We analyzed molecular and enzymatic properties of three cholinesterases (ChEs; ClAChE1, ClAChE2 and ClSChE) from Cimex lectularius. The ClAChE1 and ClAChE2 were generally present as a membrane-anchored dimeric insoluble form in the brain and ganglia. In the case of ClSChE, monomeric and dimeric soluble forms were observed. To investigate enzymatic properties, three ChEs were functionally expressed using baculovirus expression system. ClAChE1 revealed a significantly higher activity than ClAChE2 to acetylthiocholine iodide (ATChI) substrate. Kinetic analysis using two choline substrates (ATChI and butyrylthiocholine iodide) demonstrated that ClAChE2 had higher catalytic efficiency but lower substrate specificity than ClAChE1. Inhibition assay was conducted by using three inhibitors (BW284C51, eserine, Iso-OMPA) and two insecticides (chlorpyrifos-methyl and carbaryl). Two ClAChEs revealed high sensitivities to BW284C51, eserine, chlorpyrifos-methyl and carbaryl, but were not sensitive to Iso-OMPA. This inhibition profile confirmed that both ClAChEs are categorized as ChEs. Interestingly, the salivary specific cholinesterase did not show any measurable activities to choline substrates, confirming its non-synaptic function in C. lectularius

Testes‐derived unipotent male germ‐line stem (GS) cells can acquire multipotency under appropriate culture conditions to become mGS cells which can contribute to all three germ‐layers. This study was designed to investigate the epigenetic characteristics of mGS cells derived from adult mouse testes (maGS cells). The GS cells were isolated from 4 6 week DBA mouse and were cultured in Dulbecco’s modified Eagle Medium supplemented with 15% (v/v) fetal bovine serum, 1,000 U/ml LIF, 4 ng/ml GDNF at 37℃ in an humidified atmosphere of 5% CO2 in air to derive the maGS cells. The multipotency of maGS cells were verified by morphological and gene expression analyses, teratoma formation upon transplantation into nude mouse and in vitro differentiation ability. Bisulfite genomic sequencing revealed that GS cells had androgenetic DNA methylation pattern at the Igf2‐H19, Gnas‐Nespas , and Dlk1‐Dio3 imprinted gene clusters which changed to hemi‐zygotic embryonic stem (ES)‐cell like pattern in the maGS cells. Western blot analysis, using modification‐ and residue‐specific antibodies, revealed that both maGS and ES cells had similar level of histone di‐methylation at 4th and 27th lysine residue of histone 3 (H3K4me2 and H3K27me2) which represent “bivalent domain” for regulating self‐renewal and differentiation of mouse ES cells. Both maGS and ES cells also shared similar hisone modification for H3K9me2, H3K79me2, H3K9ac and H3K18ac. However, maGS cells had higher level of H3K- 36me2 and H3S10p. These data suggest that maGS and ES cells share several epigenetic characteristics but they also have their own unique epigenetic marks that may be useful as a molecular marker for their identification.

The cotton aphid, Aphis gossypii (Glover), is one of the most serious pest in the cultivation of various vegetables. A highly imidacloprid-resistant field population (CA-L) was collected from cucumber at Gangwha island in 4th August 2011. Even though neonicotinoid insecticides especially imidacloprid were sprayed six times during June and July, aphid density was too high to be counted. IEF and 2DE analyses revealed that general esterase isozyme (pI. 5) in CA-L were dramatically overexpressed and more isozyme spots identified in CA-L compared to susceptible (CA-S) strain. To identify differentially expressed genes in CA-L, comparative transcriptome analyses based on GS-FLX were conducted with total RNA extracted from CA-L, which generated ca. 143 Mb reads. Previously reported, comparative transcriptome analyses performed in imidacloprid resistant (CA-IR) and CA-S. The comparative transcriptome analyses re-investigated after all data sets were combined together. As previously reported, seven ATP-binding cassette (ABC) transporter genes were newly identified in A. gossypii, among which only ABCC9 gene was highly expressed in CA-IR and L. These results suggested that ABCC subfamily associated with imidacloprid resistance in A. gossypii.

