Botulinum Toxin: From Poison to Possible Treatment for Spasticity in Spinal Cord Injury

Botulism has been known for about three centuries, and since its discovery, botulinum toxin has been considered one of the most powerful toxins. However, throughout the 20th century, several medical applications have been discovered, among which the treatment of spasticity stands out. Botulinum toxin is the only pharmacological treatment recommended for spasticity of strokes and cerebral palsy. Although its use as an adjuvant treatment against spasticity in spinal cord injuries is not even approved, botulinum toxin is being used against such injuries. This article describes the advances that have been made throughout history leading to the therapeutic use of botulinum toxin and, in particular, its application to the treatment of spasticity in spinal cord injury.     

Isolation,identification, and bioassay of chemicals affecting nonpreference carrot-root resistance to carrot-fly larva

Roots of the carrot cultivars Vertou L.D. (resistant) and Long Chantenay (susceptible) were subjected to detailed chemical analysis to identify extracts and compounds influencing larval host-finding (preference/non-preference) behavior and to compare concentrations of these compounds in resistant and susceptible cultivars. Vertou yielded threefold less volatile material in headspace extracts of pureed roots. Extracts of chopped root in methanol, steam, hexane, and chloroform were inactive in behavioral assays. However, ether extracts were active and their hydrocarbon and carbonyl-rich fractions contained potent attractants. The principal constituent of the carbonyl-rich fraction of each cultivar was the carotatoxin complex comprising the neurotoxin falcarinol (carotatoxin), falcarindiol, and falcarindiol monoacetate, the latter compound here reported for the first time from carrot. Falcarinol (50 and 100 g) was active in a behavioral assay, and all three ingredients of the complex were potent electrophysiological stimuli, eliciting maximum single unit responses to source concentrations of 10 ng. Furthermore, the complex was more abundant by about 1000 g/root in Long Chantenay. As this comprised extra amounts of 70, 862, and 110 g of falcarinol, falcarindiol, and falcarindiol monoacetate, respectively, the observed differences seem both behaviorally and physiologically relevant. It is generally accepted that coevolution has transformed the role of many toxins into host-location cues, but this seems a relatively rare example of a neurotoxin (falcarinol) evincing, in decreased concentrations, nonpreference host resistance. This evolved response to a toxin present in large concentrations is contrasted with that totrans-2-nonenal, which paralyses and kills the larva and is present in only trace amounts in the root.     

Cooked and brined shrimps packed in a modified atmosphere have a shelf-life of >7 months at 0 °C, but spoil in 4–6 days at 25 °C

Summary Spoilage and safety of cooked, brined and modified atmosphere packed shrimps were studied at 0, 5, 8, 15 and 25 °C. Shrimps from two sources, cold and warm waters, were brined in a sodium–chloride brine containing benzoic, citric and sorbic acids. Shelf-life was above 7 months at 0 °C but only 4–6 days at 25 °C. Apparent activation energy for the effect of temperature on shelf-life was > 100 kJ mol^-1. This pronounced effect of temperature was explained by changes in spoilage microflora at different storage temperatures. Simple and empirical mathematical models for rates of spoilage were developed for the prediction of shelf-life at different temperatures. To evaluate safety, products were challenged with Listeria monocytogenes and spores of Clostridium botulinum . Above 5 °C growth responses of L. monocytogenes followed the square root model with a T_min-value of +0.2 °C. Cl. botulinum produced toxin at the time of spoilage at 25 °C but only in shrimps with < 3% water-phase salt.     

B型肉毒神经毒素的纯化及胰蛋白酶对其作用分析

目的纯化B型肉毒神经毒素,并比较未激活和用胰蛋白酶激活的B型肉毒神经毒素的分子特性,分析在酸性和碱性条件下B型肉毒毒素复合物的完整性。方法取未激活、胰蛋白酶激活和热处理激活的B型肉毒毒素复合物,采用阴离子交换柱层析纯化神经毒素(分别为A、B和C组)。在酸性条件下溶解激活的B型肉毒毒素复合物(D组)。对各组进行纯度(特异毒性)测定和SDS-PAGE分析。结果柱层析后,神经毒素纯度均比复合物的纯度高,激活的神经毒素比未激活的高2～3倍;在非还原和还原条件下,A组的SDS-PAGE显示神经毒素、重链和轻链3条带;B组显示重链和轻链2条带;C组在非还原条件下显示重链和轻链2条带,在还原条件下只显示轻链1条带。在酸性条件下,B型肉毒毒素复合物由神经毒素与非毒性成分构成,在碱性条件下,神经毒素与非毒性成分分离。结论已成功纯化了B型肉毒神经毒素,经胰蛋白酶处理后,单链裂解为双链,比活性提高。     

