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

The purpose of this study is to examine the effects of gait training using functional electrical stimulation on the improvement of hemiplegic patients' functions for balance and gait velocity. The subjects of the experiment were determined to be 10 each hemiplegic patients who had been diagnosed with stroke or brain damage six months or longer earlier assigned to an experimental group and a control group respectively. The subjects were evaluated before the experiment using Tetrax and 10M gait tests, received gait training five times a week for four weeks using functional electrical stimulation and were evaluated after the experiment in the same method as used in the evaluation before the experiment. In order to examine differences between the experimental group that received gait training using functional electrical stimulation and the control group that was treated by functional electrical stimulation and received gait training thereafter, differences between before and after the experiment were analyzed using paired sample t-tests and differences in changes after the experiment between the experimental group and the control group were analyzed using independent sample t-tests in order to compare the two groups with each other. Experimental results showed significant differences in weight bearing, balance and gait velocity between before and after the experiment in the experimental group(p<.05). In the control group, whereas weight bearing and gait velocity did not show any significant difference between before and after the experiment(p>.05), balance showed significant differences(p<.05). Weight bearing, balance and gait velocity change rates showed significant differences between the experimental group and the control group(p<.05). In conclusion, it was indicated that gait training using functional electrical stimulation is effective for enhancing stroke patients' weight bearing rates, balance abilities and gait velocity.

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.

Venom allergen-like protein 2 (Vap2) was characterized from the pinewood nematode, Bursaphelenchus xylophilus, which is a destructive pathogen in several countries including Japan, China and Korea. Among three vaps of B. xylophilus (Bx-vap)reported in GenBank, Bx-vap2 showed the highest transcript level in both pine-grown propagative stage (PGPS) and media-grown propagative stage (MGPS). Bx-vap2 also was revealed that its transcript level over 10-fold increased in PGPS. In addition, western blot using BxVap2-polyclonal antibody showed that expression level of BxVap2 was significantly increased in PGPS. In immunohistochemistry, moreover, strong signals were detected around putative subventral gland in PGPS, whereas weak signals were observed in MGPS. These experimental results suppose pathogenic function of BxVap2 and migration assay using Bx-vap2 knock-down worms by RNA interference supports this postulation.

Western blot analysis using acetylcholinesterase (AChE)-specific antibody was conducted to determine whether AChE gene (Tuace) duplication actually results in overproduction of AChE in Tetranychus urticae (TuAChE). The protein quantities of TuAChE in seven field-collected mite populations were precisely correlated with the copy numbers. To investigate the effects of each mutation on AChE insensitivity and possible fitness cost, eight variants of TuAChE were in vitro expressed using the baculovirus expression system. Kinetic analysis revealed that the Ala391Thr mutation did not change kinetic properties of AChE, whereas the Gly228Ser and Phe439Trp mutations significantly increased the insensitivity to monocrotophos. Moreover, when the Gly228Ser and Phe439Trp mutations are present together, insensitivity increased over a thousand-fold, showing that both mutations confer resistance in a synergistic manner. Presence of the mutations, however, reduced catalytic efficiency of AChE considerably, suggesting an apparent fitness cost in monocrotophosresistant mites. Reconstitution of the multiple copies of AChE having different compositions of mutations revealed that the catalytic efficiencies of the six-copy and two-copy AChEs (resembling the AD and PyriF strains of mite, respectively) were still lower but comparable to that of wildtype AChE. These finding clearly suggested that multiple rounds of Tuace duplication was needed to compensate the reduced catalytic activity of AChE caused by mutations.

World wide mushroom productions have been increased to 10-20% and more various mushrooms have been attempted to cultivate. Similar trends were also observed in Korea. More diverse mushroom varieties such as Pleurotus eryngii, Hericium erinaceus and Agrocybe aegerita have been attempted to cultivate in larger areas. In these days, 235 varieties of 33 different mushroom species have been cultivated. However only 50 varieties were protected by the law. Since 1960s, mushroom industry had been considered as one of the major export industries in Korea and mushroom production has been rapidly increased. In 2008, total mushroom production was about 198,209 ton, which were about 800 billion won (one trillion won if include mushroom factory products). Major cultivated species are Pleurotus ostreatus, P. eryngii, Flammulina velutipes, Lentinula edodes and Agaricus bisporus, which cover 90% of total production. We believed that the protection of mushroom vavarieties by the law is one of the main problems to be solved. Also, more studies to develop better mushroom production methods with low cost and improve the distribution structures are certainly required. In these days, the export of mushroom has been increased in Korea. We imported more mushrooms than exported for last 4 years, however, it has been changed that export is getting bigger in these days. Because we developed the liquid spawning and automatic cultivation systems, which lead the reduction of mushroom production cost and resulted in the increasement of export. If we develop better post-harvest system, it is certain that the mushroom export should be more important in the future. As an alternative way, some Korean companies are planning to build the plants in main export countries. Mushroom industry has promising future because mushroom is good for your health.

This study is based on specimens which had been collected from Jeollabuk-do, Chungcheonbuk-do and Gyeonsangnam-do provinces in July 1994 through September 2009. As a result of this study, Cunaxa potchensis and C. mageei were identified and newly recorded from Korea.

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.

