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Extraction of Acetylcholinestrase from the Housefly and Three Other Insect Species for In Vitro Anticholinesterase Screening (In Vitro Anticholinesterase 스크리닝을 위한 집파리 및 3종 곤충으로부터의 Acetylcholinesterase의 추출)

  • 이시혁;이준호;조광연
    • Korean journal of applied entomology
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    • v.30 no.1
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    • pp.18-28
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    • 1991
  • The optimal pH of the extraction buffer was 7.5 considiering AChE stability and its buffer capacity when AChE was isolated and extracted from the housefly(Musca domesitca L.)and three other insect species with 0.01 M sodium phosphate buffer. Also, the optimal pH of the reaction buffer was 7.5 considering enzyme activity and its buffer capacity when AChE activity was measured with the substrate in 0.1 M sodium phosphate buffer. The Potter Elvehjem type homogenizer with Teflon pestle was used to homgenize the tissues. When preparing a AChE suspension by centrifuging the homogenate, 700 g supernatant of adult head for the housefly, 700 g supernatnat of 5th instar nymphal whole body for the brown planthopper, lipid-eliminated 10,000 g supernatant of 5th instar larval whole body for the diamondback moth, and 700 g supernatant of 4th instar larval head for the tobacco cutworm were considered satisfactory as enzyme sources in view of mass preparation, extraction efficiency and stability of enzyme activity during evaluation. When AChE suspensions of 4 insect species were stored at $-18^{\circ}C$, more than 90% of activity was maintained up to 3 weeks. Km values of AChEs of the housefly, the brown planthopper, and the diamondback moth were 0.042, 0.037 and 0.043 mM, respectively and AChE-specific substrate inhibition was observed at high concentration. Km value of the tobacco cutworm ChE was 1.15 mM and BuChE characteristics was observed, though further study is needed. The optimal substrate concentration for the AChE inhibition tests was 0.5 mM for the housfly, the brown planthopper, and the diamondback moth and 12 mM for the tobacco cutworm.

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Geographic Variation in Pond Smelt (Hypomesus nipponensis) by RAPD Analysis (RAPD 분석에 의한 빙어 (Hypomesus nipponensis)의 지리적 변이)

  • Kim, Yong-Ho;Park, Su-Young;Yoon, Jong-Man
    • Korean Journal of Ichthyology
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    • v.18 no.1
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    • pp.1-11
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    • 2006
  • Genomic DNA isolated from two geographical populations of pond-smelt (Hypomesus nipponensis) was amplified for RAPD (randomly amplified polymorphic DNA) analysis. The populations were obtained from Chungju (CJ), in the inland area, and Dangjin (DJ), in the vicinity of the West Sea in Korea. Seven arbitrarily selected primers, OPB-06, OPB-10, OPB-13, OPB-17, OPC-09, OPC-17 and OPC-20, were used to generate the shared loci, polymorphic, and specific loci. Three hundred and eighty-three loci observed per primer were identified in the CJ population, and 287 were identified in the DJ population. Among them, 91 polymorphic loci or 23.8% were polymorphic in the CJ population, and 47 (16.4%) in the DJ population. The number of shared loci observed was 198 in the CJ population and 176 in the DJ population. Forty-four and 75 specific loci were detected in the CJ and DJ populations, respectively. Especially, 99 numbers of shared loci by the two populations, with an average of 14.1 per primer, were observed in the two pond-smelt populations. The average bandsharing value between the two geographical pond-smelt populations was $0.700{\pm}0.008$, ranging from 0.600 to 0.846. Compared separately, the bandsharing value of individuals within the CJ population was higher than that of the DJ population. The dendrogram obtained using the data from the seven primers indicated three genetic clusters: cluster 1, CJ 01, 02, 03, 04, 05, 06, 07, 08, 09, 10, and 11; cluster 2, DJ 01, 02, 03, 04, 05, 06, 07, 08, and 09; and cluster 3, DJ 10 and 11. The genetic distance between the two geographical populations ranged from 0.040 to 0.545. Thus, RAPD-PCR analysis revealed a significant genetic distance between the two pond-smelt populations.

