• Title/Summary/Keyword: SLT

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Relationship between Gb3 Expression and Cytotoxicity of Shiga-like Toxin I (Shiga-like Toxin I의 세포독성과 수용체 Gb3 발현과의 관계)

  • Lim, Suk-Hwan;Kim, Gi-Young;Kim, Hyung-Chun;Kim, Young-Hee;Son, Yong-Hae;Oh, Yang-Hyo;Park, Yeong-Min
    • Clinical and Experimental Pediatrics
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    • v.46 no.2
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    • pp.143-153
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    • 2003
  • Purpose : Infection with Shiga-like toxin (SLT)-producing Escherichia coli, an emerging human pathogen found particularly in young children under 5 years of age, causes a spectrum of illnesses with high morbidity and mortality, ranging from diarrhea to hemorrhagic colitis and hemolytic uremic syndrome. Host mediators play an important role in the pathogenesis of SLT-I toxicity. The experiments described here were designed to investigate the effect of SLT-I on TNF-${\alpha}$ production and to understand the effect of TNF-${\alpha}$ on GB3 expression. We also further examine the relationship between the Gb3 level and the differential susceptibility of cells to the cytotoxic action of SLT-I. Methods : The effect of purified SLT-1 from E. coli O157 : H7 (ATCC 43890) on tumor necrosis factor-${\alpha}$ (TNF-${\alpha}$) production in Raw264.7 cells was investigated. Many mediators regulate endothelial cell membrane expression of the glycolipid globotriaosyleramide (Gb3), which serves as the toxin receptor, suggesting that the host response to the toxin or other bacterial products may contribute to pathogenesis by regulating target cell sensitivity to the toxins. Therefore, the relationships between Gb3 expression and cytotoxicity against SLT-I on three types of cells were evaluated. Results : Detectable levels of TNF-${\alpha}$ were produced as early as six hours after induction and continued to increase during 48 hours by SLT-I. It was also found that Vero cells and dendritic cells (DC2.4 cells) expressed high levels of Gb3, 83% and 68%, respectively, and that Raw264.7 cells had a low level of Gb3 (29%) and appeared refractory to cytotoxicity against SLT-I. Vero cells and DC2.4 cells expressing high levels of Gb3 were highly susceptible to SLT-I. Furthermore, macrophages showed a resistance to SLT-I cytotoxicity, despite the fact that Gb3 expression was enhanced. Conclusion : These results strongly suggest that the expression of Gb3 is necessary but not sufficient to confer sensitivity of macrophages to SLT-I and further underpin the important role of SLT-I and its Gb3 receptors in the pathogenesis of E. coli O157 infection.

Regulation of Gb3 Expression on Dendritic Cells (수지상세포에 있어서 베로독소 수용체의 발현조절)

  • Lim, Suk-Hwan;Kim, Gi-Young;Kim, Hyung-Chun;Kim, Young-Hee;Son, Yong-Hae;Oh, Yang-Hyo;Park, Yeong-Min
    • Journal of Life Science
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    • v.17 no.4 s.84
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    • pp.482-492
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    • 2007
  • Infection with Shiga-like toxin (SLT)-producing Escherichia coli causes a spectrum of illnesses with high morbidity and mortality. Host mediators play an important role in the pathogenesis of SLT-I toxicity. We here investigated the effect of SLT-I on tumor necrosis $factor-{\alpha}\;(TNF-{\alpha})$ production, effect of $TNF-{\alpha}$ on glycolipid globotriaosyleramide (Gb3) expression, and relationship between Gb3 level and differential susceptibility of cells to SLT-I. In this study, we observed that detectable levels of $TNF-{\alpha}$ are produced 6 hrs after induction and continued to increase during 48 hrs by SLT-I. It was also found that Vero cells and dendritic cells expressed high levels of Gb3, 83% and 68%, respectively, and that macrophages had a low level of Gb3 (29%) and showed refractory to cytotoxicity against SLT-I. Vero cells and dendritic cells expressing high levels of Gb3 were highly susceptible to SLT-I. furthermore, macrophages showed a resistance to SLT-I cytotoxicity, despite the fact that Gb3 expression was enhanced. These results suggest that the expression of Gb3 is necessary, but not sufficient to confer sensitivity of macrophages to SLT-I and further underpin the important role of SLT-I and its receptor, Gb3, in the pathogenesis of E. coli O157 infection.