Theoretical comparison of a new and the traditional method to calculate Clostridium botulinum survival during thermal inactivation

When published isothermal survival data of Clostridium botulinum spores in the range 101–121 °C were plotted in the form of logS(t) vs t relationships, where S(t) is the momentary survival ratio, they were all non‐linear. They had a noticeable upward concavity, in violation of the assumption that sporal inactivation is a process that follows first‐order reaction order kinetics. They could be described by the power law model logS(t) = ? b(T)t ^n(T), where b(T) and n(T) are temperature‐dependent coefficients of the order of 0.1–6 and about 0.4 respectively. These coefficients were used to construct simulated survival curves under different heating regimes with a recently proposed model. The model is based on the assumption that the local slope of the non‐isothermal survival curve, or the momentary inactivation rate, is determined solely by the momentary temperature and survival ratio, which in turn are functions of the population thermal history. The survival curves calculated with this model differ considerably from those produced by the standard method based on the traditional D and Z values. The shortcomings of the standard model are that these values depend on the number of points taken for the regression, and that its predicted survival ratios depend on the selected reference temperature. The differential equation which is proposed to replace it can be solved numerically using a program such as Mathematica®. Its predictions solely depend on the observed survival patterns under isothermal conditions and not on any preconceived kinetic model. Nevertheless, the method still needs verification with experimental non‐isothermal survival data, as has already been done with Listeria and Salmonella cells. © 2001 Society of Chemical Industry     

Solid-state fermentation of Lathyrus sativus seeds using Aspergillus oryzae and Rhizopus oligosporus sp T-3 to eliminate the neurotoxin β-ODAP without loss of nutritional value

The presence of a neurotoxic amino acid, 3-N-oxalyl-L-2,3-diaminopropanoic acid (β-ODAP), in the seeds of grass pea (Lathyrus sativus) is the cause of an irreversible spastic paraparesis, neurolathyrism, after over-consumption. This is a negative factor in an otherwise very tasty, nutritious, easily cultivated and hardy food crop in Asia and Africa. The reduction of β-ODAP to less than 10% of the original content has been reached by fermenting L sativas Jamalpur seeds with Aspergillus oryzae NRRL 1988 for 48 h, followed by fermentation with Rhizopus oligosporus sp T-3 for 48 h. Other nutritional qualities were also improved in the fermented seed meal: increased content of protein, higher amino acid scores for sulphur-containing and aromatic amino acids, better resistance to high temperature and to oxidation, and drastic reduction of the flatulence factors.     

PCR assay for differentiating between Group I (proteolytic) and Group II (nonproteolytic) strains of Clostridium botulinum

Groups I (proteolytic) and II (nonproteolytic) C. botulinum are genetically and physiologically distinct groups of organisms, with both groups being involved with human botulism. Due to differences in spore heat resistance and growth characteristics, the two groups possess different types of human health risks through foods, drink, and the environment. The epidemiology of human botulism due to Groups I and II C. botulinum is poorly understood, largely due to insufficient characterization of disease isolates, and warrants thorough outbreak investigation with a particular attention to discrimination between the different physiological groups of C. botulinum. In this study, a PCR assay was developed to discriminate between Group I and Group II C. botulinum. The assay is based on the fldB associated with phenylalanine metabolism in proteolytic clostridia, and employs an internal amplification control targeted to conservative regions of 16S rrn in Groups I and II C. botulinum. The assay correctly identified all 36 Group I and 24 Group II C. botulinum strains, possessing a 100% exclusivity and inclusivity. The assay provides a substantial improvement in discriminating between the Groups I and II C. botulinum, which traditionally is based on a time-consuming and error-prone culture method. Differentiation between the physiological groups of C. botulinum is an essential step in investigation of human botulism outbreaks, and should be considered as a diagnostic corner-stone in order to improve our epidemiological understanding of human botulism.     