Causes of the Difference of Inhabited Altitudes above Sea Level of Fairy Pitta(Pitta nympha) on Jeju Island Followed by Forest Landscape Through the Comparison of Landsat Images and the Literature Review (Landsat 영상비교와 문헌연구를 통한 제주도 산림경관변화와 팔색조 서식고도 차이에 관한 연구)

  • Kim, Eun-Mi;Kwon, Jin-O;Kang, Chang-Wan;Chun, Jung-Hwa
    • Journal of the Korean Association of Geographic Information Studies
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    • v.16 no.4
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    • pp.79-90
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    • 2013
  • The altitude range of habitats in which Fairy Pitta inhabited in 1960s is different from the present in Jeju Island. We studied on the habitat environment to understand the causes of difference through the comparison of satellite image data(Landsat) between 1975 and 2002, the literature review in relation to habitats, vegetations, and forest landscapes. The area of below 600m asl.(above sea level) where is mainly Fairy Pitta inhabited at the present with a lot of forests, was massive pasture with small isolated forests nearby valley. The forests were broad-leaved evergreen forests, and second forests with poor condition in the size and forest structure. The forests around 700m asl. were also second forests with approximately 3m height trees. The forests from 800m to 1300m asl. were also disturbed by mushroom cultivation by local people. The authors believe that Fairy Pitta could not inhabited in the area above 1300m because of the poor forest conditions in the size and structure in which consist of Ilex crenata, Rhododendron mucronulatum var. ciliatum and coppice forests. Therefore it might be possible that the best forests for the Fairy Pitta habitat were located in the area of 1,000m to 1,300m above sea level in 1960s. Compared to present habitats, forests at 100m up to 800m above sea level, the authors believe that the size of habitats were smaller with less population of Fairy Pitta. Since 1960s the forest landscape of Jeju Island has been improved successfully, and because of that the population of Fairy Pitta also has been increased. To protect the Fairy Pitta and habitats in Jeju Island, it is suggested that sustainable forest management focusing on the species composition and stand structure maintain or enhance the biodiversity.

EXPRESSION OF BMP4, BMP6 FOLLOWING SINUS ELEVATION WITH DBBP IN RABBIT (가토 상악동 점막 거상 후 DBBP를 이식재로 사용시 BMP4, BMP6의 발현)

  • Lee, Hyun-Suk;Heo, Hyun-A;Pyo, Sung-Woon;Lee, Won
    • Maxillofacial Plastic and Reconstructive Surgery
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    • v.29 no.6
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    • pp.467-473
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    • 2007
  • The most important factor for successful implantation is osseointegration between the implant and bone. The expression of bone morphogenetic proteins (BMPs) inducing bone formation would differ after maxillary sinus elevation. And within the same graft material. the expression of BMPs would change with time after graft. The aim of this study was to compare the relative expressions of BMP4 and BMP6 using real-time RT-PCR when maxillary sinus elevation was performed using deproteinated bovine bone powder (DBBP) as the graft material or absorbable gelatin sponge (AGS) as the filler without any graft material. Fifteen rabbits, each weighing between 3.0 to 3.5 Kg, were divided randomly into 5 groups of 3 animals each based on their time of sacrifice 0, 3, 5, 7 and 9 days). After exposure of the maxillary sinus bilaterally, bone graft was performed in the right maxillary sinus using DBBP ($BBP^{(R)}$ Oct Inc., Cheonan, Korea) and only AGS ($Gelfoam^{(R)}$ Pharmacia & Upjohn Company, Kalamazoo, MI, U.S.A) was placed into the left without any graft material. Each group of rabbits was sacrificed at 1, 3, 5, 7, or 9 days after operation and all specimens were harvested. And the following results were obtained using real-time RT-PCR from isolated total RNA of the samples. 1. The expression of BMP4 increased at postoperative 1 and 3 days in both DBBP group and AGS group. In AGS group. it decreased at postoperative 5 days. increased again at postoperative 7 days, and decreased at postoperative 9 days. In DBBP group, it increased until postoperative 7 days and decreased at postoperative 9 days. Although the expression of BMP4 was higher in DBBP group compared with AGS group, it was not statistically significant (p>0.05). 2. The expression of BMP6 increased at postoperative 1 and 3 days in both DBBP group and AGS group. In AGS group, it decreased at postoperative 5 days, increased again at postoperative 7 days, and decreased at postoperative 9 days. In DBBP group, it increased until postoperative 7 days and decreased at postoperative 9 days. Although the expression of BMP6 was higher in AGS group compared with DBBP group, it was not statistically significant (p>0.05). 3. There was no statistically significant difference in BMP expression in both groups during same period of time. It' s probably because DBBP and AGS both functioned as a space retainer so that the BMP expression in blood clot seemed to be similar. 4. Thus, DBBP would not offer many benefits for early bone regeneration compared with AGS. The expression of BMP in early bone formation seems to be more influenced by physical carrier rather than the graft type.

