The activation of glial cells in the spinal cord has been contribute to the initiation and maintenance of pain facilitation induced by peripheral inflammation and nerve injury. The present study investigated effects of botulinum toxin type A (BoNT-A), injected subcutaneously or intracisternally, on the expression of microglia and astrocytes in rats. Complete Freund’s Adjuvant (CFA)-induced inflammation was employed as an orofacial chronic inflammatory pain model. A subcutaneous injection of 40 μL CFA into the vibrissa pad was performed under 3% isoflurane anesthesia in SD rats. Immunohistochemical analysis for changes in Iba1 (a microglia marker) and GFAP (an astrocyte marker), were performed 5 days after CFA injection. Subcutaneous injection of CFA produced increases in Iba1 and GFAP expression, in the ipsilateral superficial lamia I and II in the medullary dorsal horn of rats. Subcutaneous treatment with BoNT-A attenuated the up-regulation of Iba1 and GFAP expressions induced by CFA injection. Moreover, intracisternal injection of BoNT-A also attenuated the up-regulated Iba1 and GFAP expressions. These results suggest that the anti-nociceptive action of BoNT-A is mediated by modulation activation of glial cells, including microglia and astrocyte.

본 연구는 들깻잎과 들깻잎 생산환경을 대상으로 31개의 시료를 채취하여 S. aureus 를 분리 하였다. 분리된 S. aureus 31주의 toxicity를 평가하고자 독소유전자와 항생제 내성을 검색하였다. 그 결과 분리된 균주에서 4개의 서로다른 독소유전자 패턴은 확인하였으며 sea와 sed유전자를 동시에 보유하는 균주는 4균주 (12.9%)였고, sed 유전자를 보유하는 균주는 9균주 (29.0%)였으며 see 유전자를 보유 하는 균주는 1균주 (3.2%)였다. 한편 seb와 sec 유전자를 보유하는 균주는 없었다. 항생제 내성평가결과, 12제의 항생제(penicillin, ampicillin, oxacillin, amoxicillin-clavulanic acid, cefazolin, cephalothin, imipenem, gentamicin, tetracycline, ofloxacin, norfloxacin, and erythromycin)에 내성을 보인 균주는 7균주 (22.6%)였다. 또한 분리된 균주의 2균주 (2.6%)는 5제의 항생제(penicillin, ampicillin, amoxicillin- clavulanic acid, gentamycin, and telithromycin)에 내성을 보였고 MRSA (Methicilline Resistant Staphylococcus aureus)는 포장지, 장갑, 들깻잎에서 발견되었다. 따라서 본 연구결과는 들깻잎에 오염된 S. aureus에 의하여 독소형식중독이 발생할 가능성을 시사하며 들깻잎과 생산 환경에서 항생제 저항성 S. aureus가 검출되어 의약계뿐만 아니라 농업현장에서도 항생제내성균주 출현을 예방하는 대책이 요구된다.

Inhibitor cysteine knot (ICK) peptides exhibit ion channel blocking, insecticidal, and antimicrobial activities, but currently, no functional roles for bee-derived ICK peptides have been identified. In this study, a bee (Apis cerana) ICK peptide (AcICK) that acts as an antifungal peptide and as an insecticidal venom toxin was identified. AcICK contains an ICK fold that is expressed in the epidermis, fat body, or venom gland and is present as a 6.6-kDa peptide in bee venom. Recombinant AcICK peptide (expressed in baculovirus-infected insect cells) bound directly to Beauveria bassiana and Fusarium graminearum, but not to Escherichia coli or Bacillus thuringiensis. Consistent with these findings, AcICK showed antifungal activity, indicating that AcICK acts as an antifungal peptide. Furthermore, AcICK expression is induced in the fat body and epidermis after injection with B. bassiana. These results provide insight into the role of AcICK during the innate immune response following fungal infection. Additionally, we show that AcICK has insecticidal activity. Our results demonstrate a functional role for AcICK in bees: AcICK acts as an antifungal peptide in innate immune reactions in the body and as an insecticidal toxin in venom. The finding that the AcICK peptide functions with different mechanisms of action in the body and in venom highlights the two-pronged strategy that is possible with the bee ICK peptide.

