Clostridium botulinum type C toxin

Summary The purification and crystallization of type C botulinum toxin along with its physical characteristics are described. The shape of Clostridium botulinum type C toxin molecule is globular like a pressed ball with a 7.4 nm diameter and a 4.3 urn thickness. The molecular volume is approximately 185 nl and the molecular weight is 141 000. The toxin molecule is composed of two parts, which are separable under appropriate conditions. These parts have some differences in the electrophoretic properties, amino acid distribution, immunological, and functional characteristics. The toxin molecule can be reconstituted by association of S-S bond between the two chains. The expression of the toxicity requires that the fragments of the polypeptide chain carrying the necessary information be functionally organized for the proper development of the specific tertiary structure for active conformation.     

Temporal brow lift using botulinum toxin A

The objective of this study was to determine whether brow elevation occurs as a result of paralysis of brow depressors after botulinum toxin A injection. The study's design was a prospective case series with pretreatment and posttreatment outcome evaluation with statistical analysis at a university-based division of facial plastic surgery private clinic. Twenty-two patients of a consecutive sample desiring a cosmetic enhancement underwent injection of botulinum toxin A directed to brow depressors. Injections consisted of 7 to 10 units of botulinum toxin A (Botox, Allergan, Irvine, Calif.) into selected brow depressor muscle (lateral orbicularis oculi) bilaterally. No patients withdrew for adverse effects. All patients were evaluated 2 weeks after treatment. The outcomes were measured by change in brow elevation along vertical axis extending from both midpupil and lateral canthus to the caudal row of brow hairs with eyes at neutral gaze and the head at Frankfort plane. Preintervention and postintervention brow height was measured by the primary clinical investigator. The average brow elevation from the midpupil observed after selected injection of brow depressors with botulinum toxin A was 1.02 mm (p = 0.038). The average brow elevation from the lateral canthus observed after selected injection of brow depressors with botulinum toxin A was 4.83 mm (p<0.0001). Significant temporal brow elevation occurs as the result of paralysis of brow depressors by using botulinum toxin A injection. This procedure may be considered an alternative to surgical brow elevation.     

Binding of Clostridium botulinum type B toxin to rat brain synaptosome

Abstract Purified toxin and its subunits from Clostridium botulinum type B were labeled with ¹²⁵iodine and binding of them to rat brain synaptosomes was studied. Labeled toxin and heavy chain were shown to bind to synaptosomes and there was no significant difference in the molar quantity of bound toxin and heavy chain at several concentrations of synaptosomes, whereas labeled light chain did not bind to synaptosomes. The binding of labeled heavy chain to synaptosomes was inhibited by unlabeled toxin and heavy chain to a similar degree as that of labeled toxin. The binding of labeled toxin and heavy chain to synaptosomes were inhibited by a monoclonal antibody which is specific for the heavy chain.     

Stabilization of botulinum toxin type A during lyophilization.

Botulinum toxin for medical use is diluted to very low concentrations (nanograms per milliliter); when it is preserved by lyophilization, considerable loss of activity can occur. In the present study, conditions that gave > 90% recovery of the toxicity after lyophilization of solutions containing 20 to 1,000 mouse 50% lethal doses per ml were found. Toxicity was recovered upon drying 0.1 ml of toxin solution when the pH was maintained below 7 and bovine or human serum albumins were used as stabilizers. Various other substances tested with albumin, including glucose, sucrose, trehalose, mannitol, glycine, and cellibiose, did not increase recovery on drying.     

Stabilization of botulinum toxin type A during lyophilization.

Botulinum toxin for medical use is diluted to very low concentrations (nanograms per milliliter); when it is preserved by lyophilization, considerable loss of activity can occur. In the present study, conditions that gave > 90% recovery of the toxicity after lyophilization of solutions containing 20 to 1,000 mouse 50% lethal doses per ml were found. Toxicity was recovered upon drying 0.1 ml of toxin solution when the pH was maintained below 7 and bovine or human serum albumins were used as stabilizers. Various other substances tested with albumin, including glucose, sucrose, trehalose, mannitol, glycine, and cellibiose, did not increase recovery on drying.     

