Gene-Environment Interactions in Asthma: Genetic and Epigenetic Effects

Over the past three decades, a large number of genetic studies have been aimed at finding genetic variants associated with the risk of asthma, applying various genetic and genomic approaches including linkage analysis, candidate gene polymorphism studies, and genome-wide association studies (GWAS). However, contrary to general expectation, even single nucleotide polymorphisms (SNPs) discovered by GWAS failed to fully explain the heritability of asthma. Thus, application of rare allele polymorphisms in well defined phenotypes and clarification of environmental factors have been suggested to overcome the problem of 'missing' heritability. Such factors include allergens, cigarette smoke, air pollutants, and infectious agents during pre- and post-natal periods. The first and simplest interaction between a gene and the environment is a candidate interaction of both a well known gene and environmental factor in a direct physical or chemical interaction such as between CD14 and endotoxin or between HLA and allergens. Several GWAS have found environmental interactions with occupational asthma, aspirin exacerbated respiratory disease, tobacco smoke-related airway dysfunction, and farm-related atopic diseases. As one of the mechanisms behind gene-environment interaction is epigenetics, a few studies on DNA CpG methylation have been reported on subphenotypes of asthma, pitching the exciting idea that it may be possible to intervene at the junction between the genome and the environment. Epigenetic studies are starting to include data from clinical samples, which will make them another powerful tool for research on gene-environment interactions in asthma.

Inhibitory Effect of Algal Extracts on Mycelial Growth of the Tomato-Wilt Pathogen, Fusarium oxysporum f. sp. lycopersici

The present study was undertaken to explore the inhibitory effect of cyanobacterial extracts of Nostoc commune FA-103 against the tomato-wilt pathogen, Fusarium oxysporum f. sp. lycopersici. In an optimal medium, cell growth, antifungal activity, and antifungal compound production could be increased 2.7-fold, 4.1-fold, and 13.4-fold, respectively. A crude algal extract had a similar effect as mancozeb at the recommended dose, both in laboratory and pot tests. In vitro and in vivo fungal growth, spore sporulation and fungal infection of wilt pathogen in tomato seeds were significantly inhibited by cyanobacterial extracts. Nostoc commune FA-103 extracts have potential for the suppression of Fusarium oxysporum f. sp. lycopersici.

Inhibition of Aflatoxin Production of Aspergillus flavus by Lactobacillus casei

Lactobacillus casei KC-324 was tested for its ability to inhibit aflatoxin production and mycelial growth of Aspergillus flavus ATCC 15517 in liquid culture. Aflatoxin B1 biosynthesis and mycelial growth were inhibited in both simultaneous culture and individual antagonism assays,suggesting that the inhibitory activity was due to extracellular metabolites produced in cell-free supernatant fluids of the cultured broth of L. casei KC-324. In cell-free supernatant fluids of all media tested,deMan,Rogosa and Sharpe broth,potato dextrose broth,and Czapek-Dox broth + 1% yeast extract showed higher antiaflatoxigenic activity. In these case, fungal growths, however, was not affected as measured by mycelial dry weight. The antiaflatoxigenic metabolites from L. casei KC-324 were produced over wide range of temperatures between 25degrees C and 37degrees C. However, these metabolites were not thermostable since the inhibitory activity of the supernatant was inactivated within 30 minutes at 100degrees C and 121degrees C. The inhibitory activity was not influenced by changing pH of supernatant between 4 and 10. However,the antiaflatoxigenic activity was slightly reduced at pH 10.

Purification and Characterization of a Keratinase from a Feather-Degrading Fungus, Aspergillus flavus Strain K-03

A keratinolytic enzyme secreted by Aspergillus flavus K-03 cultured in feather meal basal medium (FMBM) containing 2% (w/v) chicken feather was purified and characterized. Keratinolytic enzyme secretion was the maximal at day 16 of the incubation period at pH 8 and 28degrees C. No relationship was detected between enzyme yield and increase of fungal biomass. The fraction obtained at 80% ammonium sulfate saturation showed 2.39-fold purification and was further purified by gel filtration in Sephadex G-100 followed by ion exchange chromatography on DEAE-Sephadex A-50, yielding an active protein peak showing 11.53-fold purification. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and zymograms indicated that the purified keratinase is a monomeric enzyme with 31 kDa molecular weight. The extracellular keratinase of A. flavus was active in a board range of pH (7~10) and temperature (30degrees C~70degrees C) profiles with the optimal for keratinase activity at pH 8 and 45degrees C. The keratinase activity was totally inhibited by protease inhibitors such as phenylmethylsulfonyl fluoride (PMSF), iodoacetic acid, and ethylenediaminetetraacetate (EDTA) while no reduction of activity by the addition of dithiothreitol (DTT) was observed. N-terminal amino acid sequences were up to 80% homologous with the fungal subtilisins produced by Fusarium culmorum. Therefore, on the basis of these characteristics, the keratinase of A. flavus K-03 is determined to be subtilisins-like.

