The development of herbicide-resistant maize: stable Agrobacterium-mediated transformation of maize using explants of type II embryogenic calli

One of the limitations to conducting maize Agrobacterium-mediated transformation using explants of immature zygotic embryos routinely is the availability of the explants. To produce immature embryos routinely and continuously requires a well-equipped greenhouse and laborious artificial pollination. To overcome this limitation, an Agrobacterium-mediated transformation system using explants of type II embryogenic calli was developed. Once the type II embryogenic calli are produced, they can be subcultured and/or proliferated conveniently. The objectives of this study were to demonstrate a stable Agrobacterium-mediated transformation of maize using explants of type II embryonic calli and to evaluate the efficiency of the protocol in order to develop herbicide-resistant maize. The type II embryogenic calli were inoculated with Agrobacterium tumefaciens strain C58C1 carrying binary vector pTF102, and then were subsequently cultured on the following media: co-cultivation medium for 1 day, delay medium for 7 days, selection medium for 4 × 14 days, regeneration medium, and finally on germination medium. The T-DNA of the vector carried two cassettes (Ubi promoter-EPSPs ORF-nos and 35S promoter–bar ORF-nos). The EPSPs conferred resistance to glyphosate and bar conferred resistance to phosphinothricin. The confirmation of stable transformation and the efficiency of transformation was based on the resistance to phosphinothricin indicated by the growth of putative transgenic calli on selection medium amended with 4 mg l^?1 phosphinothricin, northern blot analysis of bar gene, and leaf painting assay for detection of bar gene-based herbicide resistance. Northern blot analysis and leaf painting assay confirmed the expression of bar transgenes in the R₁ generation. The average transformation efficiency was 0.60%. Based on northern blot analysis and leaf painting assay, line 31 was selected as an elite line of maize resistant to herbicide.     

Diurnal and seasonal patterns of nitrogen fixation in analnus hirsuta plantation of central Korea

We used an acetylene reduction assay to measure rates of nitrogen fixation on a 38-year-oldAlnus hirsuta plantation in central Korea. The diurnal pattern of acetylene reduction changed significantly during May, August, and October, typically varying by 3-fold throughout the course of the day. Maximum rates occurred at 3 p.m. in May and October, but at 6 p.m. in August. Increasing trends were evident during the early growing season, with sustained high rates from mid-May through late September; July had the highest rates, averaging 7.2 μmole g^-1 dry nodule h^-1. The average nodule biomass for this plantation was 220 kg ha ’. Rates of acetylene reduction were related to soil temperature, but not to soil moisture content. Combining these nodule biomass calculations with seasonal average acetylene reduction rates yielded an estimate of current annual nitrogen fixation of 60 kg N ha^-1 for the plantation. This rate of annual nitrogen addition was very large in relation to the yearly nitrogen requirements of coniferous and deciduous forests in central Korea.     

Biocatalytic characterization of an endo-β-1,4-mannanase produced by <Emphasis Type="Italic">Paenibacillus</Emphasis> sp. strain HY-8

Objectives

To evaluate the biocatalytic characteristics of a new endo-β-1,4-d-mannan-degrading enzyme (ManP) from Paenibacillus sp. strain HY-8, a gut bacterium of the longicorn beetle Moechotypa diphysis.

Results

Purified ManP (32 kDa) with an N-terminal amino acid sequence of APSFAVGADFSYVPG displayed the greatest degree of biocatalytic activity toward locust bean gum (LBG) at 55 °C and pH 7.0. The enzyme degraded LBG, guar gum, ivory nut mannan, and mannooligosaccharides (M₂–M₅), but did not exhibit any hydrolytic activity against structurally unrelated substrates. The biocatalytic activity of ManP against LBG and guar gum was 695 and 450 U mg^?1, respectively. Especially, enzymatic hydrolysis of mannobiose yielded a mixture of mannose (16.6 %) and mannobiose (83.4 %), although the degree of mannobiose degradation by ManP with was relatively limited.

Conclusion

The present results suggest that ManP is an endo-β-1,4-mannanase and is distinct from various other characterized endo-β-1,4-mannanases.