BIOMECHANICS OF ABUTMENTS SUPPORTING REMOVABLE PARTIAL DENTURES UNDER UNILATERAL LOADING

  • Kim, Seong-Kyun;Heo, Seong-Joo;Koak, Jai-Young;Lee, Jeong-Taek;Roh, Hyun-Ki;Kim, Hyo-Jin;Lee, Seok-Hyung;Lee, Joo-Hee
    • The Journal of Korean Academy of Prosthodontics
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    • v.45 no.6
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    • pp.753-759
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    • 2007
  • Statement of problem. In distal extension removable partial denture, the preservation of health of abutment teeth is very important, but abutment teeth are subjected to unfavorable stress under unilateral loading specially. Purpose. The purpose of this study was to investigate the biomechanical effects of mandibular removable partial dentures with various prosthetic designs under unilateral loading, using strain gauge analysis. Material and methods. Artificial teeth of both canines were anchored bilaterally in a mandibular edentulous model made of resin. Bilateral distal extension removable partial dentures with splinted and unsplinted abutments were fabricated. Group 1: Clasp-retained mandibular removable partial denture with unsplinted abutments Group 2: Clasp-retained mandibular removable partial denture with splinted abutments by 6-unit bridge. Group 3: Bar-retained mandibular removable partial denture Strain gauges were bonded on the labial plate of the mandibular resin model, approximately 2 mm dose to the abutments. Two unilateral vertical experimental loadings (30N and 100N) were applied subsequently via miniature load cell that were placed at mandibular left first molar region. Strain measurements were performed and simultaneously monitored from a computer connected to data acquisition system. For within-group evaluations, t-test was used to compare the strain values and for between-group comparisons, a one-way analysis of variance (ANOVA) was used and Tukey test was used as post hoc comparisons. Results. The strain values of group 1 and 2 were tensile under loadings. In contrast, strain values of group 3 were compressive in nature. Strain values increased as the applied load in increased from 30N to 100N (p<.05) except for right side in group 1. Under 30N loading, in left side, group 1 showed higher strain values than groups 2 and 3 in absolute quantity (p<.05). And group 2 showed higher strain values than group 1 (p<.05). In right side, group 1 and 2 showed higher strain values than group 3 in absolute quantity (p<.05). Under 100N loading in left side, group 1 showed higher strain values than groups 2 and 3 in absolute quantity (p<.05). And group 2 showed higher strain values than group 1 (p<.05). In right side, group 1 and 2 showed higher strain values than group 3 in absolute quantity (p<.05). Under 30N loading, group 2 and 3 showed higher strain values in right side than in left side. Under 100N loading, right side strain values were higher than left side ones for all groups. Conclusion. Splinting of two isolated abutments by bridge reduced the peri-abutment strain in comparison with unsplinted abutments under unilateral loading. Bar-retained removable partial denture showed the lowest strain of three groups, and compressive nature.

