• Journal of Microbiology
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• 제41권2호
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• pp.161-164
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• 2003

Recombinant light chain of Clostridium botulinum neurotoxin type B stimulated transglutaminase activity in a dose dependent manner, Compared to native toxin, recombinant light chain showed av greater stimulatory effect on transglutaminase activity. Zn-chelating agents, inhibiting the proteolytic activity of the clostridial toxins, did not interfere with this stimulation. These results suggest that the light chain plays a major stimulatory role, which is not due to its metallopeptidase activity, but is possibly due to specific interaction with transglutaminase. More importantly, this report provides a new insight into the intracellular action of C. botulinum neurotoxins.

• Toxicological Research
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• 제13권4호
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• pp.417-421
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• 1997

Synaptobrevin is a kind of vesicle associated membrane proteins (VAMPs) which plays a secretary role in the neuronal synapse and was recently known as the biochemical target of botulinum neurotoxin type B. The structural gene of the synaptobrevin was cloned from mouse brain using RT-PCR technique and was seqrtenced. The deduced amino acid sequence showed that the synaptobrevin protein from mouse brain is exactly the same with that of the rat brain in the amino acid level. The synaptobrevin gene was subcloned into pET3a vector and expressed in E. coli. The molecular weight of the recombinant protein was 19 kDa as expected. Moreover, when the recombinant synaptobrevin protein was incubated with the native neurotoxin of Clostridium botulinum type B, it was cleaved by the toxin in a time dependent manner. This implies that the recombinant synaptobrevin protein and the native toxin are reacted in the same way as the native synaptobrevin did in the neuronal cells.

• Molecules and Cells
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• 제22권3호
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• pp.336-342
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• 2006

Real time reverse transcription (RT)-PCR was used to quantify the expression of the botulinum neurotoxin type A (BoNT/A) gene (cntA) by normalization with the expression of 16S rRNA. The method were confirmed by monitoring the mRNA levels of cntA during growth in five type A strains. In all but one of the strains the expression of cntA mRNA was maximal in the late exponential phase, and approximately 35-fold greater than in the early exponential phase. The concentration of the extracellular BoNT/A complex detected by ELISA was highest in stationary phase. Sodium nitrite and sorbic acid completely inhibited growth at 20 ppm and $4mg\;ml^{-1}$, respectively. CntA expression became lower in proportion to the concentration of sorbic acid, and this reduction was confirmed by mouse bioassay. Our results show that real time RT-PCR can be used to quantify levels of C. botulinum type A neurotoxin transcripts and to assess the effects of food additives on botulinal risk.

• Journal of Microbiology and Biotechnology
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• 제13권2호
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• pp.287-291
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• 2003

Botulinum neurotoxin type A (BONT/A) is an extremely potent toxin, which is produced by Clostridium botulinum. The light chain of this protein (BONT/A LC), which is known as a zinc endopeptidase, cleaves SNAP-25 involved in the exocytosis process. In this work, the expression of recombinant BoNT/A LC in E. coli is described. The BONT/A LC gene of C. botulinum contains a high frequency of the arginine AGA and isoleucine ATA codons that are rarely used in genes of E. coli, hampering the translation of recombinant protein. The argD and ilex tRNA genes were cloned into pACYC184 vector, resulting in pAAD131X plasmid. The translational stress of the toxin gene related to codon bias was reversed by fupplernentation of the AGA arginyl tRNA of T4 phage and AUA isoleucyl tRNA of E. coli. This system may be applicable for the expression of a variety of AT-rich heterologous genes in E. coli.

• 한국산학기술학회논문지
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• 제18권1호
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• pp.295-301
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• 2017

보툴리눔 신경독소는 콜린성 신경말단(부)을 선택적으로 공격하여 신경마비를 일으키는 신경독소로서, 그람양성을 띠고 내성포자를 형성하는 절대혐기성 세균인 보툴리눔 균(Clostridium botulinum)이 만들어낸다. 이 중 보툴리눔 A형 독소(BoNT/A)는 음식물과 물을 오염시킬 수 있으며 생물 무기나 생물 협박물질로 사용될 수도 있다. 이 때문에 독성을 탐지할 수 있는 예민한 분석방법과 중독을 치료할 수 있는 효능 있는 항독소를 개발해낼 필요성이 제기되어 왔다. 본 연구에서는 BoNT/A를 중화할 수 있는 단일클론 항체(mAb)를 생산하기 위하여 BoNT/A로 면역된 토끼의 항혈청에서 유래한 scFv 라이브러리를 인간 IgG와 융합시켰다. 그렇게 재조합된 scFvIgG 항체 단백질을 안정된 세포주에서 발현시켰고 항체 친화 크로마토그래피를 사용하여 scFvIgG mAb 단백질을 정제하였다. ELISA로 정제된 scFvIgG mAb 단백질의 효율성을 확인하였고, in vivo 실험으로 BoNT/A에 대한 중화능을 시험하였다. 독성 중화능 실험은 마우스를 사용하여 수행하였는데, 그 결과 scFvIgG 항체(10 ug)는 BoNT/A(100,000 $LD_{50}$)의 독성이 주입된 마우스를 완전히 방어하지는 못하지만 마우스의 생존 기간을 현격하게 연장시키는 것이 확인되었다. 이러한 결과들은 이 scFvIgG mAb가 BoNT/A를 중화하는 효능을 가지고 있다는 점을 제시한다.