Cloning and Nucleotide Sequence Analysis of Verotoxin Gene from Escherichia coli O157 KNIH317 Isolated in Korea

  • Park, Yong-Chjun;Shin, Hee-Jung;Kim, Young-Chang
    • Journal of Microbiology
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    • v.37 no.3
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    • pp.168-174
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    • 1999
  • Escherichia coli O157 is an important pathogenic organism which causes diarrhea, haemorrhagic colitis, and haemolytic ureamic syndrome (HUS) in human. E. coli O157 KNIH317 was isolated form patients suffering with HUS in Korea. We designed a primer set for cloning shiga-like toxin (slt) gene. The amplified PCR product was used to Southern and colony hybridization as a probe. As a result, we cloned 4.5-kb KpnI fragment containing the slt gene encoding shiga-like toxin from chromosomal DNA of E. coli O157 KNIH317. This recombinant plasmid was named pOVT45. E. coli XL1-Blue harboring pOVT45 showed cytotoxicity in Vero cells. We sequenced the slt gene of this strain. The A-subunit gene of the slt was composed of 960 base pairs with ATG initiation codon and TAA terminationcodon. The B-subunit was composed of 270 base paris with ATG initiation codon and TGA termination codon. Nucleotide sequence comparison of the slt gene exhibited 100%, 98.4%, 93.7%, and 93.7% identity with that of shiga-like toxin type II (sltII) of E. coli bacteriophage 933W, variant slt of E. coli, slt of E. coli, and variant sltII of E. coli, respectively. From these results, it was concluded that the cloned slt gene belongs to SltII family and that the strain used in this study may be a lysogeny of E. coli bcteriphage 933W.

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Shiga-like Toxin-II-Producing Escherichia coli O157:H7 infection in gnotobiotic piglets : Protection against brain vascular lesions with SLT-II antiserum (Shiga-like Toxin II 항독소에 의한 shiga-like Toxin II-Producing Escherichia coli O157:H7 감염돼지에서의 뇌혈관 병변의 방어)

  • Chae, C.;Moxley, Rodney A
    • Korean Journal of Veterinary Research
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    • v.33 no.3
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    • pp.443-454
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    • 1993
  • Shiga-like toxin-II(SLT-II)-producing Escherichia coli 0157 : H7 strain B2387이 분비하는 SLT-II가 gnotobiotic자돈에서의 뇌혈관 병변을 일으키는 pathogenesis에 관해서 실험을 했다. 제왕절개 수술로 태어난 자돈들을 두 그룹으로 나누어서, 한 그룹에는 SLT-II 중화항체를 포함한 혈청을 구강을 통해서 수동면역을 시키고, 또다른 한 그룹에는 SLT-II 중화항체가 포함되어 있지 않은 혈청을 구강을 통해서 수동면역시켰다. 24시간후 두 그룹 모두에게 SLT-II producing Escherichia coli O157 : H7 strain B2387를 구강으로 접종했다. SLT-II 중화항체가 포함되어 있지 않은 혈청으로 수동면역시킨 그룹의 자돈들은 설사와 맹결장염, 신경증상, 뇌혈관병변을 일으키고, plasma의 prostacyclin의 level이 증가했다. 하지만 SLT-II 중화항체가 포함되어 있는 혈청으로 수동면역시킨 그룹의 자돈들은 설사와 맹결장염은 유발했지만, 신경증상과 뇌혈관병변은 관찰되지 않았고, prostacyclin의 level도 증가하지 않았다. 이런 실험결과는 SLT-II 중화항체는 뇌혈관병변은 방어하지만 맹결장염은 방어하지 못한다는 의미를 나타내며, prostacylin의 증가는 뇌혈관의 endothelium의 병변을 의미한다.