Effect of Crude Polysaccharide Isolated from Mageoli Mash under Different Fermentation Conditions on Immune-Stimulating Activity (발효 조건이 막걸리 술덧 유래 다당의 면역자극 활성에 미치는 효과)

  • Rhee, Young Kyoung;Nam, So-Hyun;Kim, Hye-Ryun;Cho, Chang-Won;Lee, Young-Chul;Kim, Young-Chan;Hong, Hee-Do
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.43 no.12
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    • pp.1835-1842
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    • 2014
  • Various microorganisms are involved in the fermentation of Mageoli, Korean traditional liquor. Among them, the most predominant microorganism is yeast, including Saccharomyces cerevisae. This study investigated the effects of crude polysaccharides separated from Mageoli mash (CP-M) on activation of peritoneal macrophages in cell culture medium. Four types of yeasts, S. cerevisae 89-1-1, 98-2, 268-3, and 113-4, were used. When peritoneal macrophages were treated with $10{\mu}g/mL$ of CP-M fermented with S. cerevisae 113-4, the concentration of nitric oxide (NO) in the medium was highest ($33.3{\mu}M$). The concentrations of IL-6 and -12 were also highest at 116.3 pg/mL and 59.8 pg/mL, respectively. In the case of CP-M cultured at 15, 20, and $30^{\circ}C$ after inoculation with S. cerevisae 113-4 and fermented at different temperatures, production of NO and IL-6 by peritoneal macrophages did not change compared to the control. For CP-M obtained after fermentation at $25^{\circ}C$, however, NO concentration increased 2.7~3.3 fold and IL-6 concentration by 5.7 fold compared to the control. Furthermore, the effect of fermentation period on expression of CP-M functionality was examined. NO production by CP-M of the 5th day mash was highest, increasing 2.2 fold compared to 0-day CP-M. However, there were no significant differences in concentration of IL-6 or -12 according to different fermentation periods, although CP-M showed a large decrease after 10 days. The results show that fermentation conditions with the highest activity were observed in CP-M inoculated with S. cerevisae 113-4 and fermented at $25^{\circ}C$ for 5 days. CP-M consisted of 78.6% neutral sugar, 11.6% acidic polysaccharide, and 9.8% protein. In particular, the sugar composition of neutral sugar consisted of mannose (47.8%), glucose (29.6%), and galactose (12.7%). Based on the results, CP-M is assumed to be an extracellular polysaccharide originating in yeast with high mannose content.

A effect of calcium hydroxide endodontic materials on the differentiation and the activation of osteoclast (수산화칼슘계 근관 충전제가 파골 세포의 분화 및 활성에 미치는 영향)

  • Jang, Eun-Young;Kown, Hoon;Lee, Chang-Hee;Lee, Chang-Seop;Lee, Sang-Ho
    • Journal of the korean academy of Pediatric Dentistry
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    • v.28 no.2
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    • pp.207-218
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    • 2001
  • The purpose of this study was to investigate the direct inhibitory effect of calcium hydroxide materials on differentiation and activation of osteoclast. we used the osteoclast progenitor cells isolated from bone marrow cell of chick embryo tibia and four experimental materials [$Ca(OH)_2$ powder, Metapaste$^{(R)}$, Vitapex$^{(R)}$, Pulpdent$^{(R)}$] diluted at 0.1, 0.01, $0.05{\mu}g/ml$. There were measured both the number of differentiated osteoclast and the area of resorption lacunae. Also, we conducted MTT assay on U2OS osteoblast to examine of cytotoxic effect and obtained following result. 1. Considering the result of the inhibitory effects upon osteoclast differentiation, There were shown a significant difference increased in the following order: Metapaste$^{(R)}$, $Ca(OH)_2$ powder, Vitapex$^{(R)}$. But no significant difference was found in pulpdent group that the number of differentiated osteoclast was increased at all concentrations(p<0.05). 2. Among the three experimental groups, that is, $Ca(OH)_2$ powder, Metapaste$^{(R)}$, Vitapex$^{(R)}$ at $0.1{\mu}g/ml$ dilution that were statistically significant in reduction of the number of differentiated osteoclast. Vitapex group showed significant cytotoxic effect compared to control and another two groups exhibited no significant difference. Also, 0.2% DMSO group was shown statistically siginificant cytotoxicity (p<0.05). 3. Examining pattern and measured area of resorption lacunae in the control and the three experimental groups ,that is, $Ca(OH)_2$ powder, Metapaste$^{(R)}$, Vitapex$^{(R)}$ at $0.1{\mu}g/ml$ dilution, except $Ca(OH)_2$ powder group, statistically significant differences were found between experimental groups and control group. Also, DMSO group showed statistically significant decrease (p<0.05). From these results, we think that calcium hydroxide is responsible for suppression of hard tissue resorption by a direct inhibition of dfferentiation and activation of osteoclast.