• Food Science and Biotechnology
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• 제15권4호
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• pp.499-505
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• 2006

Clostridium botulinum spores are widely distributed in nature. Type A and proteolytic type B bacteria produce heat-resistant spores that are primarily involved in most of the food-borne botulism outbreaks associated with low-acid canned foods. Food-borne botulism results from the consumption of food in which C. botulinum has grown and produced neurotoxin. Growth and toxin production of type A and proteolytic type B in canned foods can be prevented by the use of thermal sterilization alone or in combination with salt and nitrite. The hazardousness of C. botulinum in low-acid canned foods can also be reduced by preventing post-process contamination and introducing hazard analysis and critical control point (HACCP) practices during production. Effectiveness of non-thermal technologies such as high pressure processing with elevated process temperatures on inactivation of spores of C. botulinum will be discussed.

• 대한치과의사협회지
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• 제41권12호통권415호
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• pp.826-830
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• 2003

botulinum toxin type A는 anaerobic bacterium clostridium botulinum에서 유래된 poly peptide neurotoxin으로서 사시, 안면연축, 수부다한증등에 치료목적으로 안정적으로 사용되어오다 최근에는 이마주름, 눈가주름등 주름살의 개선 목적으로 널리 사용되어 오고있다. 특히 턱얼굴외과 영역에서는 이러한 주름살의 개선이외에도 사각턱, 안면 신경의 이상으로 인한 안면 비대칭등 적용범위가 상당히 넓고 치료효과도 만족할 만하다고 하겠다. 이에 턱얼굴외과의사 나아가 치과의사들의 많은 사용을 기대하여 botulinum toxin 사용의 역사적 배경, 약리 및 작용기전, 가능한 합병증, 턱얼굴영역에서의 적용가능성 등에 대해 알아보고자 한다.

• 대한음성언어의학회:학술대회논문집
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• 대한음성언어의학회 2002년도 제16회 학술대회
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• pp.75-82
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• 2002

Botulinum toxin-A, a neurotoxin derived from Clostridia Botulinum, has been injected into the laryngeal muscle(s) for the treatment of the spasmodic dysphonia at the Voice Clinic, Yonsei Institute of Logopedics and phoniatrics since December 1995. We analyzed 355 patients with spasmodic dysphonia, using Botox register review. In the 355 patients, female is 86.8%. male is 13.2%. 305 patients (85.9%) had adductor type of spasmodic dysphonia and 35 patients (9.9%) were vocal tremor type and 15 patients were abduction and mixed type. Botulinum toxin type-A (Botox) injection using EMG was most frequently conducted as 587 cases, comparing with flexible nasopharyngoscopy gudied injection (68cases) and tele- laryn-goscopy guided injection (31cases). In the respect of frequency of Botox injection, 137 patients(38.6%) were injected one time but 1 patient was injected 17times. The mean dose of Botox is 6.2U. Clinically, initial dose of Botulinum toxin-A was high dose (7-8U) but current dose is small dose (3U). And the mean duration of Botox injection is 6.4 month. In conclusion, to optimize effect of the treatment for spasmodic dysphonia, Botulinum toxin-A injection is combined with voice therapy.

• 음성과학
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• 제3권
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• pp.5-17
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• 1998

Botulinum toxin, a neurotoxin derived from Clostridia Botulinum, has been injected into the target muscle(s) for the treatment of several kinds of voice and speech disorders at the Voice Clinic, Yonsei Institute of Logopedics and Phoniatrics since December 1995. Criteria for the diagnosis and method of injection for spasmodic dysphonia, mutational dysphonia, muscle tension dysphonia, dysphonia after total laryngectomy, and stuttering were summarized. Among 144 patients with adductor type spasmodic dysphonia, who were injected one time to maximum 8 times during the 27 months, 90% were recognized as having better than slight improvement. Even though the injected cases were small, not only the abductor type spasmodic dysphonia, but also the intractable mutational dysphonia or muscle tension dysphonia resistant to voice therapy revealed that botulinum toxin injection would be another options for treatment. Patients who cannot phonate after total laryngectomy and some forms of adulthood stutterers can also be candidates for the injection of botulinum toxin.

• 미생물학회지
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• 제20권4호
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• pp.183-188
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• 1982

Clostidium botulinum type B가 생성하는 독소를 정제할 수 있는 방법을 연구하였다. 정제과정은 독소를 ammonium sulfate로 배양액에서 침전시켜 추출한후 Polymin P를 처리하여 핵산 및 기타 단백질을 최대한 제거한 후 Sephaex G-I00에서 gel fiItration을 시키고 DEAE-Sephadex로 이온교환 크로마토그래피를 시켰다. 이러한 과정으로 정제된 독소의 회수율은 17%였으며 SDS-polyacrylamide gel electrophoresis 결과 하나의 선을 나타내 동질성을 증명하였다. 정제된 독소의 분자량은 163,000이였으며 $\beta$-mercaptoethanol을 사용하여 환원시킨 결과 분자량 106,000과 56,000의 하위 단위체로 분리되었다.