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Find video protocols related to scientific articles indexed in Pubmed.
Role of Neurotoxin Associated Proteins in the Low pH Induced Structural Changes in the Botulinum Neurotoxin Complex.
Protein J.
PUBLISHED: 11-20-2014
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Botulinum Neurotoxin (BoNT) produced by the bacterium Clostridium botulinum as a complex with NAPs causes botulism. It has been known that the NAPs protect the toxin from both extremes of pHs and proteases of the GI tract. In an attempt to emulate the physiological conditions encountered by the toxin, we examined BoNT/A, BoNT/A complex, and NAPs under different pH conditions and monitored their structural characteristics by far-UV CD and thermal denaturation analysis. BoNT/A complex showed the maximum CD signal with a mean residue weight ellipticity of -1.8 × 10(5)° cm(2)/dmol at 222 nm at both acidic and neutral pHs. Thermal denaturation analysis revealed NAPs to be the most stable amongst the three protein samples examined. Interestingly and quite uniquely, at pH 2.5, there was an increase in CD signal for BoNT complex as a function of temperature, which correlated with the NAPs profile, indicating a shielding effect of NAPs on BoNT complex at low pH. Calculation of the weighted mean of the ellipticities at the Tm for thermal unfolding of toxin and NAPs at neutral and acidic pHs showed variation with that of BoNT complex, suggesting structural reorganization in BoNT complex upon the association of NAPs and BoNT. In conclusion, this study reveals the structural behavior of BoNT complex and NAPs with pH changes substantially, which could be quite relevant for BoNT survival under extreme pH conditions in vivo.
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Current strategies for designing antidotes against botulinum neurotoxins.
Expert Opin Drug Discov
PUBLISHED: 02-13-2014
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Botulinum neurotoxins (BoNTs) are proteins responsible for the deadly paralytic disease botulism. Extreme toxicity, ease of production and lack of antidotes against BoNT makes it a category A biothreat agent, according to the United States Center of Disease Control and Prevention. The only available therapy for BoNT is an equine antitoxin antibody or/and a protracted respiratory support system. Even then, antibody treatment can only prevent further exposure of the toxin and cannot rescue already intoxicated neurons.
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Type A botulinum neurotoxin complex proteins differentially modulate host response of neuronal cells.
Toxicon
PUBLISHED: 01-24-2014
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Type A Botulinum neurotoxin (BoNT/A), the most potent poison known to mankind, is produced by Clostridium botulinum type A as a complex with neurotoxin-associated proteins (NAPs). Currently BoNT/A in purified and complex forms are both available in therapeutic and cosmetic applications to treat neuromuscular disorders. Whereas Xeomin(®) (incobotulinumtoxin A, Merz Pharmaceuticals, Germany) is free from complexing proteins, Botox(®) (onabotulinumtoxin A, Allergan, USA) contains NAPs, which by themselves have no known role in the intracellular biochemical process involved in the blockade of neurotransmitter release. Since the fate and possible interactions of NAPs with patient tissues after intramuscular injection are not known, it was the aim of this study to evaluate the binding of BoNT/A and/or the respective NAPs to cells derived from neuronal and non-neuronal human tissues, and to further explore neuronal cell responses to different components of BoNT/A. BoNT/A alone, the complete BoNT/A complex, and the NAPs alone, all bind to neuronal SH-SY5Y cells. The BoNT/A complex and NAPs additionally bind to RMS13 skeletal muscle cells, TIB-152 lymphoblasts, Detroit 551 fibroblasts besides the SH-SY5Y cells. However, no binding to these non-neuronal cells was observed with pure BoNT/A. Although BoNT/A, both in its purified and complex forms, bind to SH-SY5Y, the intracellular responses of the SH-SY5Y cells to these BoNT/A components are not clearly understood. Examination of inflammatory cytokine released from SH-SY5Y cells revealed that BoNT/A did not increase the release of inflammatory cytokines, whereas exposure to NAPs significantly increased release of IL-6, and MCP-1, and exposure to BoNT/A complex significantly increased release of IL-6, MCP-1, IL-8, TNF-?, and RANTES vs. control, suggesting that different components of BoNT/A complex induce significantly differential host response in human neuronal cells. Results suggest that host response to different compositions of BoNT/A based therapeutics may play important role in local and systemic symptoms in patients.
