Soon-Youl Lee

Sensitization of vanilloid receptor involves an increase in the phosphorylated form of the channel.

A vanilloid receptor (VR1, now known as TRPV1) is an ion channel activated by vanilloids, including capsaicin (CAP) and resiniferatoxin (RTX), which are pungent ingredients of plants. Putative endogenous activators (anandamide and metabolites of arachidonic acid) are weak activators of VR1 compared to capsaicin and RTX, and the concentrations of the physiological condition of those activators are not sufficient to induce significant activation of VR1. One way to overcome the weak activation of endogenous activators would be the sensitization of VR1, with the phosphorylation of the channel being one possibility. The phosphorylation of VR1 by several kinases has been reported, mostly by indirect evidence. Here, using an in vivo phosphorylation method, the VR1 channel was shown to be sensitized by phosphorylation of the channel itself by multiple pathways involving PKA, PKC and acid. Also, in sensitizing VR1, BK appeared to show activation of PKC for the sensitization of VR1 by phosphorylation of the channel.

Effects of Plant Extracts on Microbial Population, Methane Emission and Ruminal Fermentation Characteristics in In vitro

This study was conducted to evaluate effects of plant extracts on methanogenesis and rumen microbial diversity in in vitro. Plant extracts (Artemisia princeps var. Orientalis; Wormwood, Allium sativum for. Pekinense; Garlic, Allium cepa; Onion, Zingiber officinale; Ginger, Citrus unshiu; Mandarin orange, Lonicera japonica; Honeysuckle) were obtained from the Plant Extract Bank at Korea Research Institute of Bioscience and Biotechnology. The rumen fluid was collected before morning feeding from a fistulated Holstein cow fed timothy and commercial concentrate (TDN; 73.5%, crude protein; 19%, crude fat; 3%, crude fiber; 12%, crude ash; 10%, Ca; 0.8%, P; 1.2%) in the ratio of 3 to 2. The 30 ml of mixture, comprising McDougall buffer and rumen liquor in the ratio of 4 to 1, was dispensed anaerobically into serum bottles containing 0.3 g of timothy substrate and plant extracts (1% of total volume, respectively) filled with O2-free N2 gas and capped with a rubber stopper. The serum bottles were held in a shaking incubator at 39°C for 24 h. Total gas production in all plant extracts was higher (p<0.05) than that of the control, and total gas production of ginger extract was highest (p<0.05). The methane emission was highest (p<0.05) at control, but lowest (p<0.05) at garlic extract which was reduced to about 20% of methane emission (40.2 vs 32.5 ml/g DM). Other plant extracts also resulted in a decrease in methane emissions (wormwood; 8%, onion; 16%, ginger; 16.7%, mandarin orange; 12%, honeysuckle; 12.2%). Total VFAs concentration and pH were not influenced by the addition of plant extracts. Acetate to propionate ratios from garlic and ginger extracts addition samples were lower (p<0.05, 3.36 and 3.38 vs 3.53) than that of the control. Real-time PCR indicted that the ciliate-associated methanogen population in all added plant extracts decreased more than that of the control, while the fibrolytic bacteria population increased. In particular, the F. succinogens community in added wormwood, garlic, mandarin orange and honeysuckle extracts increased more than that of the others. The addition of onion extract increased R. albus diversity, while other extracts did not influence the R. albus community. The R. flavefaciens population in added wormwood and garlic extracts decreased, while other extracts increased its abundance compared to the control. In conclusion, the results indicated that the plant extracts used in the experiment could be promising feed additives to decrease methane gas emission from ruminant animals while improving ruminal fermentation.

Decreased pain sensitivity of Capsaicin-treated rats results from decreased VR1 expression

We investigated the neurotoxic effects of capsaicin (CAP) on pain sensitivity and on the expression of capsaicin receptor, the vanilloid receptor (VR1), in rats. High-dose application of CAP has been known to degenerate a large fraction of the sensory neurons. Although the neurotoxic effects of CAP are well documented, the effects of CAP on the vanilloid receptor (VR1) are not yet known. In this paper, we investigated the effects of high-dose application of CAP on the expression of VR1 in rats. Thermal and mechanical pain sensitivity was reduced when neonatal rats were treated with a high dose of CAP. This reduction of pain sensitivity was significantly decreased after initiating carrageenan-induced inflammation. The expression of VR1 in dorsal root ganglia (DRG) isolated from the CAP-treated rats was reduced compared to that from the vehicle-treated rats. Therefore, we can conclude that the neurotoxic effect of CAP is related to the decrease of VR1 expression.

Functional analysis of SGR4635-induced enhancement of pigmented antibiotic production in Streptomyces lividans.

