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41.
Conjugated fluorescent polymer sensor for proteolytic activity detection with designed specificity 下载免费PDF全文
A portable fluorescence assay for direct endopeptidase activity detection has been developed with the use of a cyclophane‐based conductive conjugated fluorescent polymer and peptide substrates. The substrates, carrying internal quenching amino acid, were designed to be cleaved in a sequence‐specific manner by a protease of interest. Intact substrates were incapable of quenching the fluorescence of the polymer due to steric constraints. Upon specific cleavage, the quencher became exposed and could interact with the ring structure of the fluorescent polymer, disrupting the conjugation and quenching the fluorescence along the polymer chain. The approach was developed using a model Glu‐C endopeptidase from Staphylococcus aureus strain V8, detected in the picomolar to micromolar concentration range. The developed assay was tested for the detection of endopeptidase activity of botulinum toxin. The feasibility study showed that botulinum neurotoxin (BoNT)‐A could be detected down to picomolar concentration, with limit of detection of 5 pg, or 33 amol in 5 µL of sample, and a total assay time under 2 h. The assay exhibited high specificity and no cross‐reactivity with BoNT‐B was detected. Following this proof‐of‐concept work, the assay can be further optimized and expanded to differentiate between various botulinum toxin serotypes in their active proteolytic form, or modified for detection of proteases with other specificities. © 2015 Society of Chemical Industry 相似文献
42.
Carlin F Girardin H Peck MW Stringer SC Barker GC Martinez A Fernandez A Fernandez P Waites WM Movahedi S van Leusden F Nauta M Moezelaar R Torre MD Litman S 《International journal of food microbiology》2000,60(2-3):117-135
Vegetables are frequent ingredients of cooked chilled foods and are frequently contaminated with spore-forming bacteria (SFB). Therefore, risk assessment studies have been carried out, including the following: hazard identification and characterisation — from an extensive literature review and expertise of the participants, B. cereus and C. botulinum were identified as the main hazards; exposure assessment — consisting of determination of the prevalence of hazardous SFB in cooked chilled foods containing vegetables and in unprocessed vegetables, and identification of SFB representative of the bacterial community in cooked chilled foods containing vegetables, determination of heat-resistance parameters and factors affecting heat resistance of SFB, determination of the growth kinetics of SFB in vegetable substrate and of the influence of controlling factors, validation of previous work in complex food systems and by challenge testing and information about process and storage conditions of cooked chilled foods containing vegetables. The paper illustrates some original results obtained in the course of the project. The results and information collected from scientific literature or from the expertise of the participants are integrated into the microbial risk assessment, using both a Bayesian belief network approach and a process risk model approach, previously applied to other foodborne hazards. 相似文献
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R.I Evans P.J McClure G.W Gould N.J Russell 《International journal of food microbiology》1998,40(3):8217-167
A non-proteolytic strain of Clostridium botulinum (NCIB 4270) was found to have a complex lipid composition, comprising five major phosphorus-containing lipids: phosphatidylethanolamine (PE), phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), phosphatidylserine (PS) and a glycophospholipid of unknown structure (GPL), in order of abundance. Changing the growth temperature did not alter the lipid composition either qualitatively or quantitatively. The main fatty acyl components of the lipids are 14:0, 16:0 and 16:1. When the growth temperature was lowered from 37 to 8°C, there was an increase in 14:0 from 16.4 to 37.5%, an increase in 16:1 from 10.5 to 22.5%, and a decrease in the proportion of 16:0 from 40.3 to 19.1%. There was also a decrease in the proportion of cyclopropane fatty acids (15:0cyc and 17:0cyc) from 7.3 to 0.5%, and in the equivalent chain length of the total fatty acids from 15.9 to 15.3 as the temperature was lowered. The same temperature-dependent changes occurred in the five major lipid classes examined. Despite reports of the presence of plasmalogenic forms of phospholipids (i.e. those lipids which have the acyl chain in the sn-1 position replaced by an alk-1-enyl group) in some Clostridium spp., none were detected in C. botulinum NCIB 4270 using either commercially available spray reagents or by gas-liquid chromatographic analysis of the products or acid methanolysis of total lipid extracts. It is concluded that non-proteolytic C. botulinum lacks plasmalogens, typical of other clostridia, in its membranes and instead modulates its fatty acid composition in response to temperature changes in a manner that is typical of other (non-clostridial) bacteria. 相似文献
45.
为了研究添加小米饭、糯米饭、籼米饭、再接种籼米饭以及加入葡萄糖酸内酯对传统酸肉制品中肉毒梭状芽孢杆菌、生物胺含量及产品品质的影响,对预腌和发酵期间产品的水分含量、水分活性、pH及菌相的变化进行了分析。结果表明,传统酸肉制品的水分含量介于65.21%~70.44%之间,添加不同的米饭并未促进初期pH的下降;各组均未检出肉毒梭状芽孢杆菌,且大肠菌群经发酵2d后便低于4个对数单位,经发酵5d后低于1个对数单位;除了微球菌科和真菌类增殖较慢外,耐盐性细菌、乳酸菌和乳酸菌群等微生物均生长良好,发酵5d后菌数均维持在7~8个对数单位。添加GDL组在发酵初期有降低pH和水分含量,且能抑制微球菌生长的效果,添加GDL组挥发性盐基氮和生物胺含量较低,能提高传统酸肉的贮藏品质。 相似文献
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47.