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RNA aptasensor for rapid detection of natively folded type A botulinum neurotoxin.
Talanta
PUBLISHED: 06-14-2013
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A surface plasmon resonance based RNA aptasensor for rapid detection of natively folded type A botulinum neurotoxin is reported. Using detoxified recombinant type A botulinum neurotoxin as the surrogate, the aptasensor detects active toxin within 90 min. The detection limit of the aptasensor in phosphate buffered saline, carrot juice, and fat free milk is 5.8 ng/ml, 20.3 ng/ml and 23.4 ng/ml, respectively, while that in 5-fold diluted human serum is 22.5 ng/ml. Recovery of toxin from disparate sample matrices are within 91-116%. Most significant is the ability of this aptasensor to effectively differentiate the natively folded toxin from denatured, inactive toxin, which is important for homeland security surveillance and threat assessment. The aptasensor is stable for more than 30 days and over 400 injections/regeneration cycles. Such an aptasensor holds great promise for rapid detection of active botulinum neurotoxin for field surveillance due to its robustness, stability and reusability.
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Botulinum neurotoxin: unique folding of enzyme domain of the most-poisonous poison.
J. Biomol. Struct. Dyn.
PUBLISHED: 06-08-2013
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Botulinum neurotoxin (BoNT), the most toxic substance known to mankind, is the first example of the fully active molten globule state. To understand its folding mechanism, we performed urea denaturation experiments and theoretical modeling using BoNT serotype A (BoNT/A). We found that the extent of BoNT/A denaturation from the native state (N) shows a nonmonotonic dependence on urea concentration indicating a unique multistep denaturation process, N ? I 1 [Formula: see text] I 2 [Formula: see text] U, with two intermediate states I 1 and I 2. BoNT/A loses almost all its secondary structure in 3.75?M urea (I 1), yet it displays a native-like secondary structure in 5?M urea (I 2). This agrees with the results of theoretical modeling, which helped to determine the molecular basis of unique behavior of BoNT/A in solution. Except for I 2, all the states revert back to full enzymatic activity for SNAP-25 including the unfolded state U stable in 7?M urea. Our results stress the importance of structural flexibility in the toxins mechanism of survival and action, an unmatched evolutionary trait from billion-year-old bacteria, which also correlates with the long-lasting enzymatic activity of BoNT inside neuronal cells. BoNT/A provides a rich model to explore protein folding in relation to functional activity.
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Effects of enzymatically inactive recombinant botulinum neurotoxin type A at the mouse neuromuscular junctions.
Toxicon
PUBLISHED: 04-15-2013
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Botulinum neurotoxin A (BoNT/A) is used clinically to treat several neurological and metabolic diseases. However, the mechanisms that underlie the clinical use of the toxin remain still to be elusive. BoNT/A inhibits acetylcholine (ACh) release at the motor nerve terminals (MNT) and causes neuroparalysis. The toxic effects of BoNT/A at the MNT occur in sub-pico molar range, and it is invaluable to determine the half-life and the persistence of catalytic activity of the toxin to develop therapeutics against BoNT/A intoxication. However, the use of extremely low concentrations of BoNT/A in cellular, or animal models due to high toxicity makes it difficult to determine new cellular mechanisms and binding or interacting partners of BoNT/A. In order to address this, a catalytically deactivated, non-toxic version of BoNT/A, designated as DrBoNT/A, was characterized. DrBoNT/A lacks endoprotease activity (SNAP-25 cleavage) at concentrations as high as 46,875-fold, compared to wild-type BoNT/A. Unlike BoNT/A injection (3.2 pg), injection of the recombinant product (150 ng or 3.2 pg) into mouse hind limbs failed to cause neuroparalysis as exhibited by the lack of inhibition of toe spread reflex (ability of the mouse to spread its hindlimb toes), and inhibit ACh release at the MNT. The in vitro experiments also demonstrate that DrBoNT/A uptake (at concentrations equivalent to BoNT/A), internalization and localization at the MNT remained unaltered. In addition, modeling studies support that DrBoNT/A lacked the zinc binding ability, and the ability to directly participate in the hydrolysis of SNAP-25 substrate. Collectively, we demonstrate that DrBoNT/A is non-toxic to the MNT and can be used as a surrogate tool to understand the mechanism by which BoNT/A modulates signal transduction mechanisms.