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Development of a multiplex-PCR for the rapid detection of Escherichia coli O157:H7 from raw beef (쇠고기중 Escherichia coli O157:H7 신속검출을 위한 multiplex - PCR 기법 개발)

  • Jung, Suk-chan;Jung, Byeong-yeal;Yoon, Jang-won;Cho, Yun-sang;Kim, Jong-yeom;Park, Yong-ho
    • Korean Journal of Veterinary Research
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    • v.38 no.1
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    • pp.173-181
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    • 1998
  • Esherichia coli O157 : H7의 slt I, slt II, uid A 및 eaeA 4종 유전자를 동시에 검출하기 위한 multiplex PCR 기법을 확립하고 쇠고기중 직접 E coli O157 : H7 검출시험을 실시하였다. 4 set의 primers를 이용한 multiplex PCR 기법으로 31종의 장내세균에 대한 특이성을 조사한 결과 E coli O157 : H7 에서 1,087bp (eae A), 584bp (slt II), 348bp (slt I) 또는 252bp (uid A)크기의 DNA를 동시에 특이적으로 검출할 수 있었다. E coli O157 : H7 15주는 모두 uid A 및 eae A 유전자가 동시에 검출되었고, 다른 장내세균에서는 검출되지 않았다. slt I 또는 slt II 유전자를 가지고 있는 E coli 표준균주 24종을 이용하여 multiplex PCR 기법과 Vero cell cytotoxicity assay을 비교검사한 결과 베로톡신 산생능과 PCR법의 결과는 일치하였다. mutiplex PCR 기법의 쇠고기중 검출한계는 modified EC(mEC)에서 증균없이는 E coli O157 : H7균 $10^4cells/g$ 이상에서 검출이 가능하였으나 mEC에 1차 증균후 modified TSB 증균하였을 경우에는 10cells/g이하까지도 검출이 가능하였다. 개발된 multiplex PCR 기법을 쇠고기 40종에 직접 적용한 결과 E coli O157 : H7은 검출되지 않았으나 slt I 및 slt II유전자를 가지고 있는 E coli 4종이 검출되었으며, 이들의 혈청형은 O6, O112, O115 및 O139 였다. 이 연구에서 개발된 multiplex PCR은 쇠고기중 E coli O157 : H7을 신속하고 특이적으로 검출하는데 사용할 수 있을 것으로 사료된다.

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Molecular Cloning and Expression of Shiga-Like ToxinIIGene (slt-II) from an Isolate of Healthy Korean Native Bovine Feces, Escherichia coli KSC109

  • Cha, In Ho;Kim kyoung Sook;Kim Sang Hyun;Kim Yong Hwan;Lee, Young Choon
    • Journal of Microbiology
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    • v.34 no.2
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    • pp.151-157
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    • 1996
  • By PCR amplification using the sequence of the previously cloned shiga-like toxin II DNA, a gene encoding it has been cloned from an isolate of healthy Korean native bovine feces, Escherichia coli KSC109. The nucleotide sequence s included tow open reading frames coding for 319 and 89 amino acids corresponding to A and B subunits, respectively. Comparison of the nucleotide and predicted amino acid sequences of newly cloned gene (slt-II) with those of others in the SLT-II family revealed completely identical homology with SLT-II cloned previously from bacteriophabe DNA of E. coli 933 derived from a patient with hemorrhagic colities. In addition, the sequence homology of SLT-II with SLT-II variant form bovine was more than 95% at both the nucleotide and protein levels. Overexpression of SLT-II recombinant gene by induction with IPTG using an E, coli hostvector, system was conducted and the correctly processed products with active mature form exhibited 1000-fold higher cytotoxycity for Vero cells than that form original strain.