Geoffrey Masuyer Jonathan R. Davies Pl Stenmark 《International journal of molecular sciences》2021,22(15)
The botulinum neurotoxins are potent molecules that are not only responsible for the lethal paralytic disease botulism, but have also been harnessed for therapeutic uses in the treatment of an increasing number of chronic neurological and neuromuscular disorders, in addition to cosmetic applications. The toxins act at the cholinergic nerve terminals thanks to an efficient and specific mechanism of cell recognition which is based on a dual receptor system that involves gangliosides and protein receptors. Binding to surface-anchored gangliosides is the first essential step in this process. Here, we determined the X-ray crystal structure of the binding domain of BoNT/E, a toxin of clinical interest, in complex with its GD1a oligosaccharide receptor. Beyond confirmation of the conserved ganglioside binding site, we identified key interacting residues that are unique to BoNT/E and a significant rearrangement of loop 1228–1237 upon carbohydrate binding. These observations were also supported by thermodynamic measurements of the binding reaction and assessment of ganglioside selectivity by immobilised-receptor binding assays. These results provide a structural basis to understand the specificity of BoNT/E for complex gangliosides. 相似文献
48.
Alon Ben David Ada Barnea Eran Diamant Eyal Dor Arieh Schwartz Amram Torgeman Ran Zichel 《International journal of molecular sciences》2021,22(16)
Botulinum neurotoxins (BoNTs) are the most poisonous substances in nature. Currently, the only therapy for botulism is antitoxin. This therapy suffers from several limitations and hence new therapeutic strategies are desired. One of the limitations in discovering BoNT inhibitors is the absence of an in vitro assay that correlates with toxin neutralization in vivo. In this work, a high-throughput screening assay for receptor-binding inhibitors against BoNT/A was developed. The assay is composed of two chimeric proteins: a receptor-simulating protein, consisting of the fourth luminal loop of synaptic vesicle protein 2C fused to glutathione-S-transferase, and a toxin-simulating protein, consisting of the receptor-binding domain of BoNT/A fused to beta-galactosidase. The assay was applied to screen the LOPAC1280 compound library. Seven selected compounds were evaluated in mice exposed to a lethal dose of BoNT/A. The compound aurintricarboxylic acid (ATA) conferred 92% protection, whereas significant delayed time to death (p < 0.005) was observed for three additional compounds. Remarkably, ATA was also fully protective in mice challenged with a lethal dose of BoNT/E, which also uses the SV2 receptor. This study demonstrates that receptor-binding inhibitors have the potential to serve as next generation therapeutics for botulism, and therefore the assay developed may facilitate discovery of new anti-BoNT countermeasures. 相似文献
49.
ABSTRACT: Botulinum toxin (BoNT) is a potent neurotoxin produced by toxigenic strains of Clostridium botulinum . Botulinum toxin poses a major threat since it could be employed in a deliberate attack on the U.S. food supply. Furthermore, BoNT may be liberated in any insufficiently processed food containing a reduced oxygen atmosphere. Hence, rapid and reliable detection of BoNT in foods is necessary to reduce risks posed through food contamination. We present a BoNT biosensor employing living neural cultures grown in vitro on microelectrode arrays (MEAs). An MEA is a culture dish with a grid of electrodes embedded in its surface, enabling extracellular recording of action potentials of neural cultures grown over the array. Pharmaceutical grade BoNT A was applied to the media bath of mature cortical networks cultured on MEAs. Both spontaneous and evoked activities were monitored over 1 wk to quantify changes in the neural population produced by BoNT A. Introduction of BoNT A resulted in an increased duration and number of spikes in spontaneous and evoked bursts relative to control cultures. Increases were significant within 48 h of BoNT A dosage ( P < 0.05). Application of BoNT A also induced unique oscillatory behavior within each burst that is reminiscent of early developmental activity patterns rather than the mature cultures used here. Three or more activity peaks were observed in 50% of the BoNT dosed cultures. Control cultures exhibited only a single activity peak. Thus activity of these cortical networks measured with MEAs could provide a valuable substrate for BoNT detection. 相似文献
50.
A synthetic hexapeptide (Argireline) with antiwrinkle activity 总被引:2,自引:1,他引:1
Blanes-Mira C Clemente J Jodas G Gil A Fernández-Ballester G Ponsati B Gutierrez L Pérez-Payá E Ferrer-Montiel A 《International journal of cosmetic science》2002,24(5):303-310
Botulinum neurotoxins (BoNTs) represent a revolution in cosmetic science because of their remarkable and long-lasting antiwrinkle activity. However, their high neurotoxicity seriously limits their use. Thus, there is a need to design and validate non-toxic molecules that mimic the action of BoNTs. The hexapeptide Ac-EEMQRR-NH(2) (coined Argireline) was identified as a result of a rational design programme. Noteworthy, skin topography analysis of an oil/water (O/W) emulsion containing 10% of the hexapeptide on healthy women volunteers reduced wrinkle depth up to 30% upon 30 days treatment. Analysis of the mechanism of action showed that Argireline significantly inhibited neurotransmitter release with a potency similar to that of BoNT A, although as expected, it displayed much lower efficacy than the neurotoxin. Inhibition of neurotransmitter release was due to the interference of the hexapeptide with the formation and/or stability of the protein complex that is required to drive Ca(2+)-dependent exocytosis, namely the vesicular fusion (known as SNARE) complex. Notably, this peptide did not exhibit in vivo oral toxicity nor primary irritation at high doses. Taken together, these findings demonstrate that Argireline is a non-toxic, antiwrinkle peptide that emulates the action of currently used BoNTs. Therefore, this hexapetide represents a biosafe alternative to BoNTs in cosmetics. 相似文献