어린이집 유아들이 공용으로 사용하는 수건의 미생물 오염도와 분리된 식중독 미생물의 장독소 유전자 및 장독소 생산 특성을 분석하여 어린이집 유아 사용 수건의 위생 안전성을 확보하고자 어린이가 손 씻은 후 공용으로 사용 하는 수건 22개 (사용 전 7개, 사용 중 15개)를 연구대상으로 하였다. 일반세균수는 평균 6.2 log CFU/100 cm2로 검출되었고 진균수는 평균 4.1 log CFU/100 cm2로 검출되었 으며 대장균군은 사용 전 수건의 경우 7건 중 4건 (57.1%), 사용 중인 수건의 경우 15건 모두에서 (100%) 검출되었 다. 어린이 사용 수건의 미생물 오염도가 높게 나타나 수 건의 위생적인 살균 세탁 및 상대습도가 낮은 곳에 보관하는 등의 보관 방법 개선이 필요한 것으로 판단되었다. Salmonella spp.의 경우 실험에 사용된 모든 수건에서 검출되지 않았으나, Staph. aureus는 22건 중 5건 (22.7%), B. cereus는 11건 (50.0%)에서 검출되어 B. cereus 오염이 가장 심각한 것으로 나타났다. Staph. aureus 장독소 유전 자와 독소 단백질은 모두 불검출 되었으나 분리 동정된 11균주의 B. cereus 장독소 유전자 실험 결과 hblC, hblD, hblA 유전자가 각각 72.7, 72.7, 54.5% 검출되고 nheA, nheB, nheC 유전자가 각각 81.8, 72.7, 54.5% 검출되었으며 cytK, entFM 장독소 유전자는 각각 45.5, 90.0% 검출 되었다. B. cereus 장독소 실험결과 HBL 장독소는 11균주 중 8균주 (72.7%), NHE 장독소는 5균주 (45.5%) 검출되었고 HBL과 NHE 장독소 중 하나 이상을 생산하는 B. cereus 균주는 10균주 (90.9%)로 나타났다. 수건에 오염된 B. cereus 균주의 교차오염에 따른 식중독 위험성이 상존 하는 것으로 나타나 개인별 수건 또는 종이 타올 사용을 고려하여야 할 것으로 판단되었다.

Hemorrhagic Escherichia coli (E. coli) is one of the most important agent of diarrhea in piglet. The aim of this study was to investigate the characteristics of virulence genes and genetic diversity in hemorrhagic E. coli isolated from piglets with diarrhea. Among 122 hemorrhagic E. coli, 62 isolates carried single toxin gene like heat-stable toxin (ST), heatlabile toxin (LT), verotoxin 1 (VT1), verotoxin 2 (VT2), spa and eae. The most prevalent toxin gene carried by isolates was ST gene (23 isolates), while the most common association was ST/LT (14 isolates) and ST/LT/VT2 (13 isolates). In pulsed field gel electrophoresis, isolates were not classified as one cluster by epidemiological information including isolated area, isolated year and possessed toxins.

T-2 toxin and HT-2 toxin, belong to type A trichothecences, are the most toxic mycotoxins among the trichothecene family. These mycotoxins are commonly found in cereals such as maize, wheat, barley, oats and rice, and their occurrence in food can be of concern. This review investigated the current trends of patents and researches on T-2 toxin and HT-2 toxin pertaining to natural occurrence, toxicity, metabolism, risk assessment, analytical and screening methods, and reduction/detoxification techniques. As compared with other Fusarium mycotoxins, there are limited data for natural occurrence and risk assessment, and regulatory limit and official analytical methods on T-2 toxin and HT-2 toxin in domestic and foreign countries. In particular, selective deacetylation at the C3 and/or C4 positions of T-2 toxin by carboxyesterase present in foods was reported to cause the disappearance of T-2 and the extremely high HT-2 recoveries. Currently, regulatory limits for T-2 and HT-2 are under discussion in EU. For enforcement purposes it is essential to have available precise and reliable analytical methods applicable at the regulatory levels for the T-2 toxin and HT-2 toxin and relevant commodities. In addition, a further study on natural occurrence, risk assessment and reduction/detoxification techniques will be recommended.