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Clinical Implication of Serum TNF-$\alpha$ and IL-1$\beta$ Measurement in Patients with Sepsis (패혈증환자에서 혈청 TNF-$\alpha$ 및 IL-1$\beta$)

  • Kim, Jae-Yeol;Choi, Hyung-Seok;Lee, Choon-Taek;Kim, Young-Whan;Han, Sung-Koo;Min, Kyung-Up;Kim, Yoo-Young;Shim, Young-Soo;Yoo, Chul-Gyu
    • Tuberculosis and Respiratory Diseases
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    • v.49 no.2
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    • pp.217-224
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    • 2000
  • Background : It is well known that when macrophages are stimulated with endotoxin, they produce a wide variety of cytokine mediators, including TNF-$\alpha$ and IL-1$\beta$. However, there is an alteration in the macrophages' responsiveness when they are challenged with repeated bouts of endotoxin, termed "endotoxin tolerance" which is regarded as a self-protective phenomenon from continuous stimulation. In this study, endotoxin tolerance in the peripheral blood monocytes of sepsis patients was evaluated. Methods : Fourteen patients with organism-documented sepsis were included. The severity of illness was evaluated by APACHE II score. Peripheral blood monocytes were isolated from the patients and diluted to $1{\times}10^5$ well. After stimulation with endotoxin (LPS of E. coli O114 : B4, 100 ng/ml), they were incubated at $37^{\circ}C$ in 5% $CO_2$ incubator for 24 hours. Supernatant was collected for the measurement of TNF-$\alpha$ and IL-1$\beta$ with ELISA method. Peripheral blood monocytes of seven healthy volunteers were used as control. Results : The APACHE II score (mean$\pm$SD) of the patients at the time of blood sampling was 12.2$\pm$5.7. The primary infection foci were urinary tract infection, pneumonia, subacute bacterial endocarditis, and catheter related infection, etc. The causative organisms were gram negative rods (10 cases), gram positive cocci (6 cases) with two cases of mixed infection. Serum TNF-$\alpha$ could be measured in 4 cases with 29.9$\pm$27.7 pg/ml. Serum IL-1$\beta$was measurable in only one patient. The TNF-$\alpha$ level of supernatant of cultured peripheral blood monocytes was 2,703$\pm$2,066 pg/ml in patients and 2,102$\pm$1914 pg/ml in controls. The IL-1$\beta$level of supernatant was 884$\pm$1,050 pg/ml in patients and 575$\pm$558 pg/ml in controls. There was no difference of TNF-$\alpha$ and IL-1$\beta$ level between patients and controls. Conclusion : We cannot prove the phenomenon of endotoxin tolerance in this study. Future study needs to be focused on the more severe sepsis patients who were taken for sampling earlier. Addition of serum to the culture medium could be an another valuable option for the success of this study.

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Superoxide Dismutase Gene Expression in the Endotoxin-Treated Rat Lung (내독소에 의한 백서 폐장의 Superoxide Dismutase 유전자 발현에 관한 연구)

