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Comparison of Endogenous Cellulase Genes from Four Termite Species with Different Habitats
Kyungjae Andrew Yoon,Young Ho Kim,Si Hyeock Lee 한국응용곤충학회 2013 한국응용곤충학회 학술대회논문집 Vol.2013 No.04
To search for a variety of cellulase genes from termites with different habitats consuming different foods, we collected three species (Neotermes spp., Odontotermes spp., Macrotermes spp.) from the wood and one species (Nasutitermes spp.) from the cow dung. Total RNA was isolated both from alimentary track tissues containing paunch and from other tissues, and used for the suppression subtractive hybridization (SSH). The resulting EST libraries were sequenced and searched by BLAST to identify cellulase genes. A total of 16 cellulase genes were found from the wood-dwelling termites whereas 4 cellulase genes from the cow dung-dwelling termites. Endo-beta-1,4-glucanase and beta-glucosidase were identified as the most abundant cellulase from the wood-dwelling termites and cow dung-dwelling termites, respectively. This finding suggests that cellulase profiles are significantly different depending on the termite’s habitat and food. In addition, we analyzed phylogenetic relationships among the cellulase genes along with other cellulase genes reported to date. All cellulase genes appeared to be originated from endosymbioants without any hint of horizontal gene transfer. Functional expression of endo-beta-1,4-glucanase using a baculovirus expression system is in progress to characterize its enzymatic properties.
Kyungjae Andrew Yoon,Young Han Park,Young Ho Koh,Si Hyeock Lee 한국응용곤충학회 2017 한국응용곤충학회 학술대회논문집 Vol.2017 No.10
Biological activities of bombolitins from Bombus ardens, B. consobrinus, B. terrestris and B. ussurensis (bombolitinsA, C, T and U, respectively) were examined using hemolytic, anti-microbial, anti-fungal and anti-tumor activity assays.Among the four bombolitins tested, bombolitin T showed the highest hemolytic and anti-tumor activities. All bombolitinsexhibited strong anti-microbial and anti-fungal activities, and bombolitin A specifically possessed the highest anti-microbialactivity against the Gram-negative bacteria Escherichia coli. Circular dichroism spectrometry analysis revealed that allfour bombolitins had over 61.7% and 45.5% of α-helicity in 30 mM sodium dodecyl sulfate and 50% trifluoroethanolbuffers, respectively, which form lipid-membrane-mimicking environments. Bombolitin T showed the lowest IC50 valuesof 8.5 μM and 8.8 μM against SK-OV-3 and NIH-OVCAR-3 cell lines, respectively, after 72 h of treatment, but itsrelative hemolytic activity at a concentration of 200 μM was 2.3-fold higher than that of 0.1% Triton X-100. Thisstudy provides new information on the biological and molecular properties of venom peptides of bumblebees. However,further studies on reducing cytotoxicity of bombolitins are needed for designing selective anti-tumor peptides.
Characterization of biological activities of abaecin from bumblebee Bombus ussurensis
Kyungjae Andrew Yoon,Young Ho Koh,Si Hyeock Lee 한국응용곤충학회 2017 한국응용곤충학회 학술대회논문집 Vol.2017 No.04
Abaecin is a proline-rich anti-bacterial peptide which is known to have high activity against Gram-negative bacteria and it is originally isolated from honeybee Apis mellifera. Since the anti-bacterial peptides including abaecin were identified in the hemolymph of A. mellifera infected with Gram-negative bacteria Escherichia coli, this result implies that honeybees express abaecin for defense against natural infection. Anti-microbial peptides including abaecin were also identified in the adult hemolymph of bumblebees. To investigate pharmacological and toxicological properties of abaecin from Bombus ussurensis, biological activities were evaluated by conducting anti-tumor, anti-microbial and cytotoxic activity assays. Abaecin exhibited significantly high anti-tumor activity against ovarian tumor cells SK-OV-3 and NIH; OVCAR-3 at 100 μM after 72 h treatment. Interestingly, abaecin showed none of anti-microbial activity against Gram-negative and Gram-positive bacteria. Since abaecin exhibited extremely low level of hemolytic activity against human erythrocytes, it may serve as a good model peptide for studying its potential as a selective anti-tumor drug.
Kyungjae Andrew Yoon,Young Ho Kim,Si Hyeock Lee 한국응용곤충학회 2013 한국응용곤충학회 학술대회논문집 Vol.2013 No.10
The aim of this study is to analyze the functional activity of an endo-β-1, 4-glucanase from the wood dwelling lower termite Coptotermes gestroi. Full length cDNA sequences of the endo-β-1,4-glucanase were obtained by primer walking in conjunction with Rapid Amplification cDNA Ends. With the obtained full length sequences, primers for amplifying open reading frame (ORF) excluding the signal peptide and glycophosphatidylinositol anchor were designed. Amplified endo-β-1,4-glucanase fragment was cloned and expressed using pET30(+) expression vector in BL21 E.coli strain. Expression of endo-β-1,4-glucanase was confirmed by Western blotting and the result revealed that only full ORF was expressed. The cellulase activity of protein preparations from the induced and non-induced cells was analyzed with Congo Red assay with the cellulase from Aspergillus niger (Sigma Aldrich) as a positive control. The activity of C. gestroi endo-β-1,4-glucanase was significantly higher than those observed in the positive control and the enzyme preparation from non-induced cells. Therefore, this study confirmed that C. gestroi endo-β-1,4-glucanase had a function of cellulose hydrolysis.