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Comparative immunochemical characteristics of botulinum neurotoxin type A and its associated proteins.
Toxicon
PUBLISHED: 03-04-2013
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Clostridium botulinum strains secrete their neurotoxins (BoNT) along with a group of neurotoxin-associated proteins (NAPs) that enhance the oral toxicity and provide protection to the neurotoxin against acidity, temperature and proteases in the G.I. tract. A major component of NAPs is Hn-33, a 33 kDa protein, which is also protease resistant and strongly protects BoNT. The complex form of BoNT/A is used as a commercial therapeutic formulation against many neuromuscular disorders and for cosmetic purposes. Immune response against this formulation could hinder its long-term use; therefore, it is important to characterize the immunological properties of the associated proteins. This study aims to understand the immunological reactivity of BoNT/A complex, BoNT, NAPs, and Hn-33 through a series of competitive enzyme-linked immunosorbent assays (ELISA). The results indicated that BoNT/A complex competed 6 times more with complex antibodies compared to the neurotoxin confirming that the higher immunogenicity of BoNT/A complex was indeed a result of the associated proteins with the neurotoxin complex. While the nearly identical immuno-reactivity of BoNT/A complex and Hn-33 with Hn-33 antibodies indicated that the reactivity was due to the higher immunogenicity not the abundance of Hn-33 in the complex. Both the ELISA and immuno-blot results implied that Hn-33 is primarily responsible for eliciting the antibody response in BoNT/A complex.
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Mastoparan-7 rescues botulinum toxin-A poisoned neurons in a mouse spinal cord cell culture model.
Toxicon
PUBLISHED: 02-21-2013
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Botulinum neurotoxin serotype A (BoNT/A) is the most potent poison of biological origin known to mankind. The toxicity of BoNT/A is due to the inhibition of neurotransmission at cholinergic synapses; this is responsible for the symptom of flaccid paralysis at peripheral neuromuscular junctions. At a molecular level, the BoNT/A effect is due to its inhibition of stimulated acetylcholine (ACh) release from presynaptic nerve terminals. Currently, there is no antidote available to rescue BoNT/A-poisoned synapses. Here, we report an example of rescuing botulinum-poisoned cultured mouse spinal cord neurons by treatment with Mastoparan-7 (Mas-7), which is known to be a phospholipase A2 activator compound. Mas-7, a naturally occurring bee venom peptide, was delivered to botulinum-poisoned neurons via a drug delivery vehicle (DDV) construct prepared using the recombinant non-toxic heavy chain (HC) fragment of BoNT/A itself. In this method, the BoNT/A HC component in the DDV served as a neuron specific drug targeting molecule. We found that Mas-7 delivered into BoNT/A intoxicated spinal cord cells restored over 40% their property of stimulated neurotransmitter release. Rescue from cell poisoning did not occur from inhibition of the endopeptidase activity of BoNT/A light chain (LC) against its well-known substrate, SNAP-25 that is mechanistically involved in the cholinergic neuroexocytosis process. Rather, Mas-7 induced a physiological host response apparently unrelated to SNAP-25, but linked to the phospholipase-mediated signal transduction pathway.
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Molecular composition and extinction coefficient of native botulinum neurotoxin complex produced by Clostridium botulinum hall A strain.
Protein J.
