Identification Of Toxins Research Articles

Identification of a New Diarrhetic Shellfish Poisoning Toxin, 19-epi-okadaic Acid by Liquid Chromatography with Mass Spectrometry Detection

Okadaic acid (1) (OA) and its congeners are mainly responsible for diarrhetic shellfish poisoning (DSP) syndrome. The presence of several OA derivatives have already been confirmed in Prorocentrum and Dinophysis spp. In this paper, we report on the detection and identification of a new DSP toxin, the OA isomer 19-epi-okadaic acid (2) (19-epi-OA), isolated from cultures of Prorocentrum belizeanum, by determining its retention time (RT) and fragmentation pattern using liquid chromatography coupled with mass spectrometry (LC–MS/MS).

Identification of Fumonisin B2, HT-2 Toxin, Patulin, and Zearalenone in Dried Figs by Liquid Chromatography–Time-of-Flight Mass Spectrometry and Liquid Chromatography–Mass Spectrometry

Dried figs from Turkey that were visibly moldy (or fluorescent under UV light) and thus rejected as unsuitable for human food were screened for the presence of fungal metabolites. Crude solvent extracts from individual figs were directly analyzed by liquid chromatography combined with time-of-flight mass spectrometry to generate accurate mass data for all detectable components. A comparison of these data with a metabolite database indicated the presence of fumonisins B2 and B4, patulin, HT-2 toxin, and zearalenone among various other metabolites. Portions of the same figs were reextracted and then analyzed by conventional liquid chromatography–mass spectrometry. On the basis of coincident retention times and by matching selected ion monitoring for coincident ions with that of authentic standards, the identification of fumonisin B2, HT-2 toxin, patulin, and zearalenone was confirmed.

Paradigm shifts in fungal secondary metabolite research

The 8th International Mycological Congress (IMC8; Cairns, Australia) hosted several plenary lectures, poster presentations, and even entire symposia dedicated to fungal secondary metabolites (extrolites). These advances, presented in this special issue, together demonstrated how the impact of molecular biology and genomics and the availability of sophisticated methods of analytical chemistry has resulted in paradigm shifts in our understanding of fungal secondary metabolism and its key role in fungal biology. Rather than focus on classical topics such as discovery of novel drug candidates and identification of toxins, here we address two major themes in this special issue: (1) the utility and importance of secondary metabolites and their genes in polyphasic taxonomy, phylogeny, and evolutionary history of kingdom Fungi (syn. Eumycota); and (2) the genetic processes regulating secondary metabolite biosynthesis. The history of fungal chemotaxonomy and some important classes of secondary metabolites are reviewed.

Probable Toxin Induced Polyneuropathy- Case Report of Six Individuals From A Urban Family of Lucknow

Polyneuropathies due to toxins continue to pose a challenge to the medical profession. Early suspicion of the condition, correct identification of the toxin and timely institution of therapy is not only life saving but can also greatly reduce the morbidity burden of the condition. We report six cases (2 females, 4 males) belonging to the same family and presenting with similar features of an acute to subacute onset, symmetrical, predominantly distal, graded, painful, sensori-motor polyneuropathy. All these patients complained of recurrent episodes of vomiting which started within 30 minutes to two hours of ingestion of home cooked vegetarian meal. After a variable period of 2–7 days the symptoms of polyneuropathy appeared. Electrophysiological studies demonstrated an axonal type of sensori-motor polyneuropathy. The temporal onset of symptoms, involvement of several individuals at the same time and a consistent and similar pattern of clinical findings were features highly suggestive of a toxin etiology. Using a structured and a logical approach in the background of the characteristic clinical picture the diagnostic possibilities were significantly narrowed down. Since the patients presented late and due to financial and technical constraints all tests could not be performed the toxin could not be confirmed. doi: 10.5214/ans.0972.7531.2007.140405

Approaching the Golden Age of Natural Product Pharmaceuticals from Venom Libraries: An Overview of Toxins and Toxin-Derivatives Currently Involved in Therapeutic or Diagnostic Applications

