Identification of genes implicated in toxin production in the cyanobacterium Cylindrospermopsis raciborskii

Cylindrospermopsis raciborskii is a bloom-forming cyanobacterium found in both tropical and temperate climates which produces cylindrospermopsin, a potent hepatotoxic secondary metabolite. This organism is notorious for its association with a significant human poisoning incident on Palm Island, Australia, which resulted in the hospitalization of 148 people. We have screened 13 C. raciborskii isolates from various regions of Australia and shown that both toxic and nontoxic strains exist within this species. No association was observed between geographical origin and toxin production. Polyketide synthases (PKSs) and peptide synthetases (PSs) are enzymes involved in secondary metabolite biosynthesis in cyanobacteria. Putative PKS and PS genes from C. raciborskii strains AWT205 and CYPO2OB were identified by PCR using degenerate primers based on conserved regions within each gene. Examination of the strain-specific distribution of the PKS and PS genes in C. raciborskii isolates demonstrated a direct link between the presence of these two genes and the ability to produce cylindrospermopsin. Interestingly, the possession of these two genes was also linked. They were also identified in an Anabaena bergii isolate that was demonstrated to produce cylindrospermopsin. Taken together, these data suggest a likely role for these determinants in secondary metabolite and toxin production by C. raciborskii. (C) 2001 John Wiley & Sons, Inc.

Human Fatalities from Cyanobacteria: Chemical and Biological Evidence for Cyanotoxins

An outbreak of acute liver failure occurred at a dialysis center in Caruaru, Brazil (8 degrees 17 'S, 35 degrees 58 'W), 134 km from Recife, the state capital of Pernambuco. At the clinic, 116 (89%) of 131 patients experienced visual disturbances, nausea, and vomiting after routine hemodialysis treatment on 13-20 February 1996. Subsequently, 100 patients developed acute liver failure, and of these 76 died. As of December 1996, 52 of the deaths could be attributed to a common syndrome now called Caruaru syndrome. Examination of phytoplankton from the dialysis clinic's water source, analyses of the clinic's water treatment system, plus serum and liver tissue of clinic patients led to the identification of two groups of cyanobacterial toxins, the hepatotoxic cyclic peptide microcystins and the hepatotoxic alkaloid cylindrospermopsin. Comparison of victims' symptoms and pathology using animal studies of these two cyanotoxins leads us to conclude that the major contributing factor to death of the dialyses patients was intravenous exposure to microcystins, specifically microcystin-YR, -LR, and -AR. From liver concentrations and exposure volumes, it was estimated that 19.5 mug/L microcystin was in the water used for dialysis treatments. This is 19.5 times the level set as a guideline for safe drinking water supplies by the World. Health Organization.

Toxicological aspects of treatment to remove cyanobacterial toxins from drinking water determined using the heterozygous P53 transgenic mouse model

The presence of toxic cyanobacteria in drinking water reservoirs renders the need to develop treatment methods for the 'safe' removal of their associated toxins. Chlorine has been shown to successfully remove a range of cyanotoxins including microcystins, cylindrospermopsin and saxitoxins. Each cyanotoxin requires specific treatment parameters, particularly solution pH and free chlorine residual. However, currently there has not been any investigation into the toxicological effect of solutions treated for the removal of these cyanotoxins by chlorine. Using the P53(def) transgenic mouse model mate and female C57BL/6J hybrid mice were used to investigate potential cancer inducing effects from such oral dosing solutions. Both purified cyanotoxins and toxic cell-free extract cyanobacterial solutions were chlorinated and administered over 90 and 170 days (respectively) in drinking water. No increase in cancer was found in any treatment. The parent cyanotoxins, microcystins, cylindrospermopsin and saxitoxins were readily removed by chlorine. There was no significant increase in the disinfection byproducts trihalomethanes or haloacetic acids, levels found were well below guideline values. Histological examination identified no effect of treatment solutions except male mice treated with chlorinated cylindrospermopsin (as a cell free extract). In this instance 40% of males were found to have fatty vacuolation in their livers, cause unknown. It is recommended that further toxicology be undertaken on chlorinated cyanobacterial solutions, particularly for non-genotoxic carcinogenic compounds, for example the Tg. AC transgenic mouse model. (C) 2003 Elsevier Science Ltd. All rights reserved.

Are known cyanotoxins involved in the toxicity of picoplanktonic and filamentous north atlantic marine cyanobacteria?

Eight marine cyanobacteria strains of the genera Cyanobium, Leptolyngbya, Oscillatoria, Phormidium, and Synechococcus were isolated from rocky beaches along the Atlantic Portuguese central coast and tested for ecotoxicity. Strains were identified by morphological characteristics and by the amplification and sequentiation of the 16S rDNA. Bioactivity of dichloromethane, methanol and aqueous extracts was assessed by the Artemia salina bioassay. Peptide toxin production was screened by matrix assisted laser desorption/ionization time of flight mass spectrometry. Molecular analysis of the genes involved in the production of known cyanotoxins such as microcystins, nodularins and cylindrospermopsin was also performed. Strains were toxic to the brine shrimp A. salina nauplii with aqueous extracts being more toxic than the organic ones. Although mass spectrometry analysis did not reveal the production of microcystins or other known toxic peptides, a positive result for the presence of mcyE gene was found in one Leptolyngbya strain and one Oscillatoria strain. The extensive brine shrimp mortality points to the involvement of other unknown toxins, and the presence of a fragment of genes involved in the cyanotoxin production highlight the potential risk of cyanobacteria occurrence on the Atlantic coast.