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Predictors of Success of Selective Laser Trabeculoplasty Adjusted for Intraocular Pressure Variations (단안 선택적 레이저섬유주성형술에서 안압 변동을 보정한 성공예측인자의 분석)

  • Lee, Jun Seok;Lee, Chong Eun;Seo, Sam;Lee, Kyoo Won
    • Journal of The Korean Ophthalmological Society
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    • v.59 no.12
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    • pp.1166-1172
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    • 2018
  • Purpose: To investigate the efficacy, and identify predictors of success of selective laser trabeculoplasty (SLT) in open-angle glaucoma (OAG) patients after adjusting for intraocular pressure (IOP) changes in the untreated fellow eye. Methods: This retrospective chart review included 52 eyes of 52 OAG patients who underwent SLT in one eye and were followed-up for at least 1 year after the procedure. The IOP was measured before the treatment, at 1, 2, and 3 months posttreatment, and every 3 months thereafter. To account for the possible influence of IOP fluctuations on laser outcomes, post-laser IOP values of the treated eye of each patient were also analyzed, after adjusting for IOP changes in the untreated fellow eye. Success was defined as an IOP decrease ${\geq}20%$ of the pretreatment IOP. The success rate was determined based on Kaplan-Meier survival analysis and factors predictive of success were analyzed using the Cox proportional hazard model. Results: The mean pretreatment IOP was $23.17{\pm}6.96mmHg$. The mean IOP reduction was $5.59{\pm}4.78mmHg$ (29.7%) and the success rate was 65.4% at 1 year. The adjusted mean IOP reduction was $4.70{\pm}4.67mmHg$ (23.9%) and the adjusted success rate was 53.9%. Pretreatment IOP was associated with SLT success; the higher the pretreatment IOP, the greater the post-laser IOP reduction (p = 0.025). Age and mean deviation index did not show a significant association with SLT success (p = 0.066 and p = 0.464, respectively). Conclusions: SLT is a safe and effective alternative method of IOP reduction in OAG patients. Herein, pretreatment IOP was the only factor significantly associated with SLT success. IOP fluctuations of the untreated eye should be considered for a better understanding of the impact of treatment.

Initiating Smokeless Tobacco Use across Reproductive Stages

  • Begum, Shahina;Schensul, Jean J.;Nair, Saritha;Donta, Balaiah
    • Asian Pacific Journal of Cancer Prevention
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    • v.16 no.17
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    • pp.7547-7554
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    • 2015
  • Background: The use of smokeless tobacco (SLT) among women is increasing in India, especially among those with limited education and resources. Preventing the initiation of SLT among women is critical since it has known negative consequences for oral and reproductive health. Most research on tobacco initiation in India focuses on adolescents. This paper addresses the unrecognized issues of post marital initiation among women of reproductive age, highlighting the importance of reproductive stages in women's tobacco initiation. The objective is to examine the correlates of SLT initiation among low income women in Mumbai from pre-marriage through early marriage, first pregnancy and beyond, using case examples to illustrate initiation during each of these stages. Materials and Methods: In 2011-2012, cross-sectional community level survey data were collected from a representative sample of 409 daily SLT-using married women aged 18-40 years in a low income community in Mumbai. Information on socio-demographics, initiation by reproductive stage, types of tobacco use, childhood exposure to tobacco, learning to use, and initiation influences and reasons were collected through a researcher-administered survey. Univariate and bivariate analysis assessed factors influencing initiation of SLT use by reproductive stage. In addition 42 narratives of tobacco use were collected from a purposive sample of pregnant and non-pregnant married women addressing the same questions in detail. Narratives were transcribed, translated, and coded for key concepts including initiation of tobacco use. Results: Thirty-two percent of women initiated SLT use before marriage, 44% initiated after marriage but before pregnancy, 18.1% initiated during their first pregnancy and the remainder started after their first pregnancy. Mean age of marriage among women in this study was 16 years. Younger women (i.e. age at time of the interview of less than 30 years) were 0.47 [95% CI (0.32, 0.87)] percent less likely to initiate after marriage than women aged more than 30 years. Women who got married before 18 years of age were 2.34 [95% CI (1.40, 3.93)] times more likely to initiate after marriage than their counterparts. Childhood exposure was a predictor for initiating SLT use prior to marriage but not after. Women reporting tooth and gum pain were 1.85 times more likely to initiate after marriage than their counterparts. Husband and neighbours were the most significant influences on post-marital initiation. Narratives highlighted differences in processes of initiation pre and post marriage and during pregnancy. Conclusions: Most tobacco prevention interventions are directed to adolescents in school. This study suggests that especially for low literate or illiterate women, school based interventions are ineffective. To be effective strategies to prevent SLT initiation must reach women in urban areas at or immediately after marriage and during their first pregnancy. Messages must negate culturally rooted beliefs about the health benefits of SLT in order to prevent initiation and onset of daily use.