Poisons and the toxins found in venomous and poisonous organisms have been the focus of much research over the past 70 years, most of which has been directed at understanding the biochemical and physiological mechanisms by which they elicit their dramatic pathological consequences. Much knowledge has been gained in terms of how poisons and venoms and their composite toxins give rise to the syndromes associated with envenoming and poisoning and in some isolated cases there have been a few such agents promoted for therapeutic use. However, it has only been in the past decade that an explosion of interest has occurred in mining these natural, highly evolved libraries of bioactive toxins and poisons for use in pharmacotherapeutics as drugs or drug leads as well as in diagnostic applications. We ascribe this recent phenomenon to advances in toxinology which have provided investigators with a relatively thorough understanding of the nature of venoms and their biologically active toxins: particularly with regard to the peptidomes and proteomes of venoms. This is in conjunction with our greatly improved understanding of the etiology of many human diseases and the identification of sites of potential therapeutic intervention. In this review we provide an overview of some of the toxins, toxin derivatives or poisons from animal venoms and secretions which are in various stages of development for use as pharmaceuticals or diagnostics in human diseases. As one will recognize, developments in this field suggest that toxinology is now entering a golden age in terms of the identification and use of toxins as potent novel pharmaceuticals.

Coupling Immunomagnetic Separation on Magnetic Beads with Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Detection of Staphylococcal Enterotoxin B

The growing importance of mass spectrometry for the identification and characterization of bacterial protein toxins is a consequence of the improved sensitivity and specificity of mass spectrometry-based techniques, especially when these techniques are combined with affinity methods. Here we describe a novel method based on the use of immunoaffinity capture and matrix-assisted laser desorption ionization-time of flight mass spectrometry for selective purification and detection of staphylococcal enterotoxin B (SEB). SEB is a potent bacterial protein toxin responsible for food poisoning, as well as a potential biological warfare agent. Unambiguous detection of SEB at low-nanogram levels in complex matrices is thus an important objective. In this work, an affinity molecular probe was prepared by immobilizing anti-SEB antibody on the surface of para-toluene-sulfonyl-functionalized monodisperse magnetic particles and used to selectively isolate SEB. Immobilization and affinity capture procedures were optimized to maximize the density of anti-SEB immunoglobulin G and the amount of captured SEB, respectively, on the surface of magnetic beads. SEB could be detected directly "on beads" by placing the molecular probe on the matrix-assisted laser desorption ionization target plate or, alternatively, "off beads" after its acidic elution. Application of this method to complex biological matrices was demonstrated by selective detection of SEB present in different matrices, such as cultivation media of Staphylococcus aureus strains and raw milk samples.

In this issue

In this issue

New Method for Characterizing Highly Disulfide-Bridged Peptides in Complex Mixtures: Application to Toxin Identification from Crude Venoms

Animal venoms are highly complex mixtures that can contain many disulfide-bridged toxins. This work presents an LC-MALDI approach allowing (1) a rapid classification of toxins according to their number of disulfide bonds and (2) a rapid top-down sequencing of the toxins using a new MALDI matrix enhancing in-source decay (ISD). The crude venom is separated twice by LC: the fractions of the first separation are spotted on the MALDI matrix alpha-cyano-4-hydroxycinnamic acid (CHCA) and the others using 1,5-diaminonaphthalene (1,5-DAN). CHCA spots are more convenient for obtaining a precise mass fingerprint of a large number of peptides; however, the analysis of 1,5-DAN spots allows the number of disulfide bridges to be counted owing to their partial in-plume reduction by this particular matrix. Subsequently, the disulfide bonds of all peptides present in the crude venom were reduced by an excess of tris(carboxyethyl)phosphine before the LC separation and were subjected to the same analysis in CHCA and 1,5-DAN. Toxins were sequenced using a TOF/TOF analysis of metastable fragments from CHCA spots and ISD fragmentation from 1,5-DAN spots. Novel conotoxin sequences were found using this approach. The use of 1,5-DAN for ISD top-down sequencing is also illustrated for higher molecular weight toxins such as snake cardiotoxins and neurotoxins (>6500 Da), where sequence coverage >70% is obtained from the c-ion series.

