Identification of a chemically induced point mutation mediating herbicide tolerance in annual medics (Medicago spp.)

Background and Aims: Sulfonylurea (SU) herbicides are used extensively in cereal–livestockfarming zones as effective and cheap herbicides with usefullevels of residual activity. These residues can persist beyondthe cropping year, severely affecting legumes in general, andannual medics in particular, resulting in reduced dry matterproduction, lower seed yields and decreased nitrogen fixation.A strand medic cultivar, Medicago littoralis ‘Angel’,has been developed via chemical mutagenesis with tolerance toSU soil residues. Identifying the molecular basis of the observedtolerance was the aim of this study. Methods: Two F₂ populations were generated from crosses between ‘Angel’and varieties of intolerant M. truncatula, the male-sterilemutant tap and the cultivar ‘Caliph’. Genetic mappingwith SSR (single sequence repeat) and gene-based markers allowedidentification of the trait-defining gene. Quantitative geneexpression studies showed the activity of the respective alleles. Key Results: Segregation ratios indicated the control of SU-herbicide toleranceby a single dominant gene. SU herbicides inhibit the biosynthesisof the branched-chain amino acids by targeting the acetolactatesynthase enzyme, allowing the choice of a mapping approach usingacetolactate synthase (ALS) gene homologues as candidates. SSR-markeranalysis suggested the ALS-gene homologue on chromosome 3 inM. truncatula. The ALS-gene sequences from ‘Angel’and intolerant genotypes were sequenced. In ‘Angel’,a single point mutation from C to T translating into an aminoacid change from proline to leucine was identified. The polymorphismwas used to develop a diagnostic marker for the tolerance trait.Expression of the mutant ALS allele was confirmed by quantitativeRT-PCR and showed no differences at various seedling stagesand treatments to the corresponding wild-type allele. Conclusions: The identification of the trait-defining gene and the developmentof a diagnostic marker enable efficient introgression of thiseconomically important trait in annual medic improvement programs.     

Resolution of 1,3-dioxolane derivatives using the lipase from an Acinetobacter junii SY-01

Acinetobacter junii SY-01 producing a lipase enantioselectively hydrolyzing 1,3-dioxolane derivatives was isolated from water sludge sample and the effect of solvent, acyl donor, vinyl acetate concentration, substrate concentration, operating temperature and immobilization on activity and enantioselectivity was studied for the resolution of 1,3-dioxolane derivatives through transesterification reaction using a lipase from the isolated strain. Best selectivity was obtained at lower substrate concentration (3–5 mM), higher vinyl acetate concentration (500–1000 mM) and lower temperature (30–40 °C) in the reaction mixture. Lipase immobilized onto Accurel MP-1000 (micro-porous polypropylene) gave the best results and the reactivity was about 29-fold higher than the free enzyme without the decrease of enantioselectivity. Resolution of 1,3-dioxolane derivatives was carried out in flask scale containing 100 ml solvents using the lipase immobilized onto Accurel MP-1000. In this reaction, the yield and enantiomeric excess of the remaining (2R, 4S)-alcohol were 31.2% and 98.2%, respectively. This result suggests that it can be used as an alternative method, compared to the present synthetic method, for the production of optically pure (2R, 4S)-itraconazole.     

Identification of SNP markers for common CNV regions and association analysis of risk of subarachnoid aneurysmal hemorrhage in Japanese population

Copy number variation (CNV) is emerging as a new tool for understanding human genomic variation, but its relationship with human disease is not yet fully understood. The data for a total of 317,503 genotypes were collected for a genome-wide association study of subarachnoid aneurismal hemorrhage (SAH) in a Japanese population (cases and controls, n = 497) using Illumina HumanHap300 BeadChip^®. To identify multi-allelic CNV markers, we visually inspected all genotype clusters of 317,503 SNP markers covering the whole genome using Illumina’s BeadStudio 3.0^® software. As a result, we identified 597 multi-allelic CNV markers for common (copy loss frequency > 0.05) CNV regions in a Japanese population (n = 497). The identified CNV markers shared the following characteristics: enrichment of Hardy–Weinberg disequilibria, Mendelian inconsistency among families, and high missing genotype rate. All annotated information for those markers is summarized in our database (http://www.snp-genetics.com/user/srch.htm). In addition, we performed case-control association analyses of identified multi-allelic CNV markers with the risk of subarachnoid aneurysmal hemorrhage. One SNP marker (rs1242541) within a CNV region neighboring the Sel-1 suppressor of lin-12-like protein (SEL1L) was significantly associated with a risk of SAH (P = 0.0006). We also validated the CNV around rs1242541 using real-time quantitative polymerase chain reaction (PCR). Information and methods used in this study would be helpful for accurate genotyping of SNPs on CNV regions, which could be used for association analysis of SNP markers within CNV regions.     

