Real-time PCR detection of the nontoxic nonhemagglutinin gene as a rapid screening method for bacterial isolates harboring the botulinum neurotoxin (A–G) gene complex

Botulinum neurotoxin (BoNT) producing clostridia contain genes encoding a specific neurotoxin serotype (A–G) and nontoxic associated proteins that form the toxin complex. The nontoxic nonhemagglutinin (NTNH) is a conserved component of the toxin complex in all seven toxin types. A real-time PCR assay that utilizes a locked nucleic acid hydrolysis probe to target the NTNH gene was developed to detect bacterial strains harboring the botulinum neurotoxin gene cluster. The specificity of the assay for Clostridium botulinum types A–G, Clostridium butyricum type E and Clostridium baratii type F was demonstrated using a panel of 73 BoNT producing clostridia representing all seven toxin serotypes. In addition, exclusivity of the assay was demonstrated using non-botulinum toxin producing clostridia (7 strains) and various enteric bacterial strains (n = 27). Using purified DNA, the assay had a sensitivity of 4–95 genome equivalents. C. botulinum type A was detected directly in spiked stool samples at 10²–10³ CFU/ml. Stool spiked with 1 CFU/ml was detected when the sample was inoculated into enrichment broth and incubated for 24 h. These results indicate that the NTNH real-time PCR assay can be used to screen enrichment cultures of primary specimens at earlier time points (24 h) than by toxin detection of unknown culture supernatants (up to 5 days).     

Pharmacophore-guided lead optimization: The rational design of a non-zinc coordinating,sub-micromolar inhibitor of the botulinum neurotoxin serotype a metalloprotease

Botulinum neurotoxins, responsible for the neuroparalytic syndrome botulism, are the deadliest of known biological toxins. The work described in this study was based on a three-zone pharmacophore model for botulinum neurotoxin serotype A light chain inhibition. Specifically, the pharmacophore defined a separation between the overlaps of several different, non-zinc(II)-coordinating small molecule chemotypes, enabling the design and synthesis of a new structural hybrid possessing a K_i = 600 nM (±100 nM).     

A Tenebrio molitor GPI-anchored alkaline phosphatase is involved in binding of Bacillus thuringiensis Cry3Aa to brush border membrane vesicles

Bacillus thuringiensis Cry toxins recognizes their target cells in part by the binding to glycosyl–phosphatidyl–inositol (GPI) anchored proteins such as aminopeptidase-N (APN) or alkaline phosphatases (ALP). Treatment of Tenebrio molitor brush border membrane vesicles (BBMV) with phospholipase C that cleaves out GPI-anchored proteins from the membranes, showed that GPI-anchored proteins are involved in binding of Cry3Aa toxin to BBMV. A 68 kDa GPI-anchored ALP was shown to bind Cry3Aa by toxin overlay assays. The 68 kDa GPI-anchored ALP was preferentially expressed in early instar larvae in comparison to late instar larvae. Our work shows for the first time that GPI-anchored ALP is important for Cry3Aa binding to T. molitor BBMV suggesting that the mode of action of Cry toxins is conserved in different insect orders.     

Purification and separation of the 20S immunoproteasome from the constitutive proteasome and identification of the subunits by LC–MS

The proteasome is a multicatalytic protease complex present in all eukaryotic cells, which plays a critical role in regulating essential cellular processes. During the immune response to pathogens, stimulation by γ interferon induces the production of a special form of proteasome, the immunoproteasome. Inappropriate increase of proteosomal activity has been linked to inflammatory and autoimmune diseases. Selective inhibition of the immunoproteasome specific LMP7 subunit was shown to block inflammatory cytokine secretion in human PBMC, thus making the immunoproteasome an interesting target to fight autoimmune diseases. This paper describes a method for purification and separation of the 20S immunoproteasomes from the constitutive proteasome, which is ubiquitously present in all cells, based on hydrophobic interaction chromatography. The purified immunoproteasome showed several bands, between 20–30 kDa, when subjected to polyacrylamide gel electrophoresis under denaturing conditions. The purified proteasome complexes had a molecular mass of approximately 700 kDa as estimated by gel filtration. Identification of the catalytic subunits in the immunoproteasomes was performed in Western blot with antibodies directed specifically against either the constitutive or the immunoproteasome subunits. The purified immunoproteasome possessed all three protease activities associated with the proteasome complex. LC/MS analysis confirmed the presence of the three immunoproteasome catalytic subunits in the purified immunoproteasome.     