PUBLISHED: 01-22-2013
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Seven distinct strains of Clostridium botulinum (type A to G) each produce a stable complex of botulinum neurotoxin (BoNT) along with neurotoxin-associated proteins (NAPs). Type A botulinum neurotoxin (BoNT/A) is produced with a group of NAPs and is commercially available for the treatment of numerous neuromuscular disorders and cosmetic purposes. Previous studies have indicated that BoNT/A complex composition is specific to the strain, the method of growth and the method of purification; consequently, any variation in composition of NAPs could have significant implications to the effectiveness of BoNT based therapeutics. In this study, a standard analytical technique using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and densitometry analysis was developed to accurately analyze BoNT/A complex from C. botulinum type A Hall strain. Using 3 batches of BoNT/A complex the molar ratio was determined as neurotoxin binding protein (NBP, 124 kDa), heavy chain (HC, 90 kDa), light chain (LC, 53 kDa), NAP-53 (50 kDa), NAP-33 (36 kDa), NAP-22 (24 kDa), NAP-17 (17 kDa) 1:1:1:2:3:2:2. With Bradford, Lowry, bicinchoninic acid (BCA) and spectroscopic protein estimation methods, the extinction coefficient of BoNT/A complex was determined as 1.54 ± 0.26 (mg/mL)(-1)cm(-1). These findings of a reproducible BoNT/A complex composition will aid in understanding the molecular structure and function of BoNT/A and NAPs.
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Anti-apoptotic activity of hemagglutinin-33 and botulinum neurotoxin and its implications to therapeutic and countermeasure issues.
Biochem. Biophys. Res. Commun.
PUBLISHED: 12-02-2011
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Botulinum neurotoxins (BoNTs), produced by Clostridium botulinum are the most toxic substances known to the mankind. BoNTs (seven serotypes, A-G) are produced along with a group of neurotoxin associated proteins (NAPs) in a physiologically coordinated manner, regulated by a common transcription factor for the gene cluster that encodes for the BoNT and NAPs. Hemagglutinin-33 (Hn-33) is a 33 kDa subcomponent of NAPs, which is resistant to protease digestion, and accounts for about half of the NAPs molecules in the BoNT/A complex. Natural exposures to BoNT in food poisoning cases as well as in the medical applications of BoNT as a therapeutic agent, humans are exposed to the BoNT/A complex. The toxin itself is known to block neurotransmitter release from presynaptic nerves, but the effect of NAPs is unexplored. In this article, we report an important observation of the anti-apoptotic effect of Hn-33 in Hn-33-preincubated human neuroblastoma SH-SY5Y cells. Activity of caspases, which are the central executioners of apoptosis, was substantially (78%) reduced by Hn-33. Degradation of chromosomal DNA, another biochemical hallmark of apoptosis, was blocked in Hn-33 incubated SH-SY5Y cells. Interestingly, purified BoNT/A also showed substantial anti-apoptotic activity. These findings may have significant implications to the use of BoNT as a therapeutic agent, and to devise counter measures to botulinum poisoning.
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Safety of food crops on land contaminated with trace elements.
J. Sci. Food Agric.
PUBLISHED: 03-28-2011
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Contamination of agricultural soils with trace elements (TEs) through municipal and industrial wastes, atmospheric deposition and fertilisers is a matter of great global concern. Since TE accumulation in edible plant parts depends on soil characteristics, plant genotype and agricultural practices, those soil- and plant-specific options that restrict the entry of harmful TEs into the food chain to protect human and animal health are reviewed. Soil options such as in situ stabilisation of TEs in soils, changes in physicochemical parameters, fertiliser management, element interactions and agronomic practices reduce TE uptake by food crops. Furthermore, phytoremediation and solubilisation as alternative techniques to reduce TE concentrations in soils are also discussed. Among plant options, selection of species and cultivars, metabolic processes and microbial transformations in the rhizosphere can potentially affect TE uptake and distribution in plants. For this purpose, genetic variations are exploited to select cultivars with low uptake potential, especially low-cadmium accumulator wheat and rice cultivars. The microbial reduction of elements and transformations in the rhizosphere are other key players in the cycling of TEs that may offer the basis for a wide range of innovative biotechnological processes. It is thus concluded that appropriate combination of soil- and plant-specific options can minimise TE transfer to the food chain.
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Microarray analysis of differentially regulated genes in human neuronal and epithelial cell lines upon exposure to type A botulinum neurotoxin.
Biochem. Biophys. Res. Commun.