Expression and characterization of the integral membrane domain of bacterial histidine kinase SCO3062 for structural studies

Bacterial histidine kinases play an important role in the response to external stimuli. Structural studies of the histidine kinase transmembrane domain are challenging due to difficulties in protein expression and sample preparation. After carrying out expression screening of a series of histidine kinases, we investigated sample preparation methods for obtaining high quality samples of the periplasmic and transmembrane domain (PTD) of the bacterial histidine kinase SCO3062. Various sample conditions were tested for their ability to give homogeneous NMR spectra of the SCO3062 PTD with well-resolved resonances. Circular dichroism and 3D ¹⁵N-edited NOESY spectrum results demonstrate that the SCO3062 PTD is predominantly α-helical. This method should be applicable to the NMR analysis of other transmembrane proteins.     

Synthesis and antioxidant properties of dendritic polyphenols

Three dendritic polyphenols (generation 1) were synthesized: a syringaldehyde-based dendrimer (1), a vanillin-based dendrimer (2), and an iodinated vanillin-based dendrimer (3). They all showed strong antioxidant activity according to the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical assay. The syringaldehyde dendrimer was twice and 10 times stronger than quercetin and Trolox, respectively. The vanillin-based dendrimer and its more hydrophobic iodinated derivative were also more potent antioxidants than quercetin and Trolox. The DPPH order of potency was 1 > 2, 3 > quercetin > Trolox. All three dendrimers also protected human LDL from free radical attack in a dose-dependent manner. Their order of free radical scavenging was 1 > 3 > 2 > quercetin > Trolox. The increased hydrophobic nature of the iodinated derivative may have contributed to its better LDL protection than 2. Protection of linoleic acid oxidation was studied by the β-carotene–linoleate assay. Dendrimer 1 was clearly superior to the other antioxidants in protecting the fatty acid. In case of DNA protection against free radical damage, the order of activity was 1 > quercetin > 2 > 3, Trolox. Pro-oxidant effect on copper-induced DNA oxidation showed the following order: quercetin, Trolox > 1 > 2 > 3. Results of the study show that dendritic antioxidants, even at the generation 1 level, provide promising antioxidant properties for their potential use as drug candidates for diseases associated with oxidative stress.     

Pulmonary Toxocariasis Mimicking Invasive Aspergillosis in a Patient with Ulcerative Colitis

A 45-year-old-male who had underlying ulcerative colitis and presented with fever and dry cough. Initially, the patient was considered to have invasive aspergillosis due to a positive galactomannan assay. He was treated with amphotericin B followed by voriconazole. Nevertheless, the patient deteriorated clinically and radiographically. The lung biopsy revealed eosinophilic pneumonia, and ELISA for Toxocara antigen was positive, leading to a diagnosis of pulmonary toxocariasis. After a 10-day treatment course with albendazole and adjunctive steroids, the patient recovered completely without any sequelae. Pulmonary toxocariasis may be considered in patients with subacute or chronic pneumonia unresponsive to antibiotic agents, particularly in cases with eosinophilia.     

Characterization of binding proteins that recognize oligoglucoside elicitors of phytoalexin synthesis in soybean

We are studying the cellular signaling pathway leading to pterocarpan phytoalexin biosynthesis in soybean that is induced by a branched hepta-β-glucoside originally isolated from the mycelial walls of the phytopathogenic oomycete Phytophthora sojae. Our research has focused on the specific recognition of the hepta-β-glucoside elicitor by binding proteins in soybean cells. Elicitor-binding proteins with properties expected of physiological receptors for the hepta-β-glucoside elicitor have been identified in soybean root membranes. These elicitor-binding proteins co-migrate with a plasma membrane marker (vanadate-sensitive H⁺-ATPase) on linear sucrose density gradients. Binding of a radio-iodinated derivative of the hepta-β-glucoside elicitor by membrane-localized elicitor-binding proteins is specific, reversible, saturable, and of high affinity (K_d? 1 nM). After solubilization with the nonionic detergent, n-dodecylsucrose, the elicitor-binding proteins retain their high affinity (K_d= 1.8 nM) for the radiolabeled elicitor and their binding specificity for elicitor-active oligoglucosides. A direct correlation is observed between the ability of oligoglucosides to displace labeled elicitor from the elicitor-binding proteins and the elicitor activity of the oligosaccharides. Thus, the elicitor-binding proteins recognize the same structural elements of the hepta-β-glucoside elicitor that are essential for its phytoalexin-inducing activity, suggesting that the binding proteins are physiological receptors for the elicitor. Current research is directed toward the purification of the hepta-β-glucoside elicitor-binding proteins by using ligand affinity chromatography. Purification and characterization of the hepta-β-glucoside binding proteins are among the first steps toward elucidating how the hepta-β-glucoside elicitor triggers the signal transduction pathway that ultimately leads to the synthesis of phytoalexins in soybean.     

Genome Sequence of Pseudomonas mendocina DLHK,Isolated from a Biotrickling Reactor

Pseudomonas mendocina DLHK is an aerobic bacterium isolated from a biotrickling reactor which can remove nitric oxide, a common air pollutant from combustion exhaust gas. Here, we present the draft genome of Pseudomonas mendocina DLHK.