Subunit Stoichiometry of the Clostridium botulinum Type A Neurotoxin Complex Determined Using Denaturing Capillary Electrophoresis

A denaturing capillary electrophoresis method was developed to evaluate the subunit stoichiometry of the Clostridium botulinum type A neurotoxin complex. The results indicate that the neurotoxin complex contains single copies of the 150 kDa neurotoxin and the non-toxic non-hemagglutinating subunits as well as 5–6 HA17, 4–5 HA23, 3–4 HA48, and 8–9 HA34 subunits. The calculated molecular mass for a complex with this stoichiometry is between 880 and 1,000 kDa. The molecular mass of the intact complex was determined using size-exclusion HPLC (SE-HPLC) and SE-HPLC in conjunction with multi-angle laser light scattering detection. Based on a comparison to a mixture of standard proteins, SE-HPLC analysis yielded a molecular mass of 880 kDa while light scattering analysis indicated a weight average molecular mass of 925 ± 45 kDa. The close agreement between the molecular mass values determined by the three approaches supports the subunit stoichiometry proposed for the C. botulinum type A neurotoxin complex.     

Cloning,Expression, Purification,and Characterization of Biologically Active Recombinant Hemagglutinin-33, Type A Botulinum Neurotoxin Associated Protein

Botulinum neurotoxin type A, the most toxic substance known to mankind, is produced by Clostridium botulinum type A as a complex with a group of neurotoxin-associated proteins (NAPs) through polycistronic expression of a clustered group of genes. Hemagglutinin-33 (Hn-33) is a 33 kDa subcomponent of NAPs, which is resistant to protease digestion, a feature likely to be involved in the protection of the botulinum neurotoxin from proteolysis. In order to fully understand the function of Hn-33, large amounts of Hn-33 will be needed without dealing with biosafety risks to grow large cultures of C. botulinum. There are difficulties to clone the genes with the high A + T contents produced by C. botulinum. We report here for the first time using the Gateway technology to clone functional Hn-33 that has been expressed in E. coli. The yield of the recombinant Hn-33 was about 12 mg per liter of E. coli culture. The recombinant Hn-33 folds well in aqueous solution as shown with circular dichroism spectra, resists temperature-denaturation, is totally resistant to trypsin proteolysis despite the presence of cleavage sites on the molecular surface, and maintains its biological activities comparable to the native Hn-33 hemagglutination.     

Purification and Characterization of Neurotoxin Complex from a Dual Toxin Gene Containing Clostridium Botulinum Strain PS-5

Botulinum neurotoxins are produced as a toxin complex (TC) which consists of neurotoxin (NT) and neurotoxin associated proteins. The characterization of NT in its native state is an essential step for developing diagnostics and therapeutic countermeasures against botulism. The presence of NT genes was validated by PCR amplification of toxin specific fragments from genomic DNA of Clostridium botulinum strain PS-5 which indicated the presence of both serotype A and B genes on PS-5 genome. Further, TC was purified and characterized by Western blotting, Digoxin-enzyme linked immunosorbent assay, endopeptidase activity assay, and Liquid chromatography–Mass spectrometry. The data showed the presence of serotype A specific neurotoxin. Based on the analysis of neurotoxin genes and characterization of TC, PS-5 strain appears as a serotype A (B) strain of C. botulinum which produces only serotype A specific TC in the cell culture medium.     