四川省肉毒梭菌PCR基因分型方法比较研究

目的比较分析4种肉毒毒素(botulinum neurotoxins,Bo NT)基因分型方法,为四川省监测和食物中毒中肉毒梭菌(Clostridium botulinum)分型鉴定提供可靠的方法。方法使用本实验室保存的6株肉毒梭菌(包括A、B、E型)验证4种肉毒梭菌PCR基因分型鉴定方法——美国食品药品监督管理局(FDA)多重PCR分型方法、国际标准化组织(ISO)的两种多重PCR分型方法和一种实时荧光PCR分型方法,比较方法间的差异,并初步分析差异原因。结果 3种多重PCR方法均可在一个反应中同时检测A、B、E 3种型别肉毒梭菌。ISO多重PCR方法 1中A型检测虽能获得预期条带,但结果条带不清晰。其余两种多重PCR方法在分型检测肉毒梭菌时,可获得清晰的预期条带。实时荧光PCR分型方法能在多重反应体系中同时检测到不同型的肉毒梭菌,但由于荧光标记相同,要获得分型结果需要分别检测各毒素型别。结论美国FDA多重PCR方法和ISO多重PCR方法 2操作较简单易行,可在四川省肉毒梭菌监测中推荐使用。     

Production and Stability Studies of a Neurotoxin Produced by Clostridium sp. RKD

ABSTRACT: A neurotoxigenic Clostridium sp. (RKD) isolated from intestine of decaying fish produced a neurotoxin that was neutralized by botulinum antitoxin (A+B+E) when tested by mouse protection bioassay. An amplicon of expected size (approximately 700 bp) was generated with primers specific for BoNT/B. Toxin was maximally released in the culture supernatant in the late stationary phase and was dependent on media composition. Growth was optimal in trypticase peptone yeast-extract glucose (TPYG) medium in a pH range of 7.5 to 8.0 and at a temperature between 35°C to 40°C while toxin production was optimum at 37°C (3 to 4 × 10³ minimum lethal dose per milliliter) without any protease treatment. There was no correlation between growth and toxin production when cells were grown in media containing different concentrations of NaCl (0% to 5%). Toxin in the culture supernatant was more stable (50% reduction at 50°C in 90 min) than the partially purified fraction. Toxicity was destroyed gradually after increasing the number of freeze-thaw cycles and was almost completely inactivated after 5 cycles. It was completely inactivated by overnight treatment of 1 N NaOH while it retained 1.5% activity with a similar treatment with 1 N HCl.     

我国首例由丁酸梭菌引起婴儿E型肉毒中毒实验室诊断研究

目的对一起疑似婴儿肉毒中毒进行实验室诊断研究。方法对1例疑似婴儿肉毒中毒病例的粪便、食用过的食品和生活环境涂抹共计30份标本/样品进行梭菌分离鉴定及肉毒毒素检测,对分离菌株进行产毒试验。结果将患儿粪便标本培养物上清液腹腔注射小鼠后可致小鼠出现典型肉毒毒素中毒表现(竖毛、呼吸困难并出现典型的蜂腰、四肢麻痹),继而死亡,且培养物上清液经胰酶处理后毒性增强,表现为小鼠出现中毒及死亡时间较未处理组明显缩短。但培养物上清液经100℃加热处理后再次染毒动物,小鼠未出现中毒和死亡。混合型肉毒毒素诊断血清及单价抗E型肉毒毒素诊断血清可对小鼠起到保护作用。从患儿的粪便标本中分离到G~+芽胞杆菌,该菌在哥伦比亚血平板上呈不规则半透明扁平菌落,边缘根状生长,并携带E型肉毒毒素产毒基因,16S rRNA将其鉴定为丁酸梭菌,产毒试验结果显示该菌株可产E型肉毒毒素。结论该起婴儿中毒事件是由感染产E型肉毒毒素的丁酸梭菌引起。