Three acetylcholinesterases (AChEs) were identified from the pinewood nematode, Bursaphelenchus xylophilus. Sequence comparison with known AChEs in conjunction with three-dimensional structure analysis suggested that all BxAChEs share typical characteristics of AChE at the major catalytic structures. BgAChE3 was most predominantly transcribed and then followed by AChE1 and AChE2. Immunohistochemistry using anti-BxAChEs antibodies revealed that BxAChE1 is most widely distributed whereas BxAChE2 exhibits more localized distribution in neuronal tissues. BxAChE3 was detected from entire body together with some limited tissues, including mouth parts and alimentary lining, and determined to be the only soluble AChE, suggesting its localization in hemolymph or/and extracellular space. Kinetic analysis of in vitro expressed BxAChEs revealed that BxAChE1 has the highest substrate specificity whereas BxAChE2 has the highest catalytic efficiency with BxAChE3 having the lowest catalytic efficiency. Interestingly, presence of BxAChE3 in the pool of BxAChEs significantly reduced the inhibition of BxAChE1 and BxAChE2 by inhibitors. Knockout of BxAChE3 by RNAi significantly increased the toxicity of nematicides, suggesting the protective role of BxAChE3 against these toxicants. Based on several features, including tissue distribution, expression level, substrate kinetics and inhibition property, it appeared that BxAChE1 is the major AChE with the function of postsynaptic transmission whereas BxAChE3 has been evolved to acquire the function of chemical defense, perhaps intrinsically against secondary toxic compounds from host pine trees, such as α-pinene and limonene. BxAChE2 appears to play a role in post-synaptic transmission in specialized neurons but its detailed physiological function still remains to be elucidated.

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].

Three acetylcholinesterase (ace) genes were identified from Bursaphelenchus xylophilus and named as Bxace1, Bxace2 and Bxace3. Open reading fragments of Bxace1, Bxace2 and Bxace3 were composed of 622, 625 and 588 amino acids, respectively. Sequencing comparison with Torpedo ace gene identified cholinebinding 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 Bxace3 is more actively transcribed than Bxace1 (2-3 times) and Bxace2 (9-18 times) in both propagative and dispersal stages. The three ace genes were functionally expressed using baculovirus system. Kinetic analysis using three choline substrates demonstrated that BxAce2 has higher maximum velocity than BxAce1 (ca. 2 times) and BxAce3 (ca. 100 times) whereas BxAce3 shows lower Michaelis constant than BxAce1 (12.8 times) and BxAce2 (13.6 times). In inhibition assay using five organophosphates (OPs) and three carbamates (CBs), all the three BxAces were highly inhibited by paraoxon, DDVP and chlorpyrifos-oxon but not inhibited well by fenamiphos and fosthiazate. Interestingly, inhibition assay revealed that BxAce3 is less sensitive to all insecticides tested than other two BxAces. The inhibition kinetic data obtained in this study should provide essential information for the development of OP- and CB-based nematicidal agents againt B. xylophilus.

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.

Immuno-MemBlot is a technique for detecting, analyzing, and identifying proteins, similar to the Western blot technique but differing in that protein samples are not separated electrophoretically but are spotted through circular or slot templates directly onto the membrane. Recently we developed a new Immuno-MemBlot (IMB) method applying immunoreactions and coloring procedures directly in the wells of MemBlot apparatus, which were connected by canals to perform drainage for reagent application and buffer irrigation. This IMB method was designed to get theimmunoblot results more rapidly and clearly than the previous immunoblot ones. This study is aimed to evaluate the analytical accuracy of IMB using different biological assay. In the sensitivity test of IMB the monoclonal antibody can clearly detect the 30 ng (about 12 pM) of Mucocidin peptide (35 mer), and is also available to detect at least 10 ng (about 4 pM) of Mucocidin peptide (35 mer). The IMB was effective in the quantitative analysis of methothrexate (MTX) assay for cellular apoptosis. And more, this IMB is useful to screen large number of specific samples with ease and accuracy in a short time. In the screenings for the presence of Mucocidin in saliva the quantitative comparison is conspicuous among 48 persons depend on the different conditions ofgender, drinking and smoking habits, and oral diseases. Therefore, it is presumed that, even though the target proteins were partly degraded, a specific epitope can be detected if a monoclonal antibody was still reactive. Conclusively, these data suggest that the IMB can be useful in the primary qualitativeand quantitative analysis of proteins in various fluids, i.e., blood, saliva, tear, urine, etc.

Roasting has revealed coffee’s potentials as a good source of bioactive compounds. This study was done to investigate the quantitative presence and activity of bioactive compounds including caffeine, chlorogenic acid (CGA), amino acids, and antioxidant capacity on Coffea arabica L. (Guatemala finca San Sebastian) and C. robusta L. (India Azad Hind). Analysis was performed on Green Bean (GB) Medium-Light (ML), Medium (ME) and Medium-Dark (MD) samples of both varieties. From the results, caffeine content was highest in ME samples of both varieties. GB samples of both varieties had high CGA content which decreased after increasing roasting time and temperature. Most amino acids in GB samples was highest, however, glutamic acid, valine, tyrosine, isoleucine, leucine and phenylalanine had highest quantitative increase in ME samples for both varieties. IC50 of DPPH and ABTS radical scavenging activity was highest in ML samples of both varieties. IC50 of reducing power and total phenolic content was highest in GB sample of both varieties but decreased after increasing roasting conditions. Generally Robusta had the highest quantity of bioactive compounds and antioxidant activity. From this study, the optimal roasting condition for coffee is ME above which there is a significant reduction of bioactive compounds and antioxidant activity.