Screening of Cyanobacteria (Blue-Green algae) from Rice Paddy Soil for Antifungal Activity against Plant Pathogenic Fungi

Soil cyanobacteria isolated from the rice paddy fields of 10 different locations across Korea were evaluated by agar plate diffusion test for antifungal activity. Aqueous, petroleum ether, and methanol extracts from one hundred and forty two cyanobacterial strains belonging to the 14 genera were examined for antifungal properties against seven phytopathogenic fungi causing diseases in hot pepper (Capsicum annuum L). Of total cyanobacteria, nine cyanobacteria (6.34%) exhibited antifungal effects. The nine cyanobacteria selected with positive antifungal activities were two species of Oscillatoria, two of Anabaena, three of Nostoc, one of Nodularia, and one of Calothrix. Alternaria alternata and Botrytis cinerea were inhibited by nine and eight species of cyanobacteria, respectively. Rhizopus stolonifer was suppressed by only methanol extract of Nostoc commune FK-103. In particular, Nostoc commune FK-103 and Oscillatoria tenuis FK-109 showed strong antifungal activities against Phytophthora capsici. Their antifungal activity at the late exponential growth phase is related to the growth temperature and not associated with the growth parameters such as cell biomass and chlorophyll-alpha concentration. The high inhibition levels of antibiotics were 22.5 and 31.8 mm for N. commune FK-103 and O. tenuis FK-109, respectively. The optimal temperature for antibiotic productivity was 35degrees C.

Production of Xylanolytic Enzyme Complex from Aspergillus flavus using Agricultural Wastes

Five types of agricultural wastes were used for the production of xylanolytic enzyme by Aspergillus flavus K-03. All wastes materials supported high levels of xylanase and beta-xylosidase production. A high level of proteolytic activity was observed in barley and rice bran cultures, while only a weak proteolytic activity was detected in corn cob, barley and rice straw cultures. Maximum production of xylanase was achieved in basal liquid medium containing rice barn as carbon source for 5 days of culture at pH 6.5 and 25degrees C. The xylanolytic enzyme of A. flavus K-03 showed low thermostability. The times required for 50% reduction of the initial enzyme activity were 90 min at 40degrees C, 13 min at 50degrees C, and 3 min at 60degrees C. Xylanolytic activity showed the highest level at pH 5.5~10.5 and more than 70% of the original activity was retained at pH 6.5 and 7.0. The higher stability of xylanolytic enzymes in the broad range of alkaline pH is useful for utilization of the enzymes in industrial process requiring in alkaline conditions. Moreover, the highest production of xylanolytic enzyme was obtained when 0.5% of rice bran was supplied in basal liquid medium. SDS-PAGE analysis revealed a single xylanase band of approximately 28.5 kDa from the culture filtrates.

Immobilization of Keratinase from Aspergillus flavus K-03 for Degradation of Feather Keratin

Extracellular keratinase isolated from Aspergillus flavus K-03 was immobilized on calcium alginate. The properties and reaction activities of free and immobilized keratinase with calcium alginate were characterized. The immobilized keratinase showed proteolytic activity against soluble azo-casein and azo-keratin, and insoluble feather keratin. Heat stability and pH tolerance of keratinase were greatly enhanced by immobilization. It also displayed a higher level of heat stability and an increased tolerance toward alkaline pHs compared with free keratinase. During the durability test at 40degrees C, 48% of the original enzyme activity of the immobilized keratinase was remained after 7 days of incubation. The immobilized keratinase exhibited better stability, thus increasing its potential for use in industrial application.

