycelia contained the Hc.SdhR cassette, usually as a single copy. The AGL-1 strain of A. tumefaciens was a much less efficient donor than LBA 1126; the former yielded ca. 0–30% transformation frequency, depending on fungal strain and resistance cassette used, whereas the latter yielded ca. 60–95%. [ABSTRACT FROM AUTHOR]

Succinate:quinone reductase catalyzes electron transfer from succinate to quinone in aerobic respiration. Carboxin is a specific inhibitor of this enzyme from several different organisms. We have isolated mutant strains of the bacterium Paracoccus denitrificans that are resistant to carboxin due to mutations in the succinate:quinone reductase. The mutations identify two amino acid residues, His228 in SdhB and Asp89 in SdhD, that most likely constitute part of a carboxin-binding site. This site is in the same region of the enzyme as the proposed active site for ubiquinone reduction. From the combined mutant data and structural information derived from Escherichia coli and Wolinella succinogenes quinol:fumarate reductase, we suggest that carboxin acts by blocking binding of ubiquinone to the active site. The block would be either by direct exclusion of ubiquinone from the active site or by occlusion of a pore that leads to the active site. [ABSTRACT FROM AUTHOR]

A novel selection marker gene for transformation of the white-rot basidiomycete Pleurotus ostreatus was developed by introducing a point mutation in a gene which encodes the iron-sulfur protein (Ip) subunit of succinate dehydrogenase. The mutant gene, CbxR, encodes a modified Ip subunit with an amino-acid substitution (His239 to Leu) and confers resistance to the systemic fungicide, carboxin. The DNA sequence was integrated ectopically in the chromosome of the transformants. This is the first report of a homologous marker gene which is available for the molecular breeding of an edible mushroom. [ABSTRACT FROM AUTHOR]

Succinate:quinone reductase is a membrane-bound enzyme of the citric acid cycle and the respiratory chain. Carboxin is a potent inhibitor of the enzyme of certain organisms. The bacterium Paracoccus denitrificans was found to be sensitive to carboxin in vivo, and mutants that grow in the presence of 3′-methyl carboxin were isolated. Membranes of the mutants showed resistant succinate:quinone reductase activity. The mutation conferring carboxin resistance was identified in four mutants. They contained the same missense mutation in the sdhD gene, which encodes one of two membrane-intrinsic polypeptides of the succinate:quinone reductase complex. The mutation causes an Asp to Gly replacement at position 89 in the SdhD polypeptide. P. denitrificans strains that overproduced wild-type or mutant enzymes were constructed. Enzymic properties of the purified enzymes were analyzed. The apparent K m for quinone (DPB) and the sensitivity to thenoyltrifluoroacetone was normal for the carboxin-resistant enzyme, but the succinate:quinone reductase activity was lower than for the wild-type enzyme. Mutations conferring carboxin resistance indicate the region on the enzyme where the inhibitor binds. A previously reported His to Leu replacement close to the [3Fe-4S] cluster in the iron-sulfur protein of Ustilago maydis succinate:quinone reductase confers resistance to carboxin and thenoyltrifluoroacetone. The Asp to Gly replacement in the P. denitrificans SdhD polypeptide, identified in this study to confer resistance to carboxin but not to thenoyltrifluoroacetone, is in a predicted cytoplasmic loop connecting two transmembrane segments. It is likely that this loop is located in the neighborhood of the [3Fe-4S] cluster. [ABSTRACT FROM AUTHOR]

Several commercial fungicide seed treatments were evaluated for their possible effect on the survival of Bradyrhizobium japonicum on seeds and on the nodulation and yield of soybeans in a greenhouse and a field experiment. quinolate Pro (carbendazim and oxine copper), Vitavax 200FF (carboxin and thiram), and Monceren (pencycuron) had a small effect or no effect on the survival of B. japonicum and on the nodulation and yield of soybeans. They can thus be considered compatible with soybean seed inoculation. Germipro UFB (carbendazim and iprodione), Apron 35J (metalaxyl), and Tachigaren (hymexazol) decreased B. japonicum survival and the nodulation and yield of soybeans and thus cannot be considered compatible with soybean seed inoculation. [ABSTRACT FROM AUTHOR]

Irradiation of carboxin (1) with a 500W UV lamp (filter Pyrex) in CH3CN leads to the products 3, 5–8, 12–14 depending upon the reaction conditions. All photo-products were isolated and characterized. Photooxidation occurred even if unsensitized, while photoalteration was very slow in the absence of oxygen. The main oxygenated-products 3 and 13 were also recovered under biomimetic conditions by exposure of an aqueous solution of 1 to sunlight. [ABSTRACT FROM AUTHOR]