Identification of cyanobacterial toxins in Florida's freshwater systems

Toxigenic cyanobacteria are common components of Florida's surface waters and may pose a threat to their ecology and a risk to human health. The Lake County Water Authority, the Florida Department of Health, the St. Johns River Water Management District, the South Florida Water Management District, and the Lee County Health Department have all recently monitored local waters for cyanotoxins. In 2004, 6 central Florida lakes were sampled monthly for 12 months at centrally located open water sample sites. Toxin data (n = 72 for all toxin analyses) showed that microcystins (0.1–3.6 μg/L) were present in all 12 months; multiple bloom events per lake were observed; bloom concentrations ranged from 5–7,500 μg/L; and blooms occurred throughout the entire year. Cylindrospermopsin, ranging in concentration from 0.05 to 0.2 μg/L, was reported in 22% of all samples analyzed and occurred predominantly between August and December. Anatoxin-a was not reported in 2003/2004 but was identified in 2002/2003 (30%, 0.05−7.0 μg/L). Data from the St. Johns River was similar to that of the central Florida lakes. In general, microcystins ranged between 0.1 and 31 μg/L and were detected in all samples (n = 50), while cylindrospermopsin was observed less frequently (30%, n = 48) and at lower concentrations (0.07−1.6 μg/L). Anatoxin-a was not reported (n = 48). In contrast, the major Microcystis spp. blooms that occurred in the St. Johns, the St. Lucie, and the Caloosahatchee rivers in 2005 were reported to contain relatively high levels of microcystins (rangemax. conc.= 278–5700 μg/L) and persisted for approximately 2 months. Data indicate that cyanotoxin production can be a year-round phenomenon in Florida and can occur at levels that may cause ecological and human health problems.

Modern instrumental methods in forensic toxicology.

This article reviews modern analytical instrumentation in forensic toxicology for identification and quantification of drugs and toxins in biological fluids and tissues. A brief description of the theory and inherent strengths and limitations of each methodology is included. The focus is on new technologies that address current analytical limitations. A goal of this review is to encourage innovations to improve our technological capabilities and to encourage use of these analytical techniques in forensic toxicology practice.

Identification of insect-selective and mammal-selective toxins from Parabuthus leiosoma venom

Venoms were collected from two scorpion species: Parabuthus leiosoma and Parabuthus pallidus from Kenya. Subcutaneous injection and oral toxicity tests of crude and pure fractions of scorpion venoms were done in Mus musculus (mice), Chilo partellus and Busseola fusca. The highest activity against C. partellus was found in P. leiosoma venom (LC 50 0.689 mg/50 mg body weight). Bioassay-guided purification by a combination of cation-exchange (CE) and reverse-phase high-performance liquid chromatography (RP-HPLC) led to the isolation of three toxic peptides. A lepidopteran-selective toxin ( P. leiosoma insect toxin, Plit) was isolated, and the partial N-terminal amino acid sequence (-KDGYPVDNANCKYE-) plus the molecular weight (6688.5 Da) determined. A peptide with significant insect toxicity coupled with mild effects on mice ( P. leiosoma toxin, Plt) was isolated, and the partial N-terminal amino acid sequence (-LCEKFKVQRLVELNCVD-) plus the molecular weight (6742.5 Da) was determined. Another toxin with anti-mammalian activity ( P. leisoma mammal-selective toxin, Plmt), and N-terminal partial amino acid sequence of ADVPGNYPLDKNGNRYY- plus a molecular weight of 7145.5 Da was also isolated. Comparison of the partial N-terminal amino acid sequences with other toxins revealed that Plit shows high homology to other known insect toxins. Similarly, Plmt shows high homology with several birtoxin-like anti-mammalian toxins. Plt does not exhibit homology with any known scorpion toxin with combined anti-insect and anti-mammalian activity.

Identification and characterization of a novel toxin–antitoxin module from Bacillus anthracis

Comparative genome analysis of Bacillus anthracis revealed a pair of linked genes encoding pemK (K, killer protein) and pemI (I, inhibitory protein) homologous to pem loci of other organisms. Expression of PemK in Escherichia coli and Bacillus anthracis was bacteriostatic whereas the concomitant expression of PemI reversed the growth arrest. PemK expression effectively inhibited protein synthesis with no significant effect on DNA replication. Coexpression and interaction of these proteins confirmed it to be a Type II addiction module. Thermal denaturation analysis reflected poor conformational stability of PemI as compared to PemK. Circular dichroism analysis indicated that PemI contains twice the amount of β-sheets as PemK. Gel retardation assays demonstrated that PemI binds to its upstream DNA sequence. This study reports the first evidence of an active chromosome encoded toxin–antitoxin locus in B. anthracis.