Antifungal Activity of Lactic Acid Bacteria Isolated from Kimchi Against Aspergillus fumigatus

More than 120 isolates of lactic acid bacteria obtained from Kimchi was screened for antifungal activity against Aspergillus fumigatus. Approximately 10% of the isolates showed inhibitory activity and only 4.16% (five isolates) exhibited strong activity against the indicator fungus A. fumigatus. The five isolates showed a wide rang of antifungal activity against A. flavus, Fusarium moniliforme, Penicillium commune, and Rhizopus oryzae. They were identified by 16S rDNA sequencing as Lactobacillus cruvatus, L. lactis subsp. lactis, L. casei, L. pentosus, and L. sakei. The effect of Lactobacillus on mycelial growth and fungal biomass as well as its ability to produce toxic compounds were determined. The results indicate that the three species, Lactobacillus casei, L. lactis subsp. lactis, and L. pentosus, are active against A. fumigatus.

Purification and Characterization of an Extracellular Alkaline Protease from Aspergillus niger C-15

An alkaline protease produced by Aspergillus niger C-15 was purified and characterized. The enzyme was purified 19.41-fold with a specific activity of 74150 U/mg and a recovery of 34.4% by gel filtration and ion exchange chromatography. The molecular weight of the enzyme was estimated to be 34 kDa. The optimum pH and temperature for the protease activity were pH 8.0 and 60degrees C, respectively. The enzyme activity inhibited by EDTA suggests that the preparation contains a metalloprotease. The enzyme activity of the metalloprotease was completely inhibited by 5 mM HgCl2 and FeCl3, while partially inhibited by CuSO4, and MnCl2. When polyols such as glycerol, mannitol, sorbitol and xylitol, were added to the reaction medium, most polyols tested enhanced protease activity. Especially, glycerol showed the highest effect. The alkaline metalloprotease was stable at high temperature and retained more than 90% of the initial activity at 60degrees C and 86.4% under addition of glycerol.

Preliminary Characterization of Keratinolytic Enzyme of Aspergillus flavus K-03 and Its Potential in Biodegradation of Keratin Wastes

Aspergillus flavus K-03 isolated from poultry forming soil in Korea was studied for its ability to produce extracellular proteases on basal medium containing 2% (w/v) chicken feathers. The fungus was observed to be a potent producer of such enzymes. Keratinolytic enzyme secretion was the best at 15 days of incubation period at pH 9 and temperature 40degrees C. No relationship existed between the enzyme yield and increase of biomass. Enzyme production was suppressed by exogenous sugars in descending order arabinose>maltose>mannose>fructose. But glucose did not influence the enzyme activity. The keratinolytic enzyme released by the fungus demonstrated the ability to decompose keratin substrates as chicken feather when exogenous glucose was present. The keratinolytic activity was inhibited by HgCl2 and serine-protease inhibitors such as phenymethylsulfonyl fluoride (100%), chymostain (88%), crystalline soybean trypsin inhibtor (80%), antipain (45%) and aprotinin (40%), and was not by cystein-protease and aspartyl-protease inhibitors. The enzyme activity is only partially inhibited by metallo-protease inhibitor. Thus, the enzyme secreted by A. flavus K-03 belongs to the alkaline serine-type protease.

Keratinolytic Activity of Five Aspergillus Species Isolated from Poultry Farming Soil in Korea

Various soil samples were collected from twenty-four areas of ten different poultry farms in Korea and screened for prevalence of keratinolytic fungi. Fourteen species of feather-associated fungi belonging to ten genera Acremonium, Alternaria, Aspergillus, Cladosporium, Curvularia, Fusarium, Monascus, Mucor, Penicillum, and Verticillium isolated from poultry soils were grown on keratin medium. Especially, Aspergillus spp. populations associated with the soil sample is 1x10(5) cfu/g. A. flavus, A. fumigatus, A. niger, A. nidulans, and A. terreus could utilize keratin of chicken feather and degrade it, producing sulphydryl-containing compounds detected as keratinase, cysteine and total proteins. Keratinolytic activities of five Aspergillus species also changed the pH of the medium more alkaline than those that were less keratinolytic.

Morphometric Studies on the Genus Septoria in Korea (I)

The mycoflora of Korea was rather poorly studied in the past and the fungi belonging to the genus Septoria are no exception. For this reason, taxonomic studies on Septoria have been initiated, with the eventual aim of producing a monograph of the Septoria species present in Korea. The present study circumscribes 10 species; viz., Septoria artemisiae, S. callistephi, S. chrysanthemella, S. erigerontis, S. lycopersici, S. lysimachiae, S. oenotherae, S. phlogis, S. rohlenae, and S. sonchi. Distinguishing morphological characters are described and illustrated for each species.