  • Yoo, Chul-Gyu;Suh, Gee-Young;Kim, Young-Whan;Han, Sung-Koo;Shim, Young-Soo;Kim, Keun-Youl;Han, Yong-Chol
    • Tuberculosis and Respiratory Diseases
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    • v.41 no.3
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    • pp.215-221
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    • 1994
  • Background: It is well known that oxygen free radicals(OFR) play a vital role in the various type of acute lung injury. Among various antioxidant defense mechanisms, the superoxide dismutases(SOD) are thought to be the first line of antioxidant defense by catalyzing the dismutation of two superoxide radicals to yield hydrogen peroxide and oxygen. Eukaryotic cells contain two types of intracellular SOD : cytosolic, dimeric copper/zinc- containing enzyme(CuZnSOD) and mitochondrial, tetrameric manganese-containing enzyme(MnSOD). The purpose of this study is to evaluate the time-dependent gene expression of MnSOD and CuZnSOD in the endotoxin-treated rats, and to compare with the manifestations of LPS-induced acute lung injury in rats. Methods: Total RNA from rat lung was isolated using single step phenol extraction 0, 1, 2, 4, 6, 12, 18, 24 hours after E. coli endotoxin injection(n=3, respectively). RNA was separated by formaldehyde-containing 1.2% agarose gels elctrophoresis, transblotted, baked, prehybridized, and hybridized with $^{32}P$-labeled cDNA probes for rat MnSOD and CuZnSOD, which were kindly donated by Dr. Ho(Duke University, Durham, NC, USA). The probes were labeled by nick translation. Blots were washed and autoradiography were quantitated using laser densitometry. Equivalent amounts of total RNA/gel were assessed by monitoring 28S and 18S rRNA. Results: Endotoxin caused a rise in steady-state MnSOD mRNA levels by 4h with peak mRNA accumulation by 6h. Continued MnSOD mRNA expression was observed at 12h. CuZnSOD mRNA expression was observed from 1h to 24h with peak levels by 18h. Conclusion: These results suggest that SOD palys an important defensive role in the endotoxin-induced acute lung injury in rats.

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Analysis of rpoB Gene in Rifampin-Resistant M. Tuberculosis by Direct Sequencing and Line Probe Assay (염기서열결정과 Line Probe 분석법에 의한 Rifampin내성 결핵균의 rpoB 유전자 분석)

  • Lee, Min-Ki;Kim, Yun-Seong;Lee, Hyo-Jin;Cheon, Du-Su;Yun, Sang-Myung;Park, Sam-Seok;Kim, Cheol-Min;Park, Soon-Kew
    • Tuberculosis and Respiratory Diseases
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    • v.44 no.2
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    • pp.251-263
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    • 1997
  • Background : The emergence of multidrug-resistant strains of Mycobacterium tuberculosis presents a significant challange to the treatment and control of tuberculosis, and there is an urgent need to understand the mechanisms by which strains acquire multidrug resistance. Recent advances in molecular methods for the detection of M. tuberculosis genetic targets have approached the sensitivity of culture. Furthermore the prospect of determining resistance in mycobacteria at the nucleic acid level particulary to first-line drugs like rifampin, isoniazid has provided a glimps of the next generation of sensitivity test for M. tuberculosis. Previous studies in RMP resistant M. tuberculosis have shown that mutation in $\beta$subunit of RNA polymerase is main mechanism of resistance. Method : In this study, rpoB gene for the $\beta$subunit of RNA polymerase from M. tuberculosis of 42 cultured samples (32 were RMP resistant and 10 were sensitive cases) were isolated and characterised the mutations. Direct sequencing data were compared with the results of INNO-LiPA Line Probe Assay (LiPA, Innogenetics, Belgium), commercial RMP resistance detecting kit using reverse hybridization method. Results : All of the RMP resistant samples were revealed the presence of mutation by LiPA. In 22 samples (68.8%) out of 32 RMP resistant cases, the mutation types were confirmed by the positive signal at one of 4 mutation bands in the strip. The most frequent type was R5 (S531L) which were 17 cases (77.3%). Results of direct sequencing were identified the exact characteristics of 8 mutations which were not confirmed by LiPA. S522W type point mutation and 9 base pair deletion at codon 513~515 were new identified mutations for the first time. Conclusion : Mutations in rpoB gene is the main mechanism of RMP resistance in M. tuberculosis and LiPA is a very useful diagnostic tool for the early diagnosis of RMP resistance in M. tuberculosis.

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