Almost all insect species possess two different acetylcholinesterases (AChE1 and AChE2) but it still remains unknown which AChE plays a major role in synaptic transmission. To predict the evolutionary distributions of functional AChE, relative amount and activity of two AChEs were investigated by native-PAGE in conjunction with Western blotting using AChE1- and AChE2-specific antibodies. Among 39 insect species examined, AChE1 was expressed as the main enzyme in 26 insect species across diverse taxa, suggesting that AChE1 likely plays a more crucial role in these insects. In contrast, AChE2 was predominantly expressed in remaining insect species, including paleopteran insects, suggesting that the replacement of AChE1 function with AChE2 is an old event. As expected, only AChE2 was detected in Cyclorrhaphan flies, supporting the notion that AChE2 in Cyclorrhapha has completely replaced the physiological functions of AChE1 during the evolution of Diptera. Taken together, contrary to the common belief that AChE1 is the major enzyme in most insects, many insect species across various taxa employ AChE2 as the main AChE, suggesting that functional transition from AChE1 to AChE2 has occurred rather universally and randomly with multiple independent origins within the class Insecta. This finding should provide valuable insights into which AChE has evolved to undertake the synaptic function and how functional diversification of AChE has occurred.

A quantitative sequencing (QS) protocol was established for predicting the frequencies of the A298S and G324A mutations in the diamondback moth (Plutella xylostella) type-1 acetylcholinesterase locus, putatively involved with organophosphate and carbamate insecticideresistance. The nucleotide signal ratio at each mutation site was generated from sequencing chromatograms and plotted against the corresponding resistance allele frequency. Frequency prediction equations were generatedfrom the plots by linear regression, and the signal ratios were shown to highly correlated with resistance allele frequencies (r2>0.987). QS analysis of 15 representative regional field populations of DBM in Korea revealed that the allele frequencies of both A298S and G324A were over 70% in most field populations. As judged by inhibition assay, all populations showed resistance to paraoxon, DDVP, carbaryl, and carbofuran. In addition, different DBM strains exhibited differential sensitivities to both OPs and CBs depending on the structure of inhibitor, implying that the resistance of DBM against OPs and CBs is saturated and widespread in Korea.

Large amounts of genetically modified (GM) grains, including maize, cotton and soybean, have been imported to Korea for food, feed and processing (FFP). To evaluatethe environmental impacts, particularly on non-target insects, of FFP GM grains of unknown source, it is a prerequisite to identify Cry protein types in the test GM grains and to establish proper risk assessment protocols. Imported GM maize grains were randomly obtained and their Cry toxins were analyzed by ELISA using Cry1A, Cry1F, and Cry3A antibodies. Since all tested GM maize grains contained Cry1A, Tenebrio molitor, a non-lepidopteran species, was selected as a non-target insect species. A domestic maize strain was used as a non-GM control, which did not show any differences in major nutritional composition from the GM maize grain. Slightly increased survival rate and head capsule width of T. molitor larvae were observed when reared on GM maize powder, demonstrating no sub-chronic adverse effects of GM maize on T. molitor larvae. Head capsule width of T. molitor neonate increased steadily from hatch to 70-day-old, regardless of being fed Bt or non-Bt maize. ELISA test using Cry1A-antibody revealed that concentration of Cry1A protein slowly increased in the whole body of T. molitor from 0 to 50 post-feeding days when the insects were fed GM maize but rapidly decreased within 5 days when Bt maize-fed larvae were transferred to non-Bt maize, showing that the Cry toxin is not accumulated inside the body of T. molitor once the exposure source is removed. In addition, no Cry protein was detected in the hemolymph of the larvae reared on Bt maize, suggesting little possibility of Cry toxin exposure to higher tropic level. Taken together, the imported GM-maize grains is not likely to cause any side impacts on non-target insect T. molitor.