Proteases are associated with a minor fucoxanthin chlorophyll a/c-binding protein from the diatom,Chaetoceros gracilis

We previously showed that most subunits in the oxygen-evolving photosystem II (PSII) preparation from the diatom Chaetoceros gracilis are proteolytically unstable. Here, we focused on identifying the proteases that cleave PSII subunits in thylakoid membranes. Major PSII subunits and fucoxanthin chlorophyll (Chl) a/c‐binding proteins (FCPs) were specifically degraded in thylakoid membranes. The PSI subunits, PsaA and PsaB, were slowly degraded, and cytochrome f was barely degraded. Using zymography, proteolytic activities for three metalloproteases (116, 83, and 75 kDa) and one serine protease (156 kDa) were detected in thylakoid membranes. Two FCP fractions (FCP-A and FCP-B/C) and a photosystem fraction were separated by sucrose gradient centrifugation using dodecyl maltoside‐solubilized thylakoids. The FCP-A fraction featured enriched Chl c compared with the bulk of FCP-B/C. Zymography revealed that 116, 83, and 94 kDa metalloproteases were mostly in the FCP-A fraction along with the 156 kDa serine protease. When solubilized thylakoids were separated with clear-native PAGE, zymography detected only the 83 kDa metalloprotease in the FCP-A band. Because FCP-A is selectively associated with PSII, these FCP-A-associated metalloproteases and serine protease may be responsible for the proteolytic degradation of FCPs and PSII in thylakoid membranes.     

Toxic strains of the Alexandrium ostenfeldii complex in southern South America (Beagle Channel,Argentina)

During phytoplankton monitoring in the Beagle Channel (≈54°52′ S, 67°32′ W) a previously undetected Alexandrium species was observed in coincidence with mouse bioassay toxicity. Detailed thecal plates analysis using epifluorescence and scanning electron microscopy revealed the presence of the Alexandrium ostenfeldii species complex, showing a mixture of the diagnostic features usually used to discriminate between the morphospecies A. ostenfeldii and A. peruvianum. Cells of the A. ostenfeldii complex were commonly observed during spring after the main annual diatom bloom, when temperatures and salinities were respectively around 7.5–10 °C and 30–30.5 psu, and nutrients showed a seasonal decrease. Toxin analysis by liquid chromatography–mass spectrometry revealed the production of 13-desmethyl spirolide C and 20-methyl spirolide G in cell cultures. The cellular contain of spirolides during exponential phase growth was 0.5906 ± 0.0032 and 0.1577 ± 0.0023 pg cell⁻¹ for 13-desMe-C and 20-Me-G, respectively. A third unknown compound, with a structure resembling that of spirolides was also detected in culture. Moreover, an additional compound with a similar m/z (692) than that of 13-desMe-C but presenting a higher retention time (Rt = 40.5 min) was found in high proportions in mussel samples. PSP toxins were present at low concentration in mussels but were not detected in cultures. These results extend the world-wide distribution of toxic strains of the A. ostenfeldii complex to the Beagle Channel (southern South America), where toxic events have been traditionally linked to the presence of Alexandrium catenella. This is the first confirmed occurrence of spirolides in mussels and plankton from Argentina, which highlights the importance of monitoring these toxins and their producing organisms to protect public health and improve the management of shellfish resources.     

Detection,quantification and identification of fungal extracellular laccases using polyclonal antibody and mass spectrometry

This study presents a combined method to analyze extracellular fungal laccases using a new anti-laccase antibody together with the identification of tryptic laccase peptides by mass spectrometry (nanoLC–ESI–MS/MS). The polyclonal anti-laccase antibody LccCbr2 was raised against peptides designed from the copper binding region II of fungal laccases using in silico data obtained from GenBank database. As a consequence, detection requires denaturation of the enzymes due to the stable conformation of the copper binding region II. The specificity of the antibody was shown with denatured laccase Lcc1 of Coprinopsis cinerea and laccase of Hypholoma fasciculare. LccCbr2 detected amounts as low as 5 ng of highly purified laccase, indicating a possible use of the antibody for quantification of laccase proteins. Denatured extracellular laccases from culture supernatants of the basidiomycetes C. cinerea, H. fasciculare, Lentinula edodes, Mycena sp., Piriformospora indica, Pleurotus cornucopiae, Pleurotus ostreatus, Pycnoporus cinnabarinus, Trametes versicolor and furthermore the ascomycete Verpa conica were detected with apparent molecular masses between 60 and 70 kDa by LccCbr2. The identity of extracellular laccases from C. cinerea, H. fasciculare, P. ostreatus, P. cinnabarinus and T. versicolor were verified by tryptic peptides using nanoLC–ESI–MS/MS.     

