Action of citreoviridin, a mycotoxin from Penicillium citreoviride on the gamma-aminobutyric acid metabolism of the central nervous system

Nanomaterial-Based Biosensors for Food Toxin Detection

Abstract: There is an increased interest toward the development of bioelectronic devices for food toxin (mycotoxins) detection. Mycotoxins are highly toxic secondary metabolites produced by fungi like Fusarium, Aspergillus, and Penicillium that are frequently found in crops or during storage of food including cereals, nuts, fruits, etc. The contamination of food by mycotoxins has become a matter of increasing concern. High levels of mycotoxins in the diet can cause adverse, acute, and chronic effects on human health and a variety of animal species. Side effects may particularly affect the liver, kidney, nervous system, endocrine system, and immune system. Among 300 mycotoxins known till date, there are a few that are considered to play an important part in food safety, and for these, a range of analytical methods have been developed. Some of the important mycotoxins include aflatoxins, ochratoxins, fumonisins, citreoviridin, patulin, citrinin, and zearalenon. The conventional methods of analysis of mycotoxins normally require sophisticated instrumentation, e.g., liquid chromatography with fluorescence or mass detectors, combined with extraction procedures for sample preparation. Hence, new analysis tools are necessary to attain more sensitive, specific, rapid, and reliable information about the desired toxin. For the last about two decades, the research and development of simpler and faster analytical procedures based on affinity biosensors has aroused much interest due to their simplicity and sensitivity. The nanomaterials have recently had a great impact on the development of biosensors. The functionalized nanomaterials are used as catalytic tools, immobilization platforms, or as optical or electroactive labels to improve the biosensing performance to obtain higher sensitivity, stability, and selectivity. Nanomaterials, such as carbon nanomaterials (carbon nanotubes and graphene), metal nanoparticles, nanowires, nanocomposites, and nanostructured metal oxide nanoparticles are playing an increasing role in the design of sensing and biosensing systems for mycotoxin determination. Furthermore, these nanobiosystems are also bringing advantages in terms of the design of novel food toxin detection strategies. We will focus on some of the recent results related to fabrication of nanomaterial-based biosensors for food toxin detection obtained in our laboratories.

Chemotaxonomy of Pochonia and other conidial fungi with Verticillium -like anamorphs

Abstract: Pochonins are antiviral and antiparasitic resorcylic acid lactones (RAL) structurally related to monorden. They were found in the invertebrate-associated fungus Pochonia chlamydosporia. Their production and distribution was studied by means of High Performance Liquid Chromatography with UV-visual and mass spectrometric detection (HPLC-UV/Vis and HPLCMS) in cultures of Pochonia species and further conidial fungi with Verticillium-like anamorphs that had until recently been included in Verticillium sect. Prostrata. The results support the recent generic segregation by Gams, Zare and co-workers because pochonins were found to occur exclusively in species of the genus Pochonia. With few exceptions, the production of RAL appeared to be a rather constant feature in cultures of P. chlamydosporia from around the world. According to preliminary results, secondary metabolite profiles in strains of allied genera such as Lecanicillium, Haptocillium and Rotiferophthora are different from those encountered in Pochonia. The alkaloid pseurotin A was found as main metabolite in several of the P. chlamydosporia isolates examined. As inferred from HPLC profiling data, strains of P. suchlasporia clustered into at least three chemotypes. The ex-type strain of P. suchlasporia var. catenata produced monorden, while several other strains produced metabolites whose HPLC-UV and HPLC-MS characteristics were similar to the mycotoxins, aurovertin B and citreoviridin A. Yet different metabolites were detected in a third chemotype of P. suchlasporia. Differences in secondary metabolite profiles were also found in two strains of P. bulbillosa. While the ex-type strain was found devoid of all aforementioned compounds, CBS 247.68 contained the aurovertin-related metabolites detected in part of the P. suchlasporia isolates. The sequence of the ITS nrDNA of CBS 247.68 was different from that of the type strain but identical to the sequences of P. suchlasporia var. catenata. Several strains of the latter variety showed identical sequences, despite considerable variations in their HPLC metabolite profiles. Minisatellite PCR fingerprinting was found useful to segregate Pochonia at species and strain level, pointing toward the existence of further, cryptic species. The possible chemotaxonomical importance and ecological functions of secondary metabolites in these fungi is discussed.