PUBLISHED: 01-26-2011
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Among the seven serotypes (A-G), type A botulinum neurotoxin (BoNT/A) is the most prevalent etiologic agent and the most potent serotype to cause foodborne botulism, characterized by flaccid muscle paralysis. Upon ingestion, BoNT/A crosses epithelial cell barriers to reach lymphatic and circulatory systems and blocks acetylcholine release at the pre-synaptic cholinergic nerve terminals of neuromuscular junctions (NMJs) resulting in paralysis. One of the unique features of BoNT/A intoxication is its neuroparalytic longevity due to its persistent catalytic activity. The persistent presence of the toxin inside the cell can induce host cell responses. To understand the pathophysiology and host response at the cellular level, gene expression changes upon exposure of human HT-29 colon carcinoma (epithelial) and SH-SY5Y neuroblastoma cell lines to BoNT/A complex were investigated using microarray analysis. In HT-29 cells, 167 genes were up-regulated while 60 genes were down-regulated, whereas in SH-SY5Y cells about 223 genes were up-regulated and 18 genes were down-regulated. Modulation of genes and pathways involved in neuroinflammatory, ubiquitin-proteasome degradation, phosphatidylinositol, calcium signaling in SH-SY5Y cells, and genes relevant to focal adhesion, cell adhesion molecules, adherens and gap junction related pathways in HT-29 cells suggest a massive host response to BoNT/A. A clear differential response in epithelial and neuronal cells indicates that the genes affected may play a distinct role in BoNTs cellular mode of action, involving these two types of host cells.
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Endopeptidase activities of botulinum neurotoxin type B complex, holotoxin, and light chain.
Appl. Environ. Microbiol.
PUBLISHED: 08-06-2010
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Botulinum neurotoxin (BoNT) serotype B (BoNT/B) is one of the serotypes of BoNT that causes deadly human botulism, though it is used clinically for treatment of many neuromuscular diseases. BoNT/B is produced by Clostridium botulinum, and it is secreted along with a group of neurotoxin-associated proteins (NAPs) in the form of a BoNT/B complex. The complex dissociates into a 150-kDa holotoxin and NAPs at alkaline pHs. The 150-kDa BoNT/B holotoxin can be nicked to produce a 50-kDa domain referred to as the light chain (LC) and a 100-kDa heavy chain, with the former possessing a unique endopeptidase activity. The two chains remain linked through a disulfide bond that can be reduced to separate the two chains. The endopeptidase activity is present in all three forms of the toxin (complex, purified BoNT/B holotoxin, and separated light chain), which are used by different researchers to develop detection methods and screen for inhibitors. In this research, the endopeptidase activities of the three forms, for the first time, were compared under the same conditions. The results show that enzyme activities of the three forms differ significantly and are largely dependent on nicking and disulfide reduction conditions. Under the conditions used, LC had the highest level of activity, and the complex had the lowest. The activity was enhanced by nicking of BoNT/B holotoxin and was enhanced even more by dithiothreitol (DTT) reduction after nicking. This information is useful for understanding the properties of BoNT endopeptidases and for comparing the efficacies of different inhibitors when they are tested with different forms of BoNT endopeptidase.
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Analysis of genomic differences among Clostridium botulinum type A1 strains.
BMC Genomics
PUBLISHED: 05-24-2010
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Type A1 Clostridium botulinum strains are a group of Gram-positive, spore-forming anaerobic bacteria that produce a genetically, biochemically, and biophysically indistinguishable 150 kD protein that causes botulism. The genomes of three type A1 C. botulinum strains have been sequenced and show a high degree of synteny. The purpose of this study was to characterize differences among these genomes and compare these differentiating features with two additional unsequenced strains used in previous studies.
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In vitro selection of RNA aptamers that inhibit the activity of type A botulinum neurotoxin.
Biochem. Biophys. Res. Commun.
PUBLISHED: 04-29-2010
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The category A agent, botulinum neurotoxin (BoNT), is the most toxic molecule known to mankind. The endopeptidase activity of light chain domain of BoNT is the cause for the inhibition of the neurotransmitter release and the flaccid paralysis that leads to lethality in botulism. Currently, antidotes are not available to reverse the flaccid paralysis caused by BoNT. In the present study, we have identified three RNA aptamers through SELEX-process, which bind strongly to the light chain of type A BoNT (BoNT/A) and inhibit the endopeptidase activity, with IC(50) in low nM range. Inhibition kinetic studies reveal low nM K(I) and non-competitive nature of their inhibition. Aptamers are unique group of molecules as therapeutics, and this is first report of their development as an antidote against botulism. These data on K(I) and IC(50) strongly suggest that the aptamers have strong potential as antidotes that can reverse the symptom caused by BoNT/A.