Multi-chaperone-peptide-rich mixture from colo-carcinoma cells elicits potent anticancer immunity

Background: Chaperones play an important role in inducing anti-cancer immunity. To explore the probability of using chaperone-peptide-rich complexes extracted from colo-carcinoma cells as anti-cancer vaccine, we extracted and prepared chaperone-peptide-rich complexes from CT26 cells, which were subsequently investigated on anti-cancer efficacy. Methods: The crude extracts of the CT26 cells treated with heat and Trichosanthin were precipitated with salt and dialyzed to remove proteins below 50 kDa and above 300 kDa in molecular weight; the proteins with the molecular weights in 70 kDa, 90 kDa, 95 kDa, 110 kDa and 170 kDa were collected through gel filtration and SDS-PAGE. After confirmation, the purified proteins were used to determine their effects on lymphocyte proliferation, the activities of NK and CTL, tumor suppression and the tumor-bearing mouse survival. Results: The majority of the chaperone-peptides of anti-cancer immunity in CT26 cells, including HSP70-antigen peptide, HSP90-antigen peptide, gp96-antigen peptide, HSP-110 antigen peptide, HSP170-antigen peptide, was satisfactorily extracted that the multi-chaperone-peptide-rich mixtures were obtained. All the mixtures prepared could elicit lymphocyte proliferation, enhance the activities of CTL and NK, reinforce the tumor suppression and prolong the mouse survival. Conclusions: The multi-chaperone-peptide-rich mixtures could be prepared via dialysis and gel filtration combining with SDS-PAGE. Both the heat stress and Trichosanthin could induce and increase the mixtures, of which that treated by 42 °C heat and Trichosanthin was found to possess the strongest anti-cancer efficacy.     

Deposit-feeders accumulate the cyanobacterial toxin nodularin

Blooms of toxic cyanobacteria may potentially affect food web productivity and even be a human health hazard. In the Baltic Sea, regularly occurring summer blooms of nitrogen-fixing cyanobacteria are often dominated by Nodularia spumigena, which produces the potent hepatotoxin nodularin. Evidence of sedimentation of these blooms indicates that benthic fauna can be exposed to nodularin. In a one month experiment, we simulated the settling of a summer bloom dominated by N. spumigena in sediment microcosms with three species of sediment-dwelling, deposit-feeding macrofauna, the amphipods Monoporeia affinis and Pontoporeia femorata and the bivalve Macoma balthica, and analyzed nodularin in the animals by HPLC–ESI–MS (high-performance liquid chromatography–electrospray ionization–mass spectrometry). We found nodularin in quantities of 50–120 ng g⁻¹ DW. The results show that deposit-feeding macrofauna in the Baltic Sea may contribute to trophic transfer of nodularin.     

Quantitation of bergenin in human plasma by liquid chromatography/tandem mass spectrometry

This paper reports the development and validation of an assay for quantitation of bergenin in human plasma using liquid chromatography/tandem mass spectrometry (LC–MS/MS). Bergenin and the internal standard (I.S.), 5-bromo-2,4(1H,3H)-pyrimidinedione (5-BrU), were separated by reversed phase HPLC and quantitated by MS/MS using electrospray ionization (ESI) and multiple reaction monitoring (MRM) in the negative ion mode. The most intense [M?H]^? MRM transition of bergenin at m/z 326.9 → 312.3 was used for quantitation and the transition at m/z 188.9 → 42.2 was used to monitor 5-BrU. Stability issues with bergenin required the addition of ascorbic acid to plasma samples prior to storage and analysis within 10 days storage at ?80 °C. The method was linear in the range 3–1000 ng/mL with intra- and inter-day precision of 3.94–5.96 and 1.62–8.31%, respectively, and accuracy <2.33%. The assay was successfully applied to a pharmacokinetic study in healthy volunteers after administration of a single 250 mg oral dose.     