Animaux aquatiques dangereux

Abstract: Resume Les especes aquatiques dangereuses, vertebrees et invertebrees, sont pour la plupart marines. Les especes venimeuses injectent un venin ; ce sont des eponges, des vers, des oursins et etoiles de mer, des poulpes, et, les plus dangereuses, des meduses, des mollusques (les cones), des serpents (dans la region indopacifique) et plusieurs centaines d'especes de poissons (raies-armees, vives, rascasses, poissons-pierre…). Les especes veneneuses provoquent une intoxication lorsqu'elles sont ingerees ; ce sont, notamment, des bivalves dans le cas ou ils se sont nourris de certains Dinoflagelles (algues unicellulaires), et des poissons (clupeides, murenes, thons, tetraodons ou « fugu »…). La ciguatera est un empoisonnement du a l'ingestion d'animaux marins du recif corallien, en particulier de poissons. Certains animaux sont dangereux par leurs morsures (requins, barracudas…). Les animaux et leur biologie sont presentes ; l'appareil inoculateur dans le cas des animaux venimeux est decrit. La composition du venin est indiquee quand elle est connue. Les circonstances de l'accident, la clinique et le traitement de l'empoisonnement sont decrits.

Produção, purificação e caracterização espectrométrica da micotoxina citreoviridina produzida pelo Penicillium citreonigrum em meio de cultura YES (Yeast Extract Sucrose)

Abstract: Mycotoxins are secondary metabolites produced by fungi, which may develop in agricultural commodities in the field and/or during transport and storage. Citreoviridin, a mycotoxin produced mainly by Penicillium citreonigrum, was first isolated in 1947 in Japan during the investigation of the “yellow rice” disease, which caused acute cardiac beriberi. In May 2006, an outbreak of beriberi was reported in the State of Maranhão, Brazil, and rice samples collected in the region, was shown to be positive for P. citreonigrum and citreoviridin. The main objective of this work was to establish a protocol for the production and purification of citreoviridin in order to yield enough high purity material to conduct toxicological studies that would help to elucidate the events that occurred in Maranhão. Citreoviridin was produced by P. citreonigrum in 48 flasks of 500 mL YES liquid culture medium for 8 days at 25oC. The toxin was extracted with chloroform, purified by semi-preparative HPLC from the liquid medium and the mycelium of 48 culture flasks, and fully characterized by UV/VIS, FT-IR, H and C NMR, LC-MS/MS and LC-MSD TOF. Citreoviridin concentration was determined by HPLC/PDA against a commercial standard. In average, 29.7 and 85.7 mg of citreoviridin were recovered from the liquid medium and mycelium, respectively, yielding a total of 5.5 g citreoviridin from 15.3 g of crude extract. About 12 g of this extract was purified yielding 3.25 g of purified citreoviridin (27.1% yield), which identity was confirmed by the spectrometric analyses. Chromatographic and gravimetric analysis showed that the purified citreoviridin was 100% pure.

Determination of serum proteins by means of the biuret reaction.

Abstract: In the course of an investigation of the biochemical changes following experimental liver injury we felt the need of a simple, rapid, and accurate method for determining the protein fractions in small amounts of serum. Among the simpler procedures known, the biuret reaction seemed to offer the most encouraging possibilities. Variations and improvements in the application of the biuret reaction to clinical chemistry can be traced in the works of Autenrieth (l), Hiller (2), Fine (3), Kingsley (4), and Robinson and Hogden (5). Kingsley (6) simplified the technique by adding serum directly to a “one piece” reagent. Efforts have been made to increase the stability of such biuret reagents with ethylene glycol (7), tartrate (8), and citrate (9)) We began our investigation with Kingsley’s (6) method and report briefly on the two main difficulties encountered in its use. The first is that the total protein (TP) reagent and, to a lesser extent, the albumin (ALB) reagent are not sufficiently stable. The length of time they remain so depends upon the technique of their preparation. One consequence of this variable stability is a difficulty in duplicating calibration curves with different lots of reagent. Errors may arise when results with a new reagent are read from an old calibration curve. Serious errors occur if a reagent is used after the separation of any black deposit gives evidence of deterioration. A second difficulty has been that total protein estimations made with the TP reagent and read, as prescribed, from calibration curves prepared with the ALB reagent have tended to be too low. Recorded in Table I are the results of a number of analyses in which Kingsley’s biuret procedure has been compared with the Kjeldahl method2 on both normal and ab-

Free amino acids and related compounds in dog brain: post-mortem and anoxic changes, effects of ammonium chloride infusion, and levels during seizures induced by picrotoxin and by pentylenetetrazol.