Although baculoviruses have a long history of safe use as specific, environmentally benign insect control agents, their use has been limited by several factors, especially their slow speed of action. In this study, we intended to improve the insecticidal activities of Autographa californica nucleopolyhedrovirus (AcMNPV) by expressing Kunitz-type toxin isolated from venoms of Bombus ignitus or Araneus ventricosus. For this, recombinant AcMNPVs, AcBi-KTT, AcAv-Tox1 and AcAv-Tox2 expressing Bi-KTT, Av-Tox1 and Av-Tox2, respectively, under the control of p10 gene promoter were constructed. While polyhedra produced by these recombinant viruses were identical to those of the wild-type AcMNPV in shape, their sizes were relatively smaller than those of the AcMNPV. Among recombinant viruses, AcBi-KTT and AcAv-Tox2 showed significant reduction in median lethal time (LT50) against Spodoptera exigua larvae. Especiaaly, these two viruses showed about 6.2~10-folds higher polyhedra production rate compared to that of the AcMNPV. These results suggested that Kunitz-type toxins from insect venom could be successfully applied to improve insecticidal activity of baculoviruses.

목적 : 만성 경직성 편마비 환자에서 보툴리눔 독소 근 주입 후 상지 운동기능 개선을 알아보고자 하였다. 연구방법 : 뇌졸중 발병 후 12개월 이상이 경과된 만성기 편마비 환자 18명을 대상으로 환측 상지의 요측수근굴근, 척측수근굴근, 심지굴근, 천지굴근에 보툴리눔 독소를 각각 25 단위 주사하였다. 평가는 주사 전, 주사 2주 후와 8 주 후의 상지 기능 변화 및 동작 분석을 시행하였다. 상지의 경우 수정 에쉬워스 척도와 도수 근력 평가, 뇌졸중 상 지 기능 평가, 수지의 파악력 및 측면 집기력을 평가하였고 정량 분석을 위하여 3차원 동작 분석기를 이용하여 손 목관절의 최대 굴곡-최대 신전, 손바닥 두드리기, 검지 두드리기, 30cm 추적운동의 동작을 분석하였다. 결과 : 보툴리눔 독소를 주사 후 마비측 수지의 파악력, 측면 집기력에서는 유의한 변화가 없었으나 손목관절의 신 전, 수지의 신전 근력, 뇌졸중 상지 기능 평가에서 유의한 호전이 관찰되었다(p<.05). 3차원 동작 분석기를 이용한 상지 기능의 평가에서 손목관절 최대 굴곡-손목관절 신전 동작의 관절운동범위에서 유의한 호전이 관찰되었으며 손목관절 최대 굴곡-손목관절 신전 동작 및 손바닥 두드리기와 검지 두드리기 동작에서 진동수의 유의한 증가와 변이도의 유의한 감소는 관찰되지 않았으나 진폭의 유의한 증가가 관찰 되었다(p<.05). 결론 : 만성 경직성 편마비 환자에서 환측 상지에 대한 보툴리눔 독소 치료는 상지 운동기능 개선에 유의한 효과를 보였다.

The impact of transgenic Bt maize plant contained Cry1F was evaluated on the oat aphid Rhopalosiphum padi as a non-target insect species. Slightly reduced rates of survival and alata vivipar production were observed on Bt maize than on the non-Bt maize. In addition, slightly low preference to Bt maize plant was observed. Aphid fecundity, measured as the number of offspring produced for 7 days, was higher on Bt maize than on non-Bt maize but not different significantly. ELISA test using Cry1F-antibody revealed that 26% of Cry1F protein compared to the positive control was detected from the whole body of R. padi when the insects were fed Bt maize for 50 days, showing that R. padi can carry Cry1F protein to the higher trophic level when exposed to Bt maize. Taken together, the Bt maize plant is not likely to cause any negative side impacts on non-target insect R. padi but Bt toxin can be transferred to higher predators via R. padi as it carries the toxin.