Adduct simplification in the analysis of cyanobacterial toxins by matrix‐assisted laser desorption/ionization mass spectrometry

A novel method for simplifying adduct patterns to improve the detection and identification of peptide toxins using matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry is presented. Addition of 200 microM zinc sulfate heptahydrate (ZnSO(4) . 7H(2)O) to samples prior to spotting on the target enhances detection of the protonated molecule while suppressing competing adducts. This produces a highly simplified spectrum with the potential to enhance quantitative analysis, particularly for complex samples. The resulting improvement in total signal strength and reduction in the coefficient of variation (from 31.1% to 5.2% for microcystin-LR) further enhance the potential for sensitive and accurate quantitation. Other potential additives tested, including 18-crown-6 ether, alkali metal salts (lithium chloride, sodium chloride, potassium chloride), and other transition metal salts (silver chloride, silver nitrate, copper(II) nitrate, copper(II) sulfate, zinc acetate), were unable to achieve comparable results. Application of this technique to the analysis of several microcystins, potent peptide hepatotoxins from cyanobacteria, is illustrated.

Bacterial Colitis

Bacterial colitis results in an inflammatory-type diarrhea that is characterized by bloody, purulent, and mucoid stool. These diseases have been designated as bacterial hemorrhagic enterocolitis. Associated symptoms include fever, tenesmus, and severe abdominal pain. The pathologic changes range from superficial exudative enterocolitis to a transmural enterocolitis with ulceration. Common pathologic bacteria causing bacterial colitis include Campylobacter, Salmonella, Shigella, Escherichia, and Yersinia species. The primary source of transmission is fecal-oral spread and ingestion of contaminated food and water. Although detailed history and identification of specific risk factors assist in the diagnosis, definitive diagnosis requires bacterial identification. Therefore, the physician must be familiar with the disease pathophysiology, epidemiology, and specific diagnostic modalities for clinical diagnosis and management. Specific tests are used to detect enteric pathogens and include stool and rectal swab culture, histology, and identification of specific bacterial toxins. Although many of these bacterial colitis infections are self-limiting, antibiotics should be used for high-risk patients and patients with complicated disease.

A quantitative real-time immuno-PCR approach for detection of staphylococcal enterotoxins

Bacterial intoxications represent a substantial public health concern with enterotoxins produced by Staphylococcus aureus among the most common causes of food poisoning. In addition to their role in the pathogenicity of food poisoning, staphylococcal enterotoxins have profound effects on the immune system as members of the family of pyrogenic toxin superantigens. As the classical diagnostic bioassays as well as the routinely used immunological methods are hampered by several drawbacks regarding sensitivity, specificity, and practicability, there is a need for the timely identification of toxins by highly sensitive and specific methods. To combine the versatility of an enzyme immunoassay (EIA) with the amplification power of the PCR, a quantitative real-time immuno-PCR (qRT-iPCR) was developed for the detection of staphylococcal enterotoxins A and B and compared to a commercially available EIA. A broadly applicable tool for signal amplification of pre-formed immunocomplexes was established by covalent binding of a reporter DNA to secondary detection antibodies. Therefore, the amino-modified reporter DNA was coupled successfully to N-succinimidyl-S-actyl-thioacetate-activated secondary detection antibodies. The qRT-iPCR was able to detect highly reproducibly as low as approximately 0.6 to 6 pg (4 to 40 amol/microl) of staphylococcal enterotoxin B and staphylococcal enterotoxin A, respectively. In conclusion, the qRT-iPCR approach was shown to overcome clearly the sensitivity limit of traditional immunological detection procedures for bacterial toxins, as demonstrated in this study for staphylococcal enterotoxins. The development of a stable antibody-DNA conjugate providing a universal signal amplification offers a versatile as well as a highly sensitive and specific tool for diagnostic and research purposes generally applicable for pre-formed antibody-antigen complexes.