Characterization and Cloning of the Gene Encoding Autoregulator Receptor Protein from Streptomyces longwoodensis (Streptomyces longwoodensis로부터 Autoregulator Receptor Protein 유전자의 클로닝 및 특성)

  • Yeo Soo-Hwan;Lee Sung-Bong;Kim Hyun-Soo
    • Microbiology and Biotechnology Letters
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    • v.33 no.2
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    • pp.96-105
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    • 2005
  • For screening of autoregulator receptor gene from Streptomyces longwoodensis, PCR was performed with primers of receptor gene designed on the basis of amino acid sequences of autoregulator receptor proteins with known function. PCR products were subcloned into the BamHIsite of pUC19 and transformed into the E. coli $DH5{\alpha}$. The isolated plasmid from transformant contained the fragment of 100 bp, which was detected on $2\%$ gel after BamHI treatment. The insert, 100 bp PCR product, was confirmed as the expected internal segment of gene encoding autoregulator receptor protein by sequencing. Southern and colony hybridizations with the 100 bp fragment as a probe allowed to select a genomic clone of S. longwoodensis, pSLT harboring a 4.4 kb SphI fragment. Nucleotide sequencing analyses revealed a 651 bp open reading frame(ORF) were isolated protein showing moderate homology ($35{\sim}46\%$) with the ${\Gamma}$-butyrolactone autoregulator receptors from Streptomyces sp., and this ORF was named sltR The sltR/pET-17b plasmid was constructed to overexpress the recombinant SltR protein (rSltR) in E. coli BL21 (DE3)/pLysS, and the rSltR protein was purified to homogeneity by DEAE-Sephacel column chromatography, and DEAE-5PW chromatography (HPLC). The molecular mass of the purified rSltR protein was 55 kDa by HPLC gel-filtration chromatography and 28 kDa by SDS-PAGE, indicating that the rSltR protein is present as a dimer. A binding assay with tritium-labeled autoregulators revealed that the rSltR has clear binding activity with a A-factor type autoregulator as the most effective ligand.

Analysis of Motivating Factors for Smokeless Tobacco Use in Two Indian States

  • Danawala, Saba Ashraf;Arora, Monika;Stigler, Melissa Harrell
    • Asian Pacific Journal of Cancer Prevention
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    • v.15 no.16
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    • pp.6553-6558
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    • 2014
  • Background: This study was conducted to gauge how the demographic profile of smokeless tobacco (SLT) users from Gujarat and Andhra Pradesh, India, differs from that of smokers. It also addresses how factors associated with the initiation and continuation of smokeless tobacco vary by age, gender, and education. Materials and Methods: We analyzed 2011 cross-sectional survey data collected from 4,759 respondents (smokers/SLT users/non-users) in both states. Chi-square analysis was used to make comparisons between the demographic profiles of smokers and SLT users. Multivariable logistic regression analysis was used to obtain the odds ratios (ORs) for initiation and continuation factors regressed on socio-demographic variables (age, gender, education). Results: Initiation-women were less likely than men to report "peer pressure", "fashion statement", and "stress/coping" as relevant factors for SLT use (OR: 0.45 CI: 0.30-0.70; OR: 0.42 CI: 0.24-0.74; OR: 2.47, CI: 1.47-4.15). Older age groups had lower odds of choosing "peer pressure" than the 15-24 year olds. Respondents with 11 or more years of education were more likely to report "stress/coping" than those with no education (OR: 2.82, CI: 1.06-7.48). Continuation-women were less likely than men to choose "relaxation", and "distance from family" as important continuation factors (OR: 0.50, CI:0.32-0.80; OR: 0.20, CI: 0.06-0.65). All age groups were less likely to choose "stimulation" as a factor than the youngest group. Conclusions: Along with confirming and expanding upon previous literature, the findings of this study should encourage further SLT research in women and younger age groups (15-24 and 24-44). They also confirm the need for SLT prevention and cessation interventions in India in other community-based settings, besides schools.