Nivalenol (NIV), deoxynivalenol (DON), T-2 toxin (T-2) and zearalenone (ZEN) are mycotoxins produced by some Fusarium species known to be very frequently contaminated in feed. The study for simultaneous analysis and contamination survey in animal feed carried out. All mycotoxins were analysed by using high performance liquid chromatography tandem mass with internal standard. The limits of detection (LOD) were 2.0 μg/kg,1.0 μg/kg, 1.0 μg/kg and 0.1 μg/kg for NIV, DON, T-2 and ZEN, respectively. Two hundred and thirty nine samples of feed were collected. The average concentration of DON was 212.3 μg/kg, 207.8 μg/kg and 812.1 μg/kg in chicken,pig and cattle feed, respectively. The average concentration of ZEN was 31.2 μg/kg, 35.6 μg/kg and 147.2 μg/kg for them, respectively. Especially, the levels of contamination for DON and ZEN were higher than those of NIV or T-2. And, the levels of contamination for four Fusarium mycotoxins in cattle feed appeared higher than those of pig and chicken feed. It was investigated that the high level of mycotoxin contamination in cattle feed was caused by corn gluten feed of ingredients for feed, mainly.

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.

To develop an advanced baculovirus insecticide with additional advantages, such as higher toxicity and recovering to wild-type baculovirus, a novel recombinant baculovirus, NeuroBactrus was constructed. Bacillus thuringiensis crystal protein gene (cry1-5) and an insect-specific neurotoxin gene (AaIT) were introduced into Autographa californica nucleopolyhedrovirus genome by fusion of polyhedrin-cry1-5-polyhedrin under the control of poyhedrin gene promoter, and by fusion of orf603 partial genes and AaIT under the control of early promoter of ORF3006 from Cotesia plutellae bracovirus. About 150 kDa of Polyhedrin-Cry1-5-Polyhedrin fusion protein expressed by NeuroBactrus was occluded into the polyhedra, and activated as about 65 kDa of crystal protein when treated with trypsin. RT-PCR analysis indicated that transcription of AaIT gene occurs by 2 h postinfection (p.i.) and increased at 16 h p.i.. NeuroBactrus showed high toxicity against Plutella xylostella larvae and significant reduction in median lethal time (LT50) against Spodoptera exigua larvae compared to those of wild-type AcNPV. Re-recombinants derived from NeuroBactrus, NBt-Del5 (deleted cry1-5), NBt-DelA (deleted AaIT) and NBt-Del5A (deleted cry1-5 and AaIT; wild-type baculovirus) were generated in serial passages in vitro. This result showed that the NeuroBactrus could be transferred to wild-type baculovirus along with serial passages by the homologous recombination between two polyhedrin genes and two partial orf603 genes.

The effects of pollens of Bt Chinese cabbage (Brassica campestris), expressing the insecticidal protein Cry1Ac toxin (Bt Chinese cabbage) on Bombix mori larvae, were determined in laboratory studies. When reared on an artificial diet containing pollens of Bt Chinese cabbage, decreased survival rate and body weight of B. mori larvae were observed. ELISA test using Cry1Ac-antibody implied that the Cry toxin was detected not only in the whole body and alimentary canal but also in the hemolymph and remaining body parts, suggesting that the ingested Cry1Ac protein is distributed inside the body through epithelial membrane of the midgut of B. mori. Taken together, it was suggested that the pollens of Bt Chinese cabbage adversely affect non-target insect B. mori larvae.