Two acetylcholinesterases (AChEs; BgAChE1 and BgAChE2) from Blattella germanica were functionally expressed using the baculovirus system. Kinetic analysis demonstrated that BgAChE2 had higher catalytic efficiency but lower substrate specificity than BgAChE1. Except paraoxon, BgAChE1 was generally less sensitive to inhibitors than BgAChE2. Western blot analysis using anti-BgAChE antibodies revealed that BgAChE1 was far more abundant in all examined tissues compared to BgAChE2, which is only present in the central nervous system. Both BgAChEs existed in dimeric form, covalently connected via a disulfide bridge under native conditions. Most fractions of BgAChE1 had a glycophosphatidylinositol (GPI) anchor, but a small fraction comprised a collagenlike tail. BgAChE2 appeared to have a collagen-GPI-fused tail. Based on the kinetic and molecular properties, tissue distribution and abundance, BgAChE1 was confirmed to play a major role in postsynaptic transmission.

Selenoprotein S (SelS) is widely expressed in diverse tissues where it localizes in the plasma membrane and endoplasmic reticulum. We studied the potential function of SelS in erythrocyte differentiation using K562 cells stably over-expressing SelS wild-type (WT) or one of two SelS point mutants, U188S or U188C. We found that in the K562 cells treated with 1μM Ara-C, SelS gradually declined over five days of treatment. On day 4, intracellular ROS levels were higher in cells expressing SelS-WT than in those expressing a SelS mutant. Moreover, the cell cycle patterns in cells expressing SelS-WT or U188C were similar to the controls. The expression and activation of SIRT1 were also reduced during K562 differentiation. Cells expressing SelS-WT showed elevated SIRT1 expression and activation (phosphorylation), as well as higher levels of FoxO3a expression. SIRT1 activation was diminished slightly in cells expressing SelS-WT after treatment with the ROS scavenger NAC (12 mM), but not in those expressing a SelS mutant. After four days of Ara-C treatment, SelS-WT-expressing cells showed elevated transcription of β-globin, y-globin, ε-globin, GATA-1 and zfpm-1, whereas cells expressing a SelS mutant did not. These results suggest that the suppression of SelS acts as a trigger for proerythrocyte differentiation via the ROS-mediated downregulation of SIRT1.

Bacillus thuringiensis (B. t.) strains are important microorganism because they produced a large amount of δ-endotoxin protein per bacterial cell and their toxins are highly toxic to Lepidoptera, Coleoptera, and Diptera depending on B. t. To date, more than a hundred Cry proteins have been identified and classified into 195 holotypes, based on the amino acid sequence identity. The Cry proteins or cry genes from the Korean native B. t. isolates in this study were not identified yet. The electrospray ionization of quadrupole time of flight mass spectrometry (ESI Q-TOF MS) was used to get the internal amino acid sequences of the endotoxin-spore culture mixtures of B. t. isolates, for which polymerase chain reaction (PCR) techniques were unable to detect the cognate genes. Most of Cry proteins seperated, excized, and extracted from the one dimensional - polyacrylamide gel electrophoresis (1D-PAGE), instead of 2D-PAGE, were matched with protein databases using MS-MASCOT search program. The internal amino acid sequences which were submitted to protein BLAST (basic local alignment search tool) had partially homology with the Cry protein databases. Hence, present data strongly suggest that the de novo amino acid sequencing and ESI Q-TOF/MS analysis along with MASCOT search could be used as a simple and rapid method for detection of novel Cry toxins from B. t. isolates and identification of B. t. isolates.

The diamondback moth, Plutella xylostella, is one of the most important pests of cole crops in the world and is the first insect to evolve resistance to Bt toxins in open-field populations. To search for useful molecular markers for Bt reistance monitoring, the PCR-based restriction fragment length polymorphism (RFLP) profiles of three aminopeptidase N (PxAPN1, PxAPN2 and PxAPN4) were determined for 15 representative regional field populations of P. xylostella. Most regional samples had similar RFLP patterns, whereas PxAPN1 from four regions and PxAPN4 from two regions showed different banding patterns after restriction enzyme treatment, but no differences were found in PxAPN2 among populations. The DNA sequence analysis revealed that a point mutation at the restriction site was responsible for the polymorphism of PxAPN1 but no mutations were observed in PxAPN4. Comparing amino acid sequences of PxAPNs from regional populations with reference PxAPNs (GenBank accession no. AAB70755) revealed that four regional populations possessed a point mutation in the Cry1A binding site of PxAPN1 and five regional populations possessed a deletion of eight amino acids in PxAPN4. These RFLP patterns were consistently observed in Southern regions of Korea, including Kyungsangnam-Do and Jeju-Do. The functional association of these RFLP with Bt resistance is currently under investigation

