Jae Soon Kang

Brief exposures of human body lice to sublethal amounts of ivermectin over-transcribes detoxification genes involved in tolerance

Transcriptional profiling results, using our non‐invasive induction assay {short exposure intervals (2–5 h) to sublethal amounts of insecticides [

A soluble acetylcholinesterase provides chemical defense against xenobiotics in the pinewood nematode.

The pinewood nematode genome encodes at least three distinct acetylcholinesterases (AChEs). To understand physiological roles of the three pinewood nematode AChEs (BxACE-1, BxACE-2, and BxACE-3), BxACE-3 in particular, their tissue distribution and inhibition profiles were investigated. Immunohistochemistry revealed that BxACE-1 and BxACE-2 were distributed in neuronal tissues. In contrast, BxACE-3 was detected from some specific tissues and extracted without the aid of detergent, suggesting its soluble nature unlike BxACE-1 and BxACE-2. When present together, BxAChE3 significantly reduced the inhibition of BxACE-1 and BxACE-2 by cholinesterase inhibitors. Knockdown of BxACE-3 by RNA interference significantly increased the toxicity of three nematicidal compounds, supporting the protective role of BxACE-3 against chemicals. In summary, BxACE-3 appears to have a non-neuronal function of chemical defense whereas both BxACE-1 and BxACE-2 have classical neuronal function of synaptic transmission.

Molecular properties of a venom allergen-like protein suggest a parasitic function in the pinewood nematode Bursaphelenchus xylophilus

The pinewood nematode, Bursaphelenchus xylophilus, is a destructive pest in several countries including Japan, China and Korea. Of three genes encoding the venom allergen-like protein in B. xylophilus, Bxvap-1 showed the highest transcript levels at the pine-grown propagative stage. In addition, western blot and immunohistochemical analyses using anti-BxVap-1 polyclonal antibody verified a specific increase in BxVap-1 expression levels at the pine-grown propagative stage. Using immunohistochemistry, BxVap-1 was detected around the putative oesophageal glands and metacarpus, suggesting that BxVap-1 is secreted into the host pine tree and is involved in the parasitic mechanism. To explain the parasitic role of BxVap-1, we measured the migration rate inside pine seedlings of B. xylophilus either with or without Bxvap-1 knockdown by RNA interference. Bxvap-1 knockdown resulted in a significantly lower migration rate in the >6cm region compared with the control B. xylophilus. These results suggest that BxVap-1 is involved in B. xylophilus migration, perhaps by suppressing the pine tree defence mechanism.

Construction and characterization of subtractive stage-specific expressed sequence tag (EST) libraries of the pinewood nematode Bursaphelenchus xylophilus.

To establish expressed sequence tag databases of the two life stages (the dispersal and propagative stages) of pinewood nematode Bursaphelenchus xylophilus, subtractive EST libraries that were specific to the dispersal 4th larval stage (D4S) and the pine-grown propagative mixed (PGPS) stage were constructed by suppressed subtractive hybridization, and annotated by BLASTx and Gene Ontology (GO). A total of 1112 (57.7%) contigs from the D4S-cDNA library and 1215 (46.7%) contigs from the PGPS-specific cDNA libraries had matched BLASTx hits (E<or=10(-2)), among which 913 (47.4%) and 960 (36.9%) contigs, respectively, were classified into three GO categories. A total of 14 genes were selected on the basis of stage-specific abundances and GO subcategories, and their transcription levels were analyzed by quantitative real-time PCR. We discussed the potentials of some stage-specific genes, such as sorbitol dehydrogenase, cysteine protease, venom allergen-like protein, and FMRFamide-like peptide, as diagnosis markers and novel control targets.

Rapid and accurate prediction of the frequencies of Bursaphelenchus xylophilus and B. mucronatus in mixed nematode samples using real-time species-specific PCR.

Accurate detection of Bursaphelenchus xylophilus and prediction of its frequency in crude nematode samples is often hindered by the coexistence of related nematode species, such as B. mucronatus, that are morphologically similar but non-pathogenic. To establish a detection system enabling determination of the relative frequencies of B. xylophilus and B. mucronatus from field nematode samples, we developed a real-time species-specific PCR (rtssPCR) protocol which targets the substantial sequence differences in the 5S rRNA marker gene between the two nematode species. Using standard DNA mixtures of B. xylophilus and B. mucronatus in various ratios, plots of percent species proportion vs cycle threshold value (Ct value) were generated for the prediction of species frequency. The rtssPCR protocol enables the detection of target nematode frequencies as low as 0.16% at the 95% confidence level. When nematode DNA samples were extracted from the mixed specimens of B. xylophilus and B. mucronatus in various ratios and analysed by rtssPCR, the semi-log plot was nearly identical to the plot generated from standard mixed DNA samples, demonstrating that field populations of the nematodes can be directly used for rtssPCR analysis. The rapid and accurate determination of B. xylophilus or B. mucronatus frequencies by this rtssPCR protocol makes it ideal for routine monitoring and quarantine of B. xylophilus in the field.