To develop an improved baculovirus insecticide with additional advantages, a novel recombinant baculovirus, AcB5B-AaIT was constructed. B. thuringiensis crystal protein gene (cry1-5) and insect-specific neurotoxin gene (AaIT) were introduced into Autographa californica nucleopolyhedrovirus genome by fusion of polyhedrin-cry1-5-polyhedrin under the control of polyhedrin (polh) gene promoter, and AaIT under the control of early promoter of ORF3004 from Cotesia plutellae bracovirus, respectively. About 150 kDa of Polyhedrin-Cry1-5-Polyhedrin fusion protein expressed by AcB5B-AaIT was occluded into the polyhedra produced by the recombinant virus, and activated as about 65 kDa of crystal protein when treated with gut-juice of Bombyx mori. The AcB5B-AaIT showed about 50% reduced LT50 value compared to that of the recombinant virus, Ap1Ac, expressing Cry1Ac against Plutella xylostella larvae. In addition, Spodoptera exigua larvae fed the recombinant polyhedra of AcB5B-AaIT showed about 4 fold higher refusing diet effect compared S. exigua larvae fed the recombinant polyhedra of the recombinant virus, Ap1C, expressing Cry1C. AcB5B-AaIT could be transferred to wild-type baculovirus along with serial passage by the homologous recombination between two polyhedrin genes contained in polh-cry1-5-polh fusion protein gene. These results suggested that the novel recombinant baculovirus, AcB5B-AaIT, could be applied as advanced viral insecticide.

본 연구는 생체 내 세포 및 조직배양기로서의 면역결핍동물을 개발할 목적으로 proximal lck promoter와 DT-A gene를 이용하여 형질전환생쥐을 생산하고 이 형질전환생쥐의 면역세포에서 DT-A gene이 발현되는지를 분석하였다. 형질전환생쥐와 정상생쥐로부터 thymus, spleen 및 liver에서 RNA를 추출하여 RT-PCR수행하였는데 정상생쥐의 조직에서는 어떠한 DT-A gene의 발현양상을 얻을 수 없었으나 형질전환생쥐의 thymus, spleen, liver에 DT gene의 발현을 확인할 수 있었고, Northern blotting을 이용하여 형질전환생쥐의 thymus, spleen 및 liver에서 DT-A gene이 강하게 발현되는 것으로 나타났다. 형질전환생쥐 F₁ 및 F₂ 산자의 혈액에서 T-cell 발달의 분포도를 확인하기 위해 CD4 및 CD8 ntibody를 이용하여 FACS analysis를 실시하였는데 형질전환생쥐의 혈액 내 mature T-cell인 single positive thymocyte의 수가 정상생쥐에 비해 감소하는 경향을 나타냈다. 정상생쥐의 혈액 내 T-cell 중 CD8/sup +/ T-cell의 경우 약 50%를 나타냈으나 형질전환생쥐의 경우 33%로 감소하였고, CD4/sup +/ T-cell은 정상생쥐에서 10%를 차지하고 있으나 형질전환생쥐에서는 5.9%로 감소되는 것으로 분석되었다. 그러므로 본 연구의 형질전환생쥐에서 lck promoter에 의해 초기 immature한 상태의 T-cell에서 DT-A gene이 발현되어 발육중인 T-cell이 파괴 되어 mature 상태인 CD4/sup +/ CD8/sup -/나 CD4/sup -/CD8/sup +/ cells (single-positive)들이 감소된 것으로 확인되었다.

Objective: To examine the effects of occupational therapy intervention on hand function after botulinum toxin injection in a patient with dystonia. Method: The subject was a 41 year old female and the study was carried out from February 25th to May 24th in 2002. The Jebsen Hand Function Test and Nine Hole Peg Test were used for assessment, and also hand and finger powers were assessed by grip strength, lateral pinch strength and three jaw chuck pinch strength. The scores were obtained during occupational therapy sessions at 4 weeks and 12 weeks after the injection. Modified Ashworth Scale was employed to grade spasticity before the injection, at 4 weeks, and 12 weeks after the injection. Results: Among the subtests of Jebsen Hand Function Test, the speed increased in the writing (15.4%), turning cards (64.4%), simulated feeding (92.0%), moving empty large cans (30.0%) and moving weighted large cans (41.6%), however, the speed of other subtests which were checkers(-26.8) and picking up small objects (0.0%) did not increase. The performance on Nine Hole Peg Test did not change (0.0%). Hand power of grip strength increased by 275%. Finger power increased in lateral pinch strength (40.0%) and three jaw chuck pinch strength(150.0%). Based on Modified Ashworth Scale, spasticity decreased from grade 4 before the injection to grade 1 at 4 weeks and 12 weeks after the injection in wrist and fingers. Conclusion: Occupational therapy intervention after botulinum toxin injection can improve the hand function in a patient with dystonia.