     

Association analysis of ILVBL gene polymorphisms with aspirin-exacerbated respiratory disease in asthma

Background

We previously reported that the ILVBL gene on chromosome 19p13.1 was associated with the risk for aspirin-exacerbated respiratory disease (AERD) and the percent decline of forced expired volume in one second (FEV1) after an oral aspirin challenge test. In this study, we confirmed the association between polymorphisms and haplotypes of the ILVBL gene and the risk for AERD and its phenotype.

Methods

We recruited 141 AERD and 995 aspirin-tolerant asthmatic (ATA) subjects. All study subjects underwent an oral aspirin challenge (OAC). Nine single nucleotide polymorphisms (SNPs) with minor allele frequencies above 0.05, which were present in the region from 2 kb upstream to 0.5 kb downstream of ILVBL in Asian populations, were selected and genotyped.

Results

In an allelic association analysis, seven of nine SNPs were significantly associated with the risk for AERD after correction for multiple comparisons. In a codominant model, the five SNPs making up block2 (rs2240299, rs7507755, rs1468198, rs2074261, and rs13301) showed significant associations with the risk for AERD (corrected P?=?0.001–0.004, OR?=?0.59–0.64). Rs1468198 was also significantly associated with the percent decline in FEV1 in OAC tests after correction for multiple comparisons in the codominant model (corrected P?=?0.033), but the other four SNPs in hapblock2 were not.

Conclusion

To the best of our knowledge, this is the first report of an association between SNPs on ILVBL and AERD. SNPs on ILVBL could be promising genetic markers of this condition.

     

Age dependent accumulation patterns of advanced glycation end product receptor (RAGE) ligands and binding intensities between RAGE and its ligands differ in the liver,kidney, and skeletal muscle

Background

Much evidence indicates receptor for advanced glycation end products (RAGE) related inflammation play essential roles during aging. However, the majority of studies have focused on advanced glycation end products (AGEs) and not on other RAGE ligands. In the present study, the authors evaluated whether the accumulation of RAGE ligands and binding intensities between RAGE and its ligands differ in kidney, liver, and skeletal muscle during aging.

Results

In C57BL/6 N mice aged 12 weeks, 12 months, and 22 months, ligands accumulation, binding intensities between RAGE and its ligands, activated macrophage infiltration, M1/M2 macrophage expression, glyoxalase-1expression, and signal pathways related to inflammation were evaluated. The RAGE ligands age-associated accumulation patterns were found to be organ dependent. Binding intensities between RAGE and its ligands in kidney and liver increased with age, but those in skeletal muscle were unchanged. Infiltration of activated macrophages in kidney and liver increased with age, but infiltration in the skeletal muscle was unchanged. M1 expression increased and M2 and glyoxalase-1 expression decreased with age in kidney and liver, but their expressions in skeletal muscle were not changed.

Conclusion

These findings indicate patterns of RAGE ligands accumulation, RAGE/ligands binding intensities, or inflammation markers changes during aging are organs dependent.

     

Differentiation of serum markers of homeostasis in highly qualified athletes engaged in various sports

Prognostic express diagnosis of metabolic dysregulation was tested in highly qualified athletes engaged in various Olympic sports. The method is based on laser correlation spectroscopy (LCS) of biological fluids, which has widely been approved for use in medicine and evaluation of risks for natural and anthropogenic anomalies. Evaluating the risk for metabolic dysregulations with the relevant criteria will help to objectively estimate the efficiency of preventive actions in athletes.     

Comprehensive analysis of fungal diversity and enzyme activity in <Emphasis Type="Italic">nuruk</Emphasis>, a Korean fermenting starter,for acquiring useful fungi