苦味酸法测定A型肉毒毒素中的明胶含量

目的注射用A型肉毒毒素中加入明胶作为稳定剂,建立并验证定量检测注射用A型肉毒毒素中明胶含量的方法。方法通过苦味酸与明胶特异性作用,产生强的吸收,采用紫外-可见分光光度计用外标法来检测注射用A型肉毒毒素中的明胶含量。结果通过一定浓度苦味酸与明胶作用,用分光光度计在520 nm处测定吸光度,检测限可以达到2.5 mg/L,检测范围可以达到10~100 mg/L。结论该方法具有良好的特异性、准确度、精密度和灵敏度,对于测定注射用A型肉毒毒素中的明胶含量有较好的参考价值。     

In vitro acetylcholinesterase inhibition by type A botulinum toxin

Type A botulinum toxin was studied for its ability to inhibit the action of acetyl-cholinesterase. The chromogenic substrate, indophenyl acetate, was used for assay of enzyme activity. Inhibition of enzyme function was detected through use of both 6.6 x 10(-6) mg (20 ld(50)) and 6.6 x 10(-10) mg (2 x 10(-3)ld(50)) of type A botulinal toxin. Control assays were performed by use of both homologous antitoxin and heterologous antitoxins (types B and E). Enzyme inhibition was effectively prevented by use of homologous antitoxin only. The inhibition noted was specific and reproducible for given substrate, enzyme, and toxin concentrations.     

Thermal inactivation of type E botulinum toxin

The theoretical required cooking times for inactivation of type E Clostridium botulinum toxin (5,000 ld(50) mouse units per 0.5 ml) in haddock fillets of various sizes were calculated by graphical integration of the toxin inactivation rate and heat penetration data. The results indicated that normal cooking procedures should suffice to inactivate this amount of toxin. This conclusion was substantiated by the following additional experimental observations which revealed that the original experiments had been conducted under conservative conditions. First, maximal heat stability of the toxin was found to occur at about pH 5.5, with decreasing resistance upon increasing pH. The theoretical cooking times were based on destruction of the toxin at pH 6.7. The pH of radio-pasteurized inoculated haddock, when toxin production had occurred, was on the alkaline side, at which condition the toxin is heat-labile. Second, when spoilage was discernible in radio-pasteurized inoculated haddock, the toxin titer was low, about 50 ld(50) mouse units per 0.5 ml. Third, the toxin was adequately inactivated in toxic fillets after deep-fat frying for 3 min at 375 F (190.6 C) or after pan frying for 5 min per side at 400 F (204.4 C). Fourth, in this study, residual toxin activity was assayed by intraperitoneal injection of mice. It was shown that the oral toxic dose was 50 to 100 times greater than the intraperitoneal toxic dose.     

A型肉毒毒素ELISA鉴别试验方法的建立

目的建立鉴定A型肉毒毒素的ELISA鉴别试验方法以替代传统的动物试验法。方法采用现代免疫学技术,制备马源性和兔源性抗A型肉毒毒素多克隆抗体,建立了双抗体夹心ELISA,并就此初步进行方法学验证。结果所建立的ELISA具有良好的特异性、灵敏度、精密度和耐用性,具有替代动物试验方法的良好前景。结论在进一步验证和确认之后,该方法有望正式成为可用于鉴定A型肉毒毒素的试验方法以替代动物试验法。     

注射用A型肉毒毒素的稳定性验证

目的验证注射用A型肉毒毒素(商品名:衡力)成品的稳定性。方法依据《中华人民共和国药典》2010版(三部)(简称《中国药典》)各论中注射用A型肉毒毒素成品检定标准,对注射用A型肉毒毒素成品进行稳定性长期试验(2~8℃)、加速试验(25±2)℃、高温试验(35±2)℃;同时监测在低温(-5~-20℃)冻存条件下药品到有效期终点时的质量稳定性数据及复溶后的质量稳定性数据;采用趋势分析和批内批间变异系数分析药品的稳定性。结果该药品在3年有效期内(2~8℃)保存,各项质量指标的检测结果均符合要求,批内、批间一致性亦符合要求;在室温保存条件(25±2)℃,6个月和高温(35±2)℃,15 d情况下,药品的各项质量指标同样符合《中国药典》的标准。药品在低温冻存(-5~-20℃)条件下至有效期终点,其关键质量指标均符合标准。药品复溶后在2~8℃保存6 d,其效价持续稳定,无菌检查合格。结论注射用A型肉毒毒素在≤8℃条件下保存的有效期为3年,在室温连续保存期限为6个月,高温条件连续保存时限为15 d。药品复溶后在2~8℃无菌条件可保存6 d。