Three acetylcholinesterases of the pinewood nematode, Bursaphelenchus xylophilus: insights into distinct physiological functions.

Acetylcholinesterase (AChE) plays a key role in postsynaptic transmission in most animals. Nematodes encode multiple AChEs, implying its functional diversity. To explore physiological functions of multiple AChEs, three distinct AChEs (BxACE-1, BxACE-2, and BxACE-3) were identified and characterized from the pinewood nematode. Sequencing comparison with Torpedo AChE and Caenorhabditis elegans ACEs identified choline-binding site, catalytic triad functional site, three internal disulfide bonds and aromatic residues for the catalytic gorge. Transcriptional profiling by quantitative real-time PCR revealed that BxACE-3 is more actively transcribed than BxACE-1 (2-3 times) and BxACE-2 (9-18 times) in both propagative and dispersal stages. The three BxACEs were functionally expressed using baculovirus system. Kinetic analysis of in vitro-expressed BxACEs revealed that the substrate specificity was highest in BxACE-1 whereas the catalytic efficiency was highest in BxACE-2. In inhibition assay, BxACE-3 showed the lowest inhibition rate. Taken together, it appears that both BxACE-1 and BxACE-2 play common but non-overlapping roles in synaptic transmission, whereas BxACE-3 may have non-neuronal functions. The current findings should provide valuable insights into the evolutionary process and various physiological roles of AChE.

A Combination of Biochemical and Proteomic Analyses Reveals Bx-LEC-1 as an Antigenic Target for the Monoclonal Antibody 3-2A7-2H5-D9-F10 Specific to the Pine Wood Nematode

Pine wilt disease (PWD) is one of the most devastating forest diseases in Asia and Europe. The pine wood nematode, Bursaphelenchus xylophilus, has been identified as the pathogen underlying PWD, although the pathology is not completely understood. At present, diagnosis and confirmation of PWD are time consuming tasks that require nematode extraction and microscopic examination. To develop a more efficient detection method for B. xylophilus, we first generated monoclonal antibodies (MAbs) specific to B. xylophilus. Among 2304 hybridoma fusions screened, a hybridoma clone named 3-2A7-2H5 recognized a single protein from B. xylophilus specifically, but not those from other closely related nematodes. We finally selected the MAb clone 3-2A7-2H5-D9-F10 (D9-F10) for further studies. To identify the antigenic target of MAb-D9-F10, we analyzed proteins in spots, fractions, or bands isolated from SDS-PAGE, two-dimensional electrophoresis, anion exchange chromatography, and immunoprecipitation via nano liquid chromatography electrospray ionization quadrupole ion trap mass spectrometry (nano-LC-ESI-Q-IT-MS). Peptides of galactose-binding lectin-1 of B. xylophilus (Bx-LEC-1) were commonly detected in several proteomic analyses, demonstrating that this LEC-1 is the antigenic target of MAb-D9-F10. The localization of MAb-D9-F10 immunoreactivities at the area of the median bulb and esophageal glands suggested that the Bx-LEC-1 may be involved in food perception and digestion. The Bx-LEC-1 has two nonidentical galactose-binding lectin domains important for carbohydrate binding. The affinity of the Bx-LEC-1 to d-(+)-raffinose and N-acetyllactosamine were much higher than that to l-(+)-rhamnose. Based on this combination of evidences, MAb-D9-F10 is the first identified molecular biomarker specific to the Bx-LEC-1.