Comparative functional analysis of mastoparans from social hornets Vespa crabro and Vespa analis
Kyungjae Andrew Yoon,Kyungmun Kim,Hyo-min Ahn,Ki-Gyoung Kim,Hong-Yul Seo,Young Ho Koh,Si Hyeock Lee 한국응용곤충학회 2015 한국응용곤충학회 학술대회논문집 Vol.2015 No.10
Vespa crabro and V. analis are social hornet species commonly found in Asia, including Korea. Mastoparan is one of the major venom peptides of these two hornets but its amino acid sequence defers substantially. To examine the differences in the potential toxicity and bioactivity of mastoparans between these two social hornets, differential toxicological and pharmacological activities of synthesized mastoparan were investigated. V. analis mastoparan showed a 7-fold higher hemolytic activity, suggesting its higher cytotoxic potential compared with V. crabro mastoparan. Mastoparans from both hornet species exhibited similar levels of antimicrobial activities against Staphylococcus aureus and Botrytis cinerea, whereas the mastoparan from V. analis showed more potent antimicrobial activities against Escherichia coli and Candida albicans. Nevertheless, the antimicrobial activities of mastoparans of V. crabro and V. analis were relatively lower compared with those of other wasps. Both mastoparans also exhibited some levels of antitumor activity but the activity was significantly higher in V. analis mastoparan. In summary, the hemolytic, antimicrobial, and antitumor activities of synthesized V. analis mastoparan were higher than those of V. crabro mastoparan. These differential bioactivities are likely due to the amino acid sequence differences in the mature peptides. In particular, the additional Lys residue present in V. analis mastoparan may contribute to the higher levels of bioactivity as proposed by secondary structure prediction.
Comparative transcriptomic analysis of social hornets Vespa crabro and Vespa analis
Kyungjae Andrew Yoon,Kyungmun Kim,Hyo-min Ah,Ki-Gyoung Kim,Young Ho Koh,Young Ho Koh,Si Hyeock Lee 한국응용곤충학회 2015 한국응용곤충학회 학술대회논문집 Vol.2015 No.10
The hornets Vespa crabro and V. analis are widely distributed in Asia and are known to be aggressive when disturbed, resulting in frequent stinging accidents. To investigate the differences in venom properties and toxicities between these two hornets, the transcriptomic profiles of venom glands, in conjunction with the venom components, were analyzed and compared. A total of 35 venom-specific genes were identified in both venom gland transcriptomes, but their transcriptional profiles were different between V. crabro and V. analis. In addition, the major venom components were identified and confirmed by mass spectroscopy. Prepromastoparan, vespid chemotactic precursor and vespakinin were the top three genes most prevalently transcribed in the venom gland of V. crabro, and their transcription rates were 112-, 16- and 161-fold higher, respectively, compared with those in V. analis, as judged by FPKM values. In the venom gland of V. analis, however, vespid chemotactic precursor was the most abundantly transcribed gene, followed by premastoparan and vespakinin. In general, most major venom genes were more abundantly expressed in V. crabro, whereas some minor venom genes exhibited higher transcription rates in V. analis, including muscle LIM protein, troponin, paramyosin, calponin, etc. Our findings reveal that the overall venom components of V. crabro and V. analis are similar, but that their expression profiles and levels are considerably different. The comparison of venom gland transcriptomes suggests that V. crabro likely produces venom with more highly enriched major venom components, which has potentially higher toxicity compared with V. analis venom.
Detection of the Bacterial Symbiosis in the Pea Aphid Acrythosiphon pisum.
Kyungjae Andrew Yoon,Young Ho Kim,Si Hyeock Lee 한국응용곤충학회 2012 한국응용곤충학회 학술대회논문집 Vol.2012 No.10
Many previous ecological studies on three major bacterial symbionts and a newly discovered symbiont PAXS (pea aphid X-type symbiont) in the pea aphid Acrythosiphon pisum have shown that these symbionts are associated with the expression of a variety of host phenotypes, including resistance to parasitoid Aphidius ervi and tolerance to heat stress. The principal role of all four symbionts “Candidatus Serratia symbiotica”, “Candidatus Hamiltonella defensa”, “Candidatus Regiella insecticola”, and PAXS is to protect aphids against abiotic stress by preserving the cells in which most of symbionts dwell and by reducing the rate of parasitism. In this experiment, we detected endosymbionts from four aphid clones by means of genomic DNA extraction, PCR with gene specific primer, and restriction enzyme cutting. The patterns of PCR and restriction enzyme cutting were all identical in the four aphid clones. In order to specifically identify the endosymbiont, we searched the sequences using BLAST. The BLAST search revealed that nucleotide sequences of the symbiont were 98% identical to Serratia entomophil. S. entomophil is also known to provide tolerance to heat stress, resistance to parasitoid wasps, and restoration of reproduction in aphids, suggesting its role in host protection.