Purification and characterization of a trypsin inhibitor from Senna tora active against midgut protease of podborer

Proteinaceous protease inhibitors have potential application in medicines, agriculture and biotechnology. Present study was undertaken to purify and characterize a proteinaceous protease inhibitor from a medicinal plant, Senna tora syn. Cassia tora. The inhibitor was purified by ammonium sulphate precipitation, anion exchange (Q-sepharose), affinity (trypsin-sepharose) and molecular exclusion (sephadex G-75) chromatography. Zymography and denaturing polyacrylamide gel electrophoresis revealed a single band of ∼20 kDa trypsin inhibitor. Two dimensional polyacrylamide gel electrophoresis (2D-PAGE) and Matrix-assisted laser desorption ionization (MALDI) analyses revealed the presence of 19,725 Da (pI 4.60) and ∼19,900 Da (pI 4.57) isoform proteins in purified inhibitor. Protein identification by MALDI-peptide mass fingerprinting did not reveal high MASCOT (Matrix science) scores matching with previously known inhibitors. N-terminal amino acid sequence suggested this protein as a previously unreported inhibitor. Its dissociation constant (0.23 × 10⁻⁹ M) was indicative of a high affinity trypsin inhibitor. The inhibitor was stable over a broad range of pH (4–10) and temperature (30–60 °C). The purified inhibitor effectively inhibited total protease and trypsin-like activities of podborer (Helicoverpa armigera) midgut preparation. Hence, the inhibitor and its gene(s) can find application in combating against pest and protease dependent pathogens.     

Calcium protection of PS2 complex of Phaseolus coccineus from cadmium toxicity: In vitro study

The action of 10 and 20 mM Ca against harmful Cd effect on PS2 complex isolated from leaves of Phaseolus coccineus L. cv. Pi?kny Ja? was studied. The changes in fast chlorophyll a fluorescence induction kinetics and protein composition of PS2 complex were the symptoms of Cd toxicity and Ca protection of PS2 complex. Calcium applied at 10 mM concentration prevented F₀ reduction caused by the presence of 250–1000 μM Cd in the incubation mixture, but that of (the variable chlorophyll a fluorescence) F_v, F_m, F_v/F₀, and F_v/F_m only at 250 μM Cd. Ca concentration doubling in the incubation mixture resulted in complete overcoming the toxicity of 250–1000 μM Cd to F_v and F_m. However, the protection of F_v/F₀ and the photochemical efficiency of PS2 (F_v/F_m) from 1000 μM Cd was only partial even at 20 mM Ca. A protective effect of 10 mM Ca on D1, D2 and 17 kDa proteins was found in PS2 complex exposed to 250 μM Cd, and on 43 kDa protein in the complex treated with 500 μM Cd. However, 20 mM Ca counteracted the toxicity of 500 μM Cd to the 43, 47 and 17 kDa proteins, as well as the harmful effect of 1000 μM Cd on 47 and 17 kDa ones.     

Deletion of psbJ leads to accumulation of Psb27–Psb28 photosystem II complexes in Thermosynechococcus elongatus

The life cycle of Photosystem II (PSII) is embedded in a network of proteins that guides the complex through biogenesis, damage and repair. Some of these proteins, such as Psb27 and Psb28, are involved in cofactor assembly for which they are only transiently bound to the preassembled complex. In this work we isolated and analyzed PSII from a ΔpsbJ mutant of the thermophilic cyanobacterium Thermosynechococcus elongatus. From the four different PSII complexes that could be separated the most prominent one revealed a monomeric Psb27–Psb28 PSII complex with greatly diminished oxygen-evolving activity. The MALDI-ToF mass spectrometry analysis of intact low molecular weight subunits (< 10 kDa) depicted wild type PSII with the absence of PsbJ. Relative quantification of the PsbA1/PsbA3 ratio by LC-ESI mass spectrometry using ¹⁵N labeled PsbA3-specific peptides indicated the complete replacement of PsbA1 by the stress copy PsbA3 in the mutant, even under standard growth conditions (50 μmol photons m^? 2 s^? 1). This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.     