Identification and Characterization of Expansins from Bursaphelenchus xylophilus (Nematoda: Aphelenchoididae)

Ilsong Institute of Life Science, Hallym University, Anyang 431-060, Korea(Received on August 7, 2012; Accepted on August 16, 2012)We identified two novel expansin (EXP) genes in theexpressed sequence tag database of Bursaphelenchusxylophilus, designated as Bx-EXPB2 and -EXPB3.Novel Bx-EXPBs encoded 150 amino acids and theirsimilarities in coding sequence were 70.7−84.0% to thepreviously reported EXPB1 of B. xylophilus. Bx-EXPB2and Bx-EXPB3 were clustered with Bx-EXPB1 and Bm-EXPB1, respectively, forming the independent phylo-geny with other nematode EXPs. All identified Bx-EXPBscontained the signal peptide and were only expressedduring the propagative stage, suggesting that they aresecreted to facilitate nematode migration through hostsby loosening cell walls during infection. Quantitativereal-time PCR analysis showed that the relative ac-cumulation of Bx-EXPB3 mRNAs was the highestamong the three Bx-EXPs examined and the order ofmRNA accumulation was as follows: Bx-EXPB3 > Bx-EXPB2 >> Bx-EXPB1. Homology modeling of Bx-EXPBs showed that the structurally optimum templatewas EXLX1 protein of Bacillus subtilis, whichsharedresidues essential for catalytic activity with Bx-EXPB1and Bx-EXPB2 except for Bx-EXPB3. Taken together,Bx-EXPB1 and Bx-EXPB2 may be involved migrationthrough plant tissues and play a role in pathogenesis.Keywords : Bursaphelenchus xylophilus, infections and propagation in host trees. expansin, homo-logy protein modeling, pathogenicity, pine wilt disease The pinewood nematode, Bursaphelenchus xylophilus isthe known agent causative of pine wilt disease (Mamiya,1975; Lee et al., 2011). Bursaphelenchus xylphilus areknown to be transferred from infected pine trees to newones by the pine sawyer beetle, Monochamus alternates(Mamiya, 1975). To express their potent phytopathogeni-city that is associated with infection and maintenance inthe host trees, B. xylophilus must overcome the physicalbarriers such as plant cell walls, which are mainly com-posed of polysaccharides, lipids, and proteins. The architectof plant cell wall is determined by cellulose, made of re-peating glucose molecules joined by β-1,4 glycosidic bonds(McQueen-Mason and Cosgrove, 1994; van den Brink andde Vries, 2011). The cellulose framework of the wall isinterpenetrated by a cross-linked matrix of non-cellulosemolecules, hemicelluloses, or pectin. Even though pinewoodnematode infected trees usually wither and die within acouple of months (Mamiya, 1975), we still do not understandthe molecular and cellular pathophysiology underlying theabilities of pinewood nematodes to infection and propagatein the trees. However, the presence of diverse cell walldegrading enzymes such as endoglucanases (Kikuchi et al.,2004; Smant et al., 1998), pectate lyases (Kikuchi et al.,2006; Popeijus et al., 2000) and expansins (EXPs) (Kikuchiet al., 2009) that to play crucial roles in initiation andprogression of the plant diseases are present in B. xylophilusgenomes suggested that secreted cell wall degrading enzymesfrom B. xylophilus may be important factors involved withEXPs in plants play pivotal roles in a wide variety ofdevelopmental processes by loosening of their cell walls byweakening non-covalent interactions (McQueen-Mason andCosgrove, 1994; McQueen-Mason and Cosgrove, 1995;Whitney et al., 2000), which makes cell walls susceptible todegradation and remodeling by other cell wall degradingenzymes (Cosgrove, 2000; van den Brink and de Vries,2011). While most EXPs were identified from plants, recentfindings that plant-parasitic nematodes express functionalEXPs suggested that they could be one of the major factorsregulating plant-parasite interactions (Qin et al., 2004; vanden Brink and de Vries, 2011). However, compared withEXPs in plants, the understanding of in vivo functions of

Identification of Novel Endo- β-1, 4-glucanase Isoforms from Bursaphelenchus Species (Nemtoda: Aphelenchoididae)

Abstract Bursaphelenchus xylophilus has been recognized as a causal pathogen of pine wilt disease (PWD). In order to identify relevant molecular biomarkers, we selected a variable region of endo-β-1, 4-glucanase (β 14-gcn) which was cloned from the cDNA of B. xylophilus and B. mucronatus. We then identified three novel β14-gcn isoforms: Bm-β 14-gcn and Bm-β 14-gcni from B. mucronatus, and Bx-β 14-gcn from B. xylophilus. Bm-β 14-gcn and Bx-β 14-gcn were found to be identical, whereas Bm-β 14-gcni was unique to B. mucronatus. These isoforms are Bursaphelenchus-specific and may be employed as molecular markers for the diagnosis of PWD. Our phylogenetic analysis showed that these β 14-gcns from Bursaphelenchus species were associated most closely with fungal β 14-gcns.