Identification and Characterization of an RTX Toxin in the Emerging Pathogen Kingella kingae

Kingella kingae is an emerging bacterial pathogen that is increasingly recognized as the causative agent of a variety of pediatric diseases, including septic arthritis and osteomyelitis. The pathogenesis of K. kingae disease is believed to begin with colonization of the upper respiratory tract. In the present study, we examined interactions between K. kingae and cultured respiratory epithelial cells and observed potent cytotoxicity, detected by both microscopy and lactic acid dehydrogenase (LDH) release assays. Experiments with synovial and macrophage cell lines revealed cytotoxicity for these cell types as well. Using mariner mutagenesis and a screen for loss of cytotoxicity, a genetic locus encoding an RTX toxin system was identified. Disruption of the K. kingae RTX locus resulted in a loss of cytotoxicity for respiratory epithelial, synovial, and macrophage cell lines. DNA sequence analysis demonstrated that the RTX locus is flanked by insertion elements and has a reduced G+C content compared to that of the whole genome. Two relatively less invasive Kingella species, K. oralis and K. denitrificans, were found to be noncytotoxic and to lack the RTX region, as determined by LDH release assays and Southern blotting. We concluded that K. kingae expresses an RTX toxin that has wide cellular specificity and was likely acquired horizontally. The possible roles for this toxin in the pathogenesis of K. kingae disease include breaching of the epithelial barrier and destruction of target tissues, such as synovium (joint lining).

C difficile—C’est Difficile?

C difficile—C’est Difficile?

The zebrafish ( Danio rerio) embryo as a model system for identification and characterization of developmental toxins from marine and freshwater microalgae

The zebrafish ( Danio rerio) embryo has emerged as an important model of vertebrate development. As such, this model system is finding utility in the investigation of toxic agents that inhibit, or otherwise interfere with, developmental processes (i.e. developmental toxins), including compounds that have potential relevance to both human and environmental health, as well as biomedicine. Recently, this system has been applied increasingly to the study of microbial toxins, and more specifically, as an aquatic animal model, has been employed to investigate toxins from marine and freshwater microalgae, including those classified among the so-called “harmful algal blooms” (HABs). We have developed this system for identification and characterization of toxins from cyanobacteria (i.e. “blue-green algae”) isolated from the Florida Everglades and other freshwater sources in South and Central Florida. Here we review the use of this system as it has been applied generally to the investigation of toxins from marine and freshwater microalgae, and illustrate this utility as we have applied it to the detection, bioassay-guided fractionation and subsequent characterization of developmental toxins from freshwater cyanobacteria.

Simplified enrichment and identification of environmental peptide toxins using antibody-capture surfaces with subsequent mass spectrometry detection

The development of a simplified assay for detection of congeners of the microcystin (MC) hepatotoxin is described that combines the extreme sensitivity of surface-enhanced laser desorption/ionization time-of-flight MS (SELDI TOF-MS) with the superior selectivity of immunoaffinity interactions. Using methods similar to those of conventional immunoassays, MC standards were captured and enriched on immunoreactive ProteinChips coated with an MC-antibody and analyzed by TOF-MS. Unlike with conventional immunoassays, individual congeners were resolved from mixed pools. Assay conditions were optimized for the quantification of MC from untreated raw pond water at concentrations as low as 0.025 μg L −1, well below the public health relevant guideline of 1 μg L −1.

Toxicity and toxin identification in Colomesus asellus, an Amazonian (Brazil) freshwater puffer fish

Toxicity and toxin identification in Colomesus asellus, an Amazonian (Brazil) freshwater puffer fish. By using four different techniques—mouse bioassay, ELISA, HPLC and mass spectrometry—we evaluated the toxicity in the extracts of C. asellus, a freshwater puffer fish from the rivers of the Amazon, and identified for the first time the components responsible for its toxicity. The T20G10 monoclonal antibody raised against TTX, and employed in an indirect competitive enzyme immunoassay, showed very low affinity for the C. asellus extracts, indicating that TTX and its analogs are not the main toxic components of the extracts. This antibody was efficient in detecting presence of TTX in a total extract of Sphoeroides spengleri, which is one of the most toxic puffer fish found in the Atlantic coast. Extracts of C. asellus were toxic when administered intraperitonially into mice with an average toxicity of 38.6±12 mouse unit (MU)/g, while HPLC analysis indicated a lower toxin content (7.6±0 5 MU/g). The HPLC profile showed no traces of TTX, but only the presence of PSPs (STX, GTX 2 and GTX 3). These toxins were also confirmed by electrospray ionization mass spectrometry.