Translocation of botulinum neurotoxin serotype A and associated proteins across the intestinal epithelia

Botulinum neurotoxins (BoNTs) are some of the most poisonous natural toxins. Botulinum neurotoxins associate with neurotoxin‐associated proteins (NAPs) forming large complexes that are protected from the harsh environment of the gastrointestinal tract. However, it is still unclear how BoNT complexes as large as 900 kDa traverse the epithelial barrier and what role NAPs play in toxin translocation. In this study, we examined the transit of BoNT serotype A (BoNT/A) holotoxin, complex and recombinantly purified NAP complex through cultured and polarized Caco‐2 cells and, for the first time, in the small mouse intestine. Botulinum neurotoxin serotype A and NAPs in the toxin complex were detectable inside intestinal cells beginning at 2 h post intoxication. Appearance of the BoNT/A holotoxin signal was slower, with detection starting at 4–6 h. This indicated that the holotoxin alone was sufficient for entry but the presence of NAPs enhanced the rate of entry. Botulinum neurotoxin serotype A detection peaked at approximately 6 and 8 h for complex and holotoxin, respectively, and thereafter began to disperse with some toxin remaining in the epithelia after 24 h. Purified HA complexes alone were also internalized and followed a similar time course to that of BoNT/A complex internalization. However, recombinant HA complexes did not enhance BoNT/A holotoxin entry in the absence of a physical link with BoNT/A. We propose a model for BoNT/A toxin complex translocation whereby toxin complex entry is facilitated by NAPs in a receptor‐mediated mechanism. Understanding the intestinal uptake of BoNT complexes will aid the development of new measures to prevent or treat oral intoxications.     

A quantitative HPLC–MS method for the simultaneous determination of testosterone, 11-ketotestosterone and 11-β hydroxyandrostenedione in fish serum

A simple and novel HPLC–MS method for the simultaneous quantification of testosterone, 11-ketotestosterone, and 11β-hydroxyandrostenedione in fish serum was developed and validated. Separation was achieved on a C-18 column using a water–acetonitrile mobile-phase with a cycle time of 12 min. Ion detection was performed using ESI positive SIM at [M+H] (m/z 303, 303, 289). The linear ranges (0.2–50 ng/ml), limits of detection (0.1–0.2 ng/ml) and quantification (0.2–0.5 ng/ml) were established. The method was validated by measuring the three androgens in goldfish sera, displaying comparable values to those reported by other analytical techniques (RIA, EIA).     

Chemical composition,antioxidant and macromolecule damage protective effects of Picrorhiza kurroa Royle ex Benth

In the present study, we identified the chemical constituents of 70% hydroalcoholic fraction of Picrorhiza kurroa by LC–ESI–MS/MS which showed the presence of iridoid glucosides such as picroside I, picroside II, picroside III, picroside IV, kutkoside, pikuroside and flavonoids like apocynin and vanillic acid. P. kurroa exhibited DPPH radical scavenging and metal chelating activities with IC₅₀ of 75.16 ± 3.2 and 55.5 ± 4.8 μg/mL and also showed potent reducing power and total antioxidant activities. The extract inhibited macromolecule damage such as H₂O₂ induced plasmid DNA damage and AAPH induced oxidation of bovine serum albumin and lipid peroxidation of rat hepatic tissues.     

One-step purification of YLLIP2 isoforms from Candida sp. 99–125 by polyethyleneimine modified poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) monolith

The extracellular lipase Yarrowia lipolytica (YLLIP2) crude extract was efficiently separated and purified from Candida sp. 99–125 by one-step ion-exchange chromatography on polyethyleneimine (PEI) functionalized monolithic columns. The preparative conditions for the functionalization of monoliths were optimized, including PEI molecular mass, PEI concentration, modification time and temperature. The monolithic skeleton was prepared in situ by polymerization of glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EGDMA) with a volume ratio of 8:2. Heptane was used as the porogen. PEI 30 kDa with the concentration of 10% (v/v) was applied for the modification of the monolith at 55 °C for 12 h. Lipase (EC.3.1.1.3) from Candida sp. 99–125 was separated to four isoforms (isoform A, isoform B, isoform C and isoform D). As analyzed on non-denaturing PAGE and MALDI-TOF–MS, the four isoforms are homogenous and have the same molecular mass of approximate 38 kDa. The monoliths can afford direct crude lipase loading without increasing too much back pressure, which explores the great potential of the application of monoliths for one-single step fast separation and purification of complicated proteins.