Nuruk is a fermenting starter that is involved in the production of alcoholic beverages, and has been used in South Korea for a very long time. To analyze the fungal diversity, we collected a total of 59 nuruk samples from several companies and persons in 2013 to 2014, and obtained 364 isolates. All of the single isolated fungi were identified, both morphologically and molecularly, based on the sequences of ribosomal RNA gene [18S, ITS1-5.8S-ITS2, and 26S (D1/D2 region)]. In 46 nuruk samples out of 59 (78%), Saccharomycopsis fibuligera, a dimorphic yeast, was most frequently isolated. Among the filamentous fungi, Aspergillus and Lichtheimia were found in more than 50% of the samples with lower colony forming unit (CFU/g of sample) than those of yeasts. The yeasts S. fibuligera and Wickerhamomyces anomalus were counted with maximum 1.3–1.8 × 10⁸ CFU/g. Among Mucorales fungi, Lichtheimia and Mucor were isolated in much higher numbers than Rhizopus and Rhizomucor. Overall, the home-made nuruks tend to contain more diverse filamentous fungi than the commercial nuruks. To acquire industrially useful filamentous fungi and yeasts, we analyzed the enzyme activities of α-amylase, glucoamylase and acid protease associated with brewing properties for 131 strains. Aspergillus oryzae and S. fibuligera had high α- and glucoamylase activities and most isolates of Lichtheimia ramosa had high acid protease activity. For further applications, 27 fungal strains were chosen based on isolation frequencies from nuruk, and the ability to produce useful enzyme.     

Lectin precipitation using phytohemagglutinin-L(4) coupled to avidin-agarose for serological biomarker discovery in colorectal cancer

N-acetylglucosaminyltransferase V (GnT-V) has been reported to be upregulated in malignant cancer cells, and its targets have been sought after with regard to biomarker identification. The low capacity and high false positive rates of 2-DE gel-based lectin blots using phytohemagglutinin-L(4) (L-PHA) prompted us to develop a novel protocol for identifying GnT-V targets, in which serum proteins were subjected to immunodepletion, alkylation, and lectin precipitation using L-PHA coupled to avidin-agarose bead complexes, and tryptic digestion. Proteins captured by L-PHA conjugates were analyzed by a nano-LC-FT-ICR/LTQ MS. Here, we report 26 candidate biomarkers for colorectal cancer (CRC) that show 100% specificity and sensitivities of greater than 50%. Not only can these candidate proteins be used as analytes for validation, but the novel protocol described herein can be applied to biomarker discovery in nonCRCs.     

Bikaverin and fusaric acid from Fusarium oxysporum show antioomycete activity against Phytophthora infestans

Aims: To isolate and identify antioomycete substances from Fusarium oxysporum EF119 against Phytophthora infestans and to investigate their antimicrobial activities against various plant pathogenic bacteria, oomycetes and true fungi. Methods and Results: Two antioomycete substances were isolated from liquid cultures of F. oxysporum EF119, which shows a potent disease control efficacy against tomato late blight caused by P. infestans. They were identified as bikaverin and fusaric acid by mass and nuclear magnetic resonance spectral analyses. They inhibited the mycelial growth of plant pathogenic oomycetes and fungi. Fusaric acid also effectively suppressed the cell growth of various plant pathogenic bacteria, but bikaverin was virtually inactive. Treatment with bikaverin at 300 μg ml^?1 suppressed the development of tomato late blight by 71%. Fusaric acid provided effective control against tomato late blight and wheat leaf rust over 67% at concentrations more than 100 μg ml^?1. Conclusions: Both bikaverin and fusaric acid showed in vitro and in vivo antioomycete activity against P. infestans. Significance and Impact of the Study: Fusarium oxysporum EF119 producing both bikaverin and fusaric acid may be used as a biocontrol agent against tomato late blight caused by P. infestans.     

Morphological variation of Enterobacter sp. BL-2 in acetate-mediated pH environment for excretive production of cationic microbial polyglucosamine biopolymer

Enterobacter sp. BL-2 excretively produced unique cationic polyglucosamine biopolymer PGB-1 comprised of more than 95% D-glucosamine in an acetate-mediated culture condition. The excretion of the biopolymer PGB- was closely associated with the cellular morphology Enterobacter sp. BL-2, a feature highly dependable on the pH of the medium. The initially formed uneven and irregular surface cells were aggregated into the cell-biopolymer network structure connected by the adhesion modules of the cell-bound biopolymer. The excretive production of the biopolymer PGB-1 coincided with the disruption of the cell-biopolymer network, most actively at the medium pH of 8.0.