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The zinc-dependent protease activity of the botulinum neurotoxins.
Toxins (Basel)
PUBLISHED: 04-01-2010
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The botulinum neurotoxins (BoNT, serotypes A-G) are some of the most toxic proteins known and are the causative agents of botulism. Following exposure, the neurotoxin binds and enters peripheral cholinergic nerve endings and specifically and selectively cleaves one or more SNARE proteins to produce flaccid paralysis. This review centers on the kinetics of the Zn-dependent proteolytic activities of these neurotoxins, and briefly describes inhibitors, activators and factors underlying persistence of toxin action. Some of the structural, enzymatic and inhibitor data that are discussed here are available at the botulinum neurotoxin resource, BotDB (http://botdb.abcc.ncifcrf.gov).
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Zinc tolerance and uptake by Arabidopsis halleri ssp. gemmifera grown in nutrient solution.
Environ Sci Pollut Res Int
PUBLISHED: 03-19-2010
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Zinc is an essential micronutrient element but its concentrations found in contaminated soils frequently exceed those required by the plant and soil organisms, and thus create danger to animal and human health. Phytoremediation is a technique, often employed in remediation of contaminated soils, which aims to remove heavy metals or other contaminants from soils or waters using plants. Arabidopsis (A.) halleri ssp. gemmifera is a plant recently found to be grown vigorously in heavy metal contaminated areas of Japan and it contained remarkably high amount of heavy metals in its shoots. However, the magnitude of Zn accumulation and tolerance in A. halleri ssp. gemmifera need to be investigated for its use as a phytoremediation plant.
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Clostridial neurotoxins as a drug delivery vehicle targeting nervous system.
Biochimie
PUBLISHED: 03-09-2010
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Several neuronal disorders require drug treatment using drug delivery systems for specific delivery of the drugs for the targeted tissues, both at the peripheral and central nervous system levels. We describe a review of information currently available on the potential use of appropriate domains of clostridial neurotoxins, tetanus and botulinum, for effective drug delivery to neuronal systems. While both tetanus and botulinum neurotoxins are capable of delivering drugs the neuronal cells, tetanus neurotoxin is limited in clinical use because of general immunization of population against tetanus. Botulinum neurotoxin which is also being used as a therapeutic reagent has strong potential for drug delivery to nervous tissues.
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Molecular basis of activation of endopeptidase activity of botulinum neurotoxin type E.
Biochemistry
PUBLISHED: 02-25-2010
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Botulinum neurotoxins (BoNTs) are a group of large proteins that are responsible for the clinical syndrome of botulism. The seven immunologically distinct serotypes of BoNTs (A-G), each produced by various strains of Clostridium botulinum, act on the neuromuscular junction by blocking the release of the neurotransmitter acetylcholine, thereby resulting in flaccid muscle paralysis. BoNTs are synthesized as single inactive polypeptide chains that are cleaved by endogenous or exogenous proteases to generate the active dichain form of the toxin. Nicking of the single chain BoNT/E to the dichain form is associated with 100-fold increase in toxicity. Here we investigated the activation mechanism of botulinum neurotoxin type E upon nicking and subsequent reduction of disulfide bond. It was observed that nicking of BoNT/E significantly enhances its endopeptidase activity and that at the physiological temperature of 37 degrees C the reduced form of nicked BoNT/E adopts a dynamically flexible conformation resulting from the exposure of hydrophobic segments and facilitating optimal cleavage of its substrate SNAP-25. Such reduction-induced increase in the flexibility of the polypeptide folding provides a rationale for the mechanism of BoNT/E endopeptidase against its intracellular substrate, SNAP-25, and complements current understanding of the mechanistics of interaction between the substrate and BoNT endopeptidase.
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Immunological characterization of the subunits of type A botulinum neurotoxin and different components of its associated proteins.