The Gram-positive mycelium-producing bacterium Streptomyces undergoes complex morphological differentiation after autolytic degradation of the vegetative mycelium. Cell-wall breakdown during growth stimulates cell development and secondary metabolite production by Streptomyces. N-acetylglucosamine (GlcNAc) produced by cell-wall lysis acts as a signal molecule, triggering the production of secondary metabolites in S. coelicolor A3(2). Here, we report that introduction of multiple copies of the GlcNAc-internalizing gene (sgr4635, encoding nagE2) of S. griseus activates actinorhodin and undecylprodigiosin production during the late growth of S. lividans in the absence of GlcNAc. Furthermore, the repressor-type transcriptional regulator DasR binds to two operator sites upstream of sgr4635. Our findings indicate that sgr4635 induces DasR-mediated antibiotic production by internalizing the GlcNAc accumulated from cell-wall lysis.

Isolation and Characterization of Novel Denitrifying Bacterium Geobacillus sp. SG-01 Strain from Wood Chips Composted with Swine Manure.

Nitrate contamination in ground and surface water is an increasingly serious environmental problem and only a few bacterial strains have been identified that have the ability to remove nitrogen pollutants from wastewater under thermophilic conditions. We therefore isolated thermophilic facultative bacterial strains from wood chips that had been composted with swine manure under aerated high temperature conditions so as to identify strains with denitrifying ability. Nine different colonies were screened and 3 long rod-shaped bacterial strains designated as SG-01, SG-02, and SG-03 were selected. The strain SG-01 could be differentiated from SG-02 and SG-03 on the basis of the method that it used for sugar utilization. The 16S rRNA genes of this strain also had high sequence similarity with Geobacillus thermodenitrificans 465T (99.6%). The optimal growth temperatures (55°C), pH values (pH 7.0), and NaCl concentrations (1%) required for the growth of strain SG-01 were established. This strain reduced 1.18 mM nitrate and 1.45 mM nitrite in LB broth after 48 h of incubation. These results suggest that the G. thermodenitrificans SG-01 strain may be useful in the removal of nitrates and nitrites from wastewater generated as a result of livestock farming.

Sphingomonas aquatica sp. nov., isolated from tap water

A Gram-stain-negative, strictly aerobic, rod-shaped bacterium, designated W1-2-1T, was isolated from tap water in South Korea. The strain was characterized by a polyphasic approach to clarify its taxonomic position. Strain W1-2-1T grew at 18-42 °C and at pH 6.0-10.0 on R2A medium. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the isolate belongs to the genus Sphingomonas and is most closely related to the Sphingomonas oligophenolica JCM 12082T (97.2 % similarity), Sphingomonas asaccharolyticaNBRC 15499T (96.8 %), Sphingomonas desiccabilis CP1DT (96.8 %), Sphingomonas pruniNBRC 15498T (96.8 %), Sphingomonas hankookensis ODN7T (96.4 %) and Sphingomonas yabuuchiae DSM 14562T (95.8 %). Chemotaxonomic data [major ubiquinone - Q10, major polyamine - homospermidine, major fatty acids - summed feature 8 (C18  : 1ω7c/ω6c), C16 : 0 and C14 : 0 2-OH and presence of sphingoglycolipid] supported the affiliation of the strain to the genus Sphingomonas. The G+C content of genomic DNA was 67.1 mol%. However, low level of DNA-DNA relatedness value between strain W1-2-1T and S. oligophenolica JCM 12082T and the results of physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain W1-2-1T from other Sphingomonas species with validly published names. Therefore, the isolate represents a novel species, for which the name Sphingomonas aquatica sp. nov. (type strain W1-2-1T=KACC 18309T=LMG 28596T) is proposed.

Marmoricola ginsengisoli sp. nov. and Marmoricola pocheonensis sp. nov. isolated from a ginseng-cultivating field.

Two novel actinobacteria, designated strains Gsoil 097T and Gsoil 818T, isolated from soil of a ginseng field, South Korea, were characterized by a polyphasic approach to clarify their taxonomic positions. They were Gram-reaction-positive, aerobic, non-spore-forming and rod-shaped. Phylogenetic analysis based on 16S rRNA gene sequences indicated that both isolates belong to the genus Marmoricola and were related most closely to Marmicola solisilvae KIS18-7T (99.1 and 98.3 % similarity, respectively), Marmicola terrae JOS5-1T (97.9 and 97.9 %), Marmicola scoriae Sco-D01T (97.8 and 97.1 %) and Marmicola aequoreus SST-45T (97.5 and 97.0 %). The G+C content of the genomic DNA was 68.8 and 70.0 mol%, respectively. Both strains were characterized chemotaxonomically as having ll-2,6-diaminopimelic acid in the cell-wall peptidoglycan, MK-8(H4) as the predominant menaquinone and C17 : 1ω6c, C18 : 1ω9c, C18 : 0 10-methyl and iso-C16 : 0 as major fatty acids. These chemotaxonomic data supported the affiliation of both strains to the genus Marmoricola. However, levels of DNA-DNA relatedness between the two strains and closely related type strains of Marmoricola species were less than 30 %. Moreover, the results of physiological and biochemical tests allowed the phenotypic differentiation of strains Gsoil 097T and Gsoil 818T from other Marmoricola species with validly published names. Therefore, the two isolates represent two novel species, for which the names Marmoricola ginsengisoli sp. nov. (type strain Gsoil 097T = KACC 14267T = DSM 22772T) and Marmoricola pocheonensis sp. nov. (type strain Gsoil 818T = KACC 14275T = DSM 22773T) are proposed.