Abstract: PREVIOUS studies in this laboratory have been concerned with chemical changes in the brain associated with convulsive activity (STONE et al., 1960) as revealed by analysis of brain tissue after in vivo fixation with liquid air. The development by MOORE and STEIN (1954) of ion exchange procedures for the separation of amino acids has made it feasible to extend the study of cerebral constituents during convulsions to include many free amino acids and related substances. These compounds are of particular interest in view of recent investigations by GEIGER and his coworkers (1960 a, 6) indicating that protein turnover in the brain is accelerated during convulsive activity induced by pentylenetetrazol. Accordingly, the cerebral levels of a large number of nitrogenous constituents have been determined in control experiments and during seizures induced by picrotoxin or by pentylenetetrazol. In an attempt to classify different types of seizures on the basis of specific chemical patterns (STONE et al., 1960), the seizures induced by these two agents have been tentatively classed together as representing a type in which distinctive metabolic changes have not yet been observed. As an aid in the interpretation of any changes which might be observed during seizures or other conditions, it was considered essential to study also post-mortem changes and the effects of anoxia and of ammonium chloride infusion.

The role of gaba metabolism in the convulsant and anticonvulsant actions of aminooxyacetic acid

Abstract: — At high dosage levels AOAA acted as a convulsant agent in mice and rats but in lower amounts it was an effective anticonvulsant agent against INH-induced seizures, by tripling the time to the onset of the convulsions. AOAA elevated brain GABA levels as a result of a preferential inhibition of the GABA-T enzyme system but, contrary to previous reports, the activity of the GAD enzyme system was also inhibited, even by relatively low dosage levels of AOAA. The state of excitability of the brain following the administration of AOAA was related, within the limits of the present study, to changes in GAD activity and GABA levels, but additional data are required before the relationship can be properly evaluated.

Actions of Tremorgenic Fungal Toxins on Neurotransmitter Release

Abstract: The neurochemical effects of the tremorgenic mycotoxins Verruculogen and Penitrem A, which produce a neurotoxic syndrome characterised by sustained tremors, were studied using sheep and rat synaptosomes. The toxins were administered in vivo, either by chronic feeding (sheep) or intraperitoneal injection 45 min prior to killing (rat), and synaptosomes were subsequently prepared from cerebrocortical and spinal cord/medullary regions of rat, and corpus striatum of sheep. Penitrem A (400 mg mycelium/kg) increased the spontaneous release of endogenous glutamate, GABA (gamma-aminobutyric acid), and aspartate by 213%, 455%, and 277%, respectively, from cerebrocortical synaptosomes. Verruculogen (400 mg mycelium/kg) increased the spontaneous release of glutamate and aspartate by 1300% and 1200%, respectively, but not that of GABA from cerebrocortical synaptosomes. The spontaneous release of the transmitter amino acids or other amino acids was not increased by the tremorgens in spinal cord/medullary synaptosomes. Penitrem A pretreatment reduced the veratrine (75 microM) stimulated release of glutamate, aspartate, and GABA from cerebrocortical synaptosomes by 33%, 46%, and 11%, respectively, and the stimulated release of glycine and GABA from spinal cord/medulla synaptosomes by 67% and 32% respectively. Verruculogen pretreatment did not alter the veratrine-induced release of transmitter amino acids from cerebrocortex and spinal cord/medulla synaptosomes. Penitrem A pretreatment increased the spontaneous release of aspartate, glutamate, and GABA by 68%, 62%, and 100%, respectively, from sheep corpus striatum synaptosomes but did not alter the synthesis and release of dopamine in this tissue. Verruculogen was shown to cause a substantial increase (300-400%) in the miniature-end-plate potential (m.e.p.p.) frequency at the locust neuromuscular junction. The response was detectable within 1 min, rose to a maximum within 5-7 min, and declined to the control rate over a similar period. No change in the amplitude of the m.e.p.p.'s was observed. These effects of the tremorgens on transmitter release are interpreted in terms of their mode of action.

Glutamic acid decarboxylase inhibition and ultrastructural changes by the convulsant drug allylglycine.

Abstract: The effects in vivo and vitro of the convulsant drug allylglycine on the activity of glutamic acid decarboxylase (GAD), aminobutyrate aminotransferase, glutamine synthetase and aspartate- and alanine-aminotransferases of the rat cerebral cortex were studied. The most significant result was the finding of an inhibition of GAD, during the period of convulsion, which was even greater by addition in vitro of the drug. This inhibition is not by way of the cofactor, pyridoxal phosphate. Preincubation of the homogenate with allylglycine enhanced the inhibition, while preincubation in buffer-substrate produced a protective effect. The inhibition of GAD was correlated with a decrease of 40 per cent in the concentration of γ-aminobutyric acid in the cerebral cortex. In the convulsant rat, ultra-structural alterations of some nerve endings of the cerebral cortex were observed. After cell fractionation, such altered nerve endings were preferentially found in the GAD-rich (nonaminergic) fraction of isolated nerve endings. The possible mechanism of the convulsions induced by allyglycine is discussed and a specific effect on GAD-rich inhibitory nerve endings is postulated.