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.

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.

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.

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.

The damage of citrus fruit by yellow tea thrips, Scirtothrips dorsalis Hood, was first confirmed in 2008, and has since been one of the serious pests of citrus in Jeju. This was reported on damage symptom on tangor cultivar, Setoka (Tangor Norin No. 8) fruits cultivated in a greenhouse and the characteristic of spatial disttribution of S. dorsalis caught on yellow sticky traps was conducted in three commercial citrus orchards. The feeding habits by S. dorsalis cause rind blemishes on the navel part of fruit. The damage is characterized by being scarped and figure like covered cloud on satsum mandarine fruit, but on by a brown ring of rough russeting that occurs on navel part of Setoka fruit. The season of damage occurrence was from middle of June to late of July. There was a highly significant relationship between the average number of thrips per trap and the maximum number caught on a trap. The overall differences between the linear regression models obtained from mean-variance relationship for each surveyed fields were tested by type Ⅲ error and contrast method. The characteristic of spatial distribution of S. dorsalis was better described by Taylor's power law than Iwao's patchiness regression, and the dispersion index which is the slope (=1.72) of linear regression model showed the aggregated distribution pattern. The sequential sampling stop lines at fixed precision level of 0.20, 0.25 and 0.30 were calculated.

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.

Mitis-salivarius sucrose bacitracin(MSB) medium is widely used in the selective isolation of mutans streptococci(MS), a designation for a group of oral cariogenic species. Recently, we have isolated three bacterial strains grown on MSB agar from human dental plaques. The three strains exhibited biochemical characteristics similar to those of the biotype IV of MS, with the exception that they manifested a positive reaction for arginine deaminase. The objective of this study was to identify and characterize these three clinical isolates. The bacteria were identified with biochemical tests as well as by 16S rDNA cloning and sequencing. In order to compare the antibiotics susceptibility of the clinical isolates with that of type strain, the minimum inhibitory concentrations of 9 antibiotics were determined using broth dilution assays. The results identified all of our three clinical isolates as Enterococcus faecalis. All E. faecalis strains were found to be susceptible to penicillin G, amoxicillin, augmentin, and vancomycin, but were resistant to ciprofloxacin, cefuroxim axetil, and clindamycin. Our findings indicate that E. faecalis is capable of growing on MSB agar, and suggest that the MSB medium be improved so that only MS should be recoverable on the medium, as originally devised for their selection.

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.

분갈(Pueraria thomsonii Benth)은 전통적으로 중국 의학에서 발열, 급성 이질, 설사, 당뇨병 및 심혈관 질환 치료를 위한 약재로 사용되어 왔다. 이러한 분갈의 꽃인 분갈화의 피부 효능은 아직까지 밝혀진 바 없어, 본 연구에서는 멜라닌세포인 B16F1 세포주와 섬유아세포인 HS68 세포주를 이용하여 분갈화의 피부 효능을 검증하고자 하였다. 분갈화의 에탄올 추출물이 멜라닌세포의 멜라닌 합성을 농도 의존적으로 감소시킴을 확인하고, 분갈화 추출물의 효능 성분을 추적하고자 high performance liquid chromatography (HPLC)를 이용하여 분석하였다. 그 결과, 분갈화에 함유된 이소플라본류 화합물인 tectorigenin, tectoridin, tectorigenin 7-O-xylosylglucoside 3종 성분을 확인하였다. 3종 성분 모두 독성을 보이지 않는 농도에서 멜라닌세포의 멜라닌 생합성을 농도 의존적으로 감소시켰으며, 이러한 멜라닌 생합성 감소는 tyrosinase와 microphthalmia-associated transcription factor (MITF) 유전자 발현을 감소시키는 것에 기인함을 확인하였다. 멜라닌 합성저해의 또 다른 기전을 확인하기 위하여, 섬유아세포에서 유래되는 멜라닌 합성억제 인자인 DKK-1의 발현에 대한 영향을 측정한 결과, 분갈화 추출물, tectoridin, tectorigenin 7-O-xylosylglucoside 는 DKK-1의 발현을 농도 의존적으로 감소시킨 반면, tectorigenin은 DKK-1의 발현을 농도 의존적으로 증가 시키는 것을 확인하였다. 이상의 연구 결과를 바탕으로, 분갈화 성분 중 tectorigenin은 멜라닌세포에서의 멜라닌 합성 억제와 섬유아세포에서의 멜라닌 생성 억제 인자 분비를 촉진하는 효과적인 미백 개선제로 활용할 수 있을 것으로 판단된다.

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.

별불가사리(Asterina pectinifera Muller and Troschel)는 우리나라 전국 연안에서 흔히 볼 수 있는 토속종으로, 패류 양식장에 피해를 주는 불가사리류 중의 하나이다. 별불가사리 퇴치를 위해 대부분 건조하여 비료로 활용하고 있으며, 고부가가치 창출을 위한 다양한 연구가 진행되었으나 실제 활용은 미미한 실정이다. 따라서, 별불가사리로부터 피부 유용성분을 밝혀 새로운 활용 방안을 모색하고자 하였다. 성분연구를 통해 별불가사리로부터 2종의 polyhydroxysteroid와 1종의 saponin을 분리하였으며, 이의 구조를 각각 5α-cholestane-3β,6α,7α,8,15α,16β,26-heptol, 5α-cholestane-3β,4β,6α,7α,8,15β,16β,26-octol 및 asterosaponin P1으로 동정하였다. 이 물질들의 피부 효능을 확인한 결과 asterosaponin P1이 표피 줄기세포의 증식을 촉진시키고, 각질형성세포에서 히알루론산을 합성하는 효소인 hyaluronan synthase-2와 hyaluronan synthase-3 유전자의 발현을 증가시킴을 확인하였다. 또한, asterosaponin P1은 섬유아세포에서 진피의 주요 콜라겐인 type 1 콜라겐의 생합성을 촉진하는 효능을 보였다. 이상의 결과들로부터, 별불가사리로부터 분리한 asterosaponin P1은 노화에 동반되는 피부 증상을 개선하는 항노화 화장품 소재로 활용될 수 있을 것으로 판단된다.

This study aims to assess forest healing programs to middle-aged people in Korea with metabolic syndrome as a method to control the syndrome through prevention and health improvement rather than treatment. In order to develop healing programs in the urban forests for metabolic syndrome patients, environment condition of the forests and moods of participants were compared. Thermal environments and the concentration of phytoncides were analyzed by the site. Saneum Healing Forest had a lower temperature but a higher humidity than Seoul Forest. Seoul Forest had higher PMV and PPD levels than Saneum Healing Forest, providing patients with freshness. This seems to be due to the seasonal factor of autumn. As for the total emissions of phytoncide, mountain forest generated more than urban forest. Nine components out of investigated twenty turned out to be generated more in the urban forests. The atmospheric composition of phytoncides, volatile organic compounds that are released from vegetation, was analyzed at both sites. Profile of Mood States (POMS) was measured before and after the healing program. The POMS suggested that forest environments reduce stress and increase comfort, calm, and feelings of refreshment. The tendency towards positive mood state in the forest recommend that middle-aged Metabolic syndrome patients participate in healing programs in the forests.