Cordycepin (3’-deoxyadenosin), a polyadenylation specific inhibitor, is the main functional component in Cordyceps militaris which is one of the top three famous traditional Chinese medicine. It has been shown to possess many pharmacological activities including immunologically stimulating, anti-cancer, anti-bacterial, and anti-virus, anti-infection effects. However, its anti-cancer molecular mechanisms are poorly understood. In this study, the apoptotic effects by cordycepin were investigates in human leukemia cells. Treatment of cordycepin significantly inhibited cells growth in a concentrationdependent manner by inducing apoptosis, as evidenced by morphological change and apoptotic cell death such as formation of apoptotic bodies, DNA fragmentation and increased populations of sub-G1. Induction of apoptosis by cordycepin was associated with modulation of Bcl-2 and inhibitor of apoptosis proteins (IAP) family expression. Cordycepin also increased reactive oxygen species (ROS) generation, activation of casepase-3, caspase-8, caspase-9, cleavage of poly(ADP-ribose) polymerase (PARP), β-catenin and phospholipase C (PLC)-γ1 protein. The quenching of ROS generation by N-acetyl-L-cysteine administration, a scavenger of ROS, reversed the cordycepin-induced apoptosis effects. Theresults suggested that cordycepin may be a potential chemotherapeutic agent for the treatment of leukemia patients [This work was supported by Blue-Bio Industry RIC at Dong-Eui University as a RIC (08-06-07) program of ITEP under Ministry of Knowledge Economy].

The traditional use of insects as food continues to be widespread in tropical and subtropical countries and to provide significant nutritional, economic and ecological benefits for rural communities. Specially, Bee brood serves as a food source to humans in many countries although limited data exists concerning its nutrient composition. Bee brood (pupa and larvae) were analyzed for Carbohydrate, Saturated fatty acid, Cholesterol, protein, fat, fiber, minerals, and vitamins. Bee brood was high in protein(46.4%～46.73%), fat(18.84%～ 20.75%),carbohydrate(24.66 %～35.79 %), Folic acid(222.30 ㎍/100g), and vitamins. Differentially, folic acid had been contained by high density in pupa of drone. While low in iron, bee brood was a good source of folic acid, and carbohydrate. The fat was composed mostly of saturated and mono-unsaturated fatty acids. The present data suggest bee brood to be an excellent source of many valuable nutrients including energy, amino acids, many essential minerals, and B-vitamins. These data suggest bee brood could be a valuable source of nutrients to various populations.

Periodontal ligament (PDL) tissue is a connective tissue that is interposed between the roots of the teeth and the inner wall of the alveolar bone socket. PDL is always exposed to physiologic mechanical force such as masticatory force and PDL cells play important roles during orthodontic tooth movement by synthesizing and secreting different mediators involved in bone remodeling. The Wnt/β-catenin signaling pathway was recently shown to play a significant role in the control of bone formation. In the present study, we applied cyclic tensile stress of 20% elongation to cultured human PDL cells and assessed its impact after six days upon components of the Wnt/β-catenin signaling pathway. RTPCR analysis showed that Wnt1a, Wnt3a, Wnt10b and the Wnt receptor LRP5 were down-regulated, whereas the Wnt inhibitor DKK1 was up-regulated in response to these stress conditions. In contrast, little change was detected in the mRNA expression of Wnt5a, Wnt7b, Fz1, and LRP6. By western blotting we found decreased expression of the β-catenin and p-GSK-3β proteins. Our results thus show that mechanical stress suppresses the canonical Wnt/β-catenin signaling pathway in PDL cells.