Toxicon
PUBLISHED: 08-13-2009
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Botulinum neurotoxins (BoNTs) constitute a family of seven structurally similar but antigenically distinct proteins produced by different strains of Clostridium botulinum. Type A botulinum neurotoxin (BoNT/A) is produced along with 6 neurotoxin associated proteins (NAPs) including hemagglutinin (Hn-33) through polycistronic expression of a clustered group of genes to form a complex (BoNT/AC). The presence of NAPs enhances the oral toxicity of the neurotoxin significantly. Hn-33 makes up the largest fraction of NAPs in BoNT/AC and strongly protects BoNT/A against proteases of the GI tract. BoNT in its complex form is also used in therapeutic and cosmetic applications to treat several neuromuscular disorders. In this study immunological reactivity of BoNT/A in its purified and complex forms, neurotoxin associated proteins, and Hn-33 have been examined using enzyme-linked immunosorbent assay (ELISA). Antibodies raised against the whole complex reacted 60 times better with the complex and 35 times better with Hn-33 and NAPs compared to the purified neurotoxin suggesting stronger immunogenicity of NAPs over that of purified neurotoxin and a higher potential of BoNT/AC and its associated proteins to induce host immune response. This observation also suggests that Hn-33 and other NAPs could potentially be employed as adjuvants for development of vaccines against botulism and could be a good surrogate for botulinum diagnostics. ELISA binding curves of BoNT/AC and BoNT/A with antibodies raised against BoNT/A indicate that BoNT/A in its purified and complex forms induces equal immunogenic response and a 2.5-fold higher immunogenic response compared to BoNT/A light and heavy chains. We have also discovered a new protein, an intimin analog, present within the complex preparation of BoNT/A which shows dramatically high immunoreactivity.
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The identification and biochemical characterization of drug-like compounds that inhibit botulinum neurotoxin serotype A endopeptidase activity.
Toxicon
PUBLISHED: 08-01-2009
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A robust, high-throughput, two-tiered assay for screening small molecule inhibitors against botulinum neurotoxin serotype A was developed and employed to screen 16,544 compounds. Thirty-four compounds were identified as potent hits employing the first-tier assay. Subsequently, nine were confirmed as actives by our second-tier confirmatory assay. Of these, one displayed potent inhibitory efficacy, possessing an IC(50)=16 microM (+/-1.6 microM) in our in vitro assay. This inhibitor (0831-1035) is highly water-soluble, and possesses an IC(50)=47 microM (+/-7.0 microM) in our primary cell culture assay (with virtually no cytotoxicity up to 500 microM), suggesting that this inhibitor is a good candidate for further development as a therapeutic countermeasure to treat botulism resulting from botulinum neurotoxin serotype A intoxication. An enzyme kinetics study indicated that this inhibitor exhibits mixed non-competitive inhibition, with a K(I)=9 microM.
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An efficient drug delivery vehicle for botulism countermeasure.
BMC Pharmacol.
PUBLISHED: 07-13-2009
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Botulinum neurotoxin (BoNT) is the most potent poison known to mankind. Currently no antidote is available to rescue poisoned synapses. An effective medical countermeasure strategy would require developing a drug that could rescue poisoned neuromuscular synapses and include its efficient delivery specifically to poisoned presynaptic nerve terminals. Here we report a drug delivery strategy that could directly deliver toxin inhibitors into the intoxicated nerve terminal cytosol.
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Molecular identification of glutathione S-transferase gene and cDNAs of two isotypes from northern quahog (Mercenaria mercenaria).
Comp. Biochem. Physiol. B, Biochem. Mol. Biol.
PUBLISHED: 02-03-2009
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Glutathione S-transferases (GSTs) are universally xenobiotic detoxifying enzymes which have been shown to play a unique detoxifying role in northern quahogs. GST consists of two distinct domains: N-terminal domain which contributes residues to form G-site (GSH-binding site) and C-terminal domain which provides residues to form a hydrophobic H-site (second substrate-binding site). In this study, glutathione S-transferases (GSTs) gene and cDNAs of two isotypes from the northern quahog (Mercenaria mercenaria) were cloned and characterized for the first time. Two full-length GST cDNAs were obtained and named as GST Pi-1 and Pi-2, respectively. Both cDNAs have an open reading frame of 624 bp, which encodes a 207-amino acid protein. Multiple sequences alignment analysis shows that the deduced amino acid sequences of GST Pi-1 and Pi-2 have high homology with other Pi class GSTs. The evolutionary relationship assessment indicates that the two deduced amino acid sequences are closely related to Pi class GSTs. The GST Pi-1 gene is an intronless gene so that it has the same sequence as the transcript. The digested peptide fragments of the purified northern quahog GSTs were analyzed by tandem mass spectrometry and the results confirm the existence of the translation products of the GST Pi-1 and Pi-2 transcripts. The predicted three-dimensional structures of GST Pi-1 and Pi-2 showed that both proteins have two domains, N-terminal domain and C-terminal domain. The conserved phenylalanine-48 serving as the ball in ball and socket style interface in all reported Pi class GSTs was found in both proteins. These results suggest that GST Pi-1 and Pi-2 from the northern quahog belong to the Pi class GSTs which may be involved in polychlorinated biphenyl (PCB) dechlorination.