Phenylobacterium aquaticum sp. nov., isolated from the reservoir of a water purifier

A Gram-reaction-negative, strictly aerobic, non-motile, non-spore-forming, rod-shaped bacterial strain designated W2-3-4T was isolated from the reservoir of a water purifier. This bacterium was characterized to determine its taxonomic position by using a polyphasic approach. Strain W2-3-4T grew well at 25-30 °C on nutrient and R2A agars. On the basis of 16S rRNA gene sequence similarity, strain W2-3-4T was shown to belong to the family Caulobacteraceaeand to be related to Phenylobacterium conjunctumFWC21T (98.0 % sequence similarity) and Phenylobacterium haematophilum CCUG 26751T (97.2 %). Lower sequence similarities were found with the type strains of all other recognized members of the genus Phenylobacterium (95.7-97.1 %). The G+C content of the genomic DNA was 68.7 mol%. The major respiratory quinone was Q-10 and the major fatty acids were summed feature 8 (comprising C18 : 1ω7c and/or C18 : 1ω6c), C16 : 0, C18 : 1ω7c 11-methyl and summed feature 3 (comprising C16 : 1ω7c and/or C16 : 1ω6c). The polar lipids were phosphatidylglycerol, an unknown phospholipid, four unknown glycolipids and three unidentified polar lipids. DNA-DNA relatedness values between strain W2-3-4Tand its closest phylogenetically neighbours were below 7 %. Strain W2-3-4T could be differentiated genotypically and phenotypically from recognized species of the genus Phenylobacterium. The isolate therefore represents a novel species, for which the name Phenylobacterium aquaticum sp. nov. is proposed, with the type strain W2-3-4T (=KACC 18306T=LMG 28593T).

Characterization of Transgenic Lettuce (Lactuca sativa L.) Using a BL1 Gene Encoding Bromelain Isolated from Pneapple

To clarify the roles of bromelain in plants, we isolated BL1 gene encoding bromelain from pineapple stem tissues and sequenced. The full length cDNA is 933 bp and encodes a polypeptide of 311 amino acid residues. The cDNA is most similar 94% at the amino acid level to bromelain previously isolated from pineapple (BAA21929). Explants of Lactuca sativa were co-cultivated with Agrobacterium tume-faciences LBA 4404 strains containing nptII and BL1 gene for transformation. Through initial selection of regenerated explants by culturing on a kanamycin and carbenicillin containing MS medium, multiple shoots were obtained after 2 months of culture. For a complementary step of selection, putative transgenic shoots were transferred to 1/2 Ms basal medium supplemented with 100 mg/L kanamycin and 500 mg/L carbenicillin. The selected shoots were obtained T1 generation seeds with emasculation, and tested with PCR analysis using 35S promoter and BL1 specific primers whether BL1 gene was introduced to genome of the plants. These results confirmed that produced the specific PCR bands in the putative transgenic lines. Additionally the Northern blot and endo protease activity showed that transcripts of BL1 gene were detected in transgenic lines. Theses results suggest that BL1 gene be successfully integrated and transcripted in the transgenic lettuce plants.

Role of Rab11 on Membrane Trafficking of Rat Vanilloid Receptor, TRPV1

Abstract Vanilloid receptor, TRPV1 (transient receptor potential vanilloid 1) is a non-selective cation channel that responds to a variety of pain-eliciting material including capsaicin, pH, heat. Although, membrane trafficking of TRPV1 was not much known so far, TRPV1 was reported to interact with FIP3 (family of Rab11 interacting protein 3). FIP3 was identified as one of Rab11 interacting proteins that is recently reported important in membrane trafficking of several channel proteins directly or indirectly. Therefore, in this study, we examined the role of Rab11 in the membrane trafficking of TRPV1 using cell biological and biochemical techniques. Rab11 was found really colocalized with TRPV1 based on the result of confocal microscopy. However, GST-pulldown assay, one of biochemical technique, found that Rab11 did not interact with TRPV1. Although Rab11 does not interact with TRPV1 directly, we hypothesized that Rab11 is indeed involved in the membrane trafficking of TRPV1. In order to examine further the role of Rab11 in the membrane trafficking of TRPV1, the expression of TRPV1 on the membrane was examined when the expression of Rab11 was decreased down to about 50% by siRNA technique and found decreased significantly. From this result, we can conclude that Rab11 is involved in the membrane trafficking of TRPV1 in a way of including FIP3.