Thrombin-induced platelet microbicidal protein (tPMP) is a small cationic peptide that exerts potent in vitro microbicidal activity against a broad spectrum of human pathogens, including Staphylococcus aureus and Streptococcus rattus BHT. Earlier evidence has suggested that tPMP targets and disrupts the bacterial membrane. However, it is not yet clear whether membrane disruption itself is sufficient to kill the bacteria or whether subsequent, presumably intracellular, events are also involved in this process. In this study, we investigated the microbicidal activity of rabbit tPMP toward S. rattus BHT cells in the presence or absence of a pretreatment with antibiotics that differ in their mechanisms of action. The streptocidal effects of tPMP on control cells (no antibiotic pretreatment) were rapid and concentration-dependent. Pretreatment of S. rattus BHT cells with either penicillin or amoxicillin (inhibitors of bacterial cell wall synthesis) significantly enhanced the anti-S. rattus BHT effects of tPMP compared with the effects against the respective control cells over most tPMP concentration ranges tested. On the other hand, pretreatment of S. rattus BHT cells with tetracycline or doxycycline (30S ribosomal subunit inhibitors) significantly decreased the streptocidal effects of tPMP over a wide peptide concentration range. Furthermore, pretreatment with rifampin (an inhibitor of DNA-dependent RNA polymerase) essentially blocked the killing of S. rattus BHT by tPMP at most concentrations compared with the respective control cells. These results suggest that tPMP exerts anti-S. rattus BHT activity through mechanisms involving both the cell membrane and intracellular targets.

Sacbrood virus(SBV) causes a fatal disease(sacbrood) of honeybee larvae, which fail to pupate, change color and shape, and finally die. The complete nucleotide sequence of SBV has recently been determined, and with these data, we now report a Reverse Transcription-PCR(RT-PCR) test for the direct, rapid, and sensitive detection of these viruses. To detect the SBV infection in Korea, we collect beekeepers from various apiaries, which the RT-PCR technique was used. And we designed SBV specific primers in conserved region of the viral genome in the GenBank database. We confirmed the SBV amplicon using cloning and sequence. Homology between determined sequences of SBV korean strain and published virus sequences were 97% in DNA sequence, and 100% in amino acid sequence. We describe the first time that presence of sacbrood virus(SBV) in Korea honey bee colonies using RT-PCR. We also developed and validated a RT-PCR assay for the detection of SBV in Korea.

Viruses of the honeybee, Apis mellifera L. are known to reside at low levels in colonies, typically showing no apparent signs of infection. Chronic paralysis virus(CBPV) is known to induce significant losses in honey bee colonies. The pathology is characterized by clusters of trembling, flightless, crawling bees and by individual bees, sometimes hairless, standing at the hive entrance. A minusstrand-specific RT-PCR was used to assess viral replication. This is the first report on the infection of CBPV in Korea. Using (-)RT-PCR, 27 apiaries in korea were screened for the honeybee viruses, with positive colonies being analysed for viral genetic diversity. We got 550-nt PCR product from CBPV genomic RNA. Nucleotide sequences were aligned to the complete CBPV genomic RNA sequence deposited in the GenBank database and was revealed 96%(AM-CBPV) identity, respectively. Sequence comparison with other CBPV and honeybee virus.

Mortality of honeybees is a serious problem that beekeepers have to face periodically in Korea and worldwide. The presence of RNA viruses, in addition to other pathogens may be one of its possible causes. In this work, we detected Deformed wing virus(DWV), Israle Acute Paralysis Virus (IAPV), Black queen cell virus (BQCV), Cloudy wing virus(CWV), Kashmir bee virus(KBV), Sacbrood virus(SBV), Chronic bee paralysis virus(CBPV) in samples of korea honeybees with or without Varroa destructor and Nosema apis. The detection of viruses in all provinces, simultaneous co-infection of colonies by several viruses and the fact that 96.3% of the samples were infected with one or more virus, indicates they are widely spread in the region. Using uniplex and multiplex RT-PCR we screened honey bee colonies for the presence of several bee viruses, including DWV, IAPV, BQCV, KBV, CWV, and described the detection of mixed virus infections in bees from these colonies. Conclusively, investigated disease of the bee, and confirmed new virus that lead to bee disease, this is thought by valuable thing as data for development of beekeeping industry such as CCD(Colony Collapse Disorder)'s cause searching examination.