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Effects of tree vegetation and waste amendments on the fractionation of Cr, Cu, Ni, Pb and Zn in polluted mine soils.
Sci. Total Environ.
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Soils at a depleted copper mine in Touro (Galicia, Spain) are physically and chemically degraded and have also polluted the surrounding area. Due to these environmental problems and the large area of these mine soils, the reclamation strategies carried out at Touro have consisted of planting trees (pine or eucalyptus), amending with waste material (sewage sludge and paper mill residues), or using both treatments. Tree planting has been carried out for 21 years and waste amending for 10. Two different zones were selected in the mine (the settling pond and mine tailing) in order to evaluate the effect of the different reclamation practices on the chemical fractions of Cr, Cu, Ni, Pb and Zn. The results showed that soils in the untreated sites were polluted by Cr and Cu. Planting pines and eucalyptus on mine soils decreased the concentration of these heavy metals in non-mobile soil fractions. Amendments also attenuated pollution by Cr and Cu as the wastes that were used had lower concentrations than the untreated mine soils. Planting trees increased Ni, Pb and Zn retention in the non-mobile fractions, preventing them from being leached into surrounding areas. However, caution should be exercised when adding organic wastes, as they can lead to increase concentrations of Ni, Pb and Zn and their phytoavailable form. The results also showed that changes in the chemical fractionation of heavy metals in soils was more influenced by the clay percentage and both dissolved and soil organic carbon (SOC and DOC) than by soil pH or cation exchange capacity.
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Development of a fluorescence internal quenching correction factor to correct botulinum neurotoxin type A endopeptidase kinetics using SNAPtide.
Anal. Chem.
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Botulinum neurotoxins (BoNTs), which are highly toxic proteins responsible for botulism, are produced by different strains of Clostridium botulinum. These various strains of bacteria produce seven distinct serotypes, labeled A-G. Once inside cells, the zinc-dependent proteolytic light chain (LC) degrades specific proteins involved in acetylcholine release at neuromuscular junctions causing flaccid paralysis, specifically synaptosomal-associated protein 25 (SNAP-25) for botulinum neurotoxin type A (BoNT/A). BoNT endopeptidase assays using short substrate homologues have been widely used and developed because of their ease of synthesis, detection limits, and cost. SNAPtide, a 13-amino acid fluorescence resonance energy transfer (FRET) peptide, was used in this study as a SNAP-25 homologue for the endopeptidase kinetics study of BoNT/A LC. SNAPtide uses a fluorescein isothiocyanate/4-((4-(dimethylamino)phenyl)azo) benzoic acid (FITC/DABCYL) FRET pair to produce a signal upon substrate cleavage. Signal quenching can become an issue after cleavage since quencher molecules can quench cleaved fluorophore molecules in close proximity, reducing the apparent signal. This reduction in apparent signal provides an inherent error as SNAPtide concentrations are increased. In this study, fluorescence internal quenching (FIQ) correction factors were derived using an unquenched SNAPtide peptide to quantify the signal quenching over a range of SNAPtide concentrations and temperatures. The BoNT/A LC endopeptidase kinetics at the optimally active temperature (37 °C) using SNAPtide were studied and used to demonstrate the FIQ correction factors in this study. The FIQ correction factors developed provide a convenient method to allow for improved accuracy in determining and comparing BoNT/A LC activity and kinetics using SNAPtide over a broad range of concentrations and temperatures.
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