Produção e qualidade dos frutos de pessegueiro e nectarineira em clima subtropical do estado de São Paulo

Tendo em vista que o conhecimento do comportamento de uma cultura, em determinada região, determina o sucesso da produção, a qualidade do produto final e a garantia de aceitação pelo mercado consumidor, o presente trabalho teve como objetivo avaliar a produção e as características físicas e físico-químicas de frutos de cultivares de pessegueiro e nectarineira, cultivados ou não tradicionalmente em São Paulo, mas que possam vir a apresentar uma opção de diversificação aos fruticultores do município de Botucatu-SP, e regiões climáticas semelhantes. O experimento foi realizado durante três ciclos agrícolas: 2006/2007, 2007/2008 e 2008/2009, na Faculdade de Ciências Agronômicas, UNESP, Botucatu-SP. O clima da região é do tipo mesotérmico, Cwa, ou seja, subtropical úmido com chuvas no verão e seca no inverno. As cultivares de pessegueiro avaliadas foram: Turmalina, Cascata 968, Cascata 848, Cascata 587, Conserva 693, Precocinho, Diamante Mejorado, Oro Azteca, CP-9553CYN e Tropic Beauty. A cultivar de nectarineira avaliada foi 'Sun Blaze'. Foram avaliadas características de produção e qualidade dos frutos. Os resultados obtidos permitiram concluir que a cultivar Turmalina foi a mais produtiva. As cultivares Cascata 968 e Diamante Mejorado apresentaram o maior teor de sólidos solúveis, e as cultivares Cascata 587 e CP 9553 CYN, a maior relação sólidos solúveis/acidez titulável.

Analyse quantitative des cyanotoxines d'eau douce par LDTD-APCI-MS/MS

Avec la hausse mondiale de la fréquence des floraisons de cyanobactéries (CB), dont certaines produisent des cyanotoxines (CT), le développement d’une méthode de détection/quantification rapide d’un maximum de CT s’impose. Cette méthode permettrait de faire un suivi quotidien de la toxicité de plans d’eau contaminés par des CB et ainsi d’émettre rapidement des avis d’alerte appropriés afin de protéger la santé publique. Une nouvelle technologie utilisant la désorption thermique induite par diode laser (LDTD) couplée à l’ionisation chimique sous pression atmosphérique (APCI) et reliée à la spectrométrie de masse en tandem (MS/MS) a déjà fait ses preuves avec des temps d'analyse de l’ordre de quelques secondes. Les analytes sont désorbés par la LDTD, ionisés en phase gazeuse par APCI et détectés par la MS/MS. Il n’y a donc pas de séparation chromatographique, et la préparation de l’échantillon avant l’analyse est minimale selon la complexité de la matrice contenant les analytes. Parmi les quatre CT testées (microcystine-LR, cylindrospermopsine, saxitoxine et anatoxine-a (ANA-a)), seule l’ANA-a a généré une désorption significative nécessaire au développement d’une méthode analytique avec l’interface LDTD-APCI. La forte polarité ou le poids moléculaire élevé des autres CT empêche probablement leur désorption. L’optimisation des paramètres instrumentaux, tout en tenant compte de l’interférence isobarique de l’acide aminé phénylalanine (PHE) lors de la détection de l’ANA-a par MS/MS, a généré une limite de détection d’ANA-a de l’ordre de 1 ug/L. Celle-ci a été évaluée à partir d’une matrice apparentée à une matrice réelle, démontrant qu’il serait possible d’utiliser la LDTD pour effectuer le suivi de l’ANA-a dans les eaux naturelles selon les normes environnementales applicables (1 à 12 ug/L). Il a été possible d’éviter l’interférence isobarique de la PHE en raison de sa très faible désorption avec l’interface LDTD-APCI. En effet, il a été démontré qu’une concentration aussi élevée que 500 ug/L de PHE ne causait aucune interférence sur le signal de l’ANA-a.

Leaf nutritional levels in peach and nectarine grown in subtropical climate

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Características dos frutos de cultivares de pessegueiros e nectarineira, no município de Botucatu – SP

The research had as purpose to evaluate the fruits physical-chemical characteristics of peach cultivars (CP-9553 CYN, CP951 C, Tropic Beauty, Cascata 953, Cascata 797, Cascata 587, Precocinho, Conserva 693, Diamante Mejorado, Turmalina e Marli and Sun Blaze nectarine at Botucatu, São Paulo, Brazil region. It were evaluated the fruits fresh matter, longitudinal and transversal diameters, and the crop period. In relation the physical-chemical characteristics of fruits, it was evaluated the texture, the pH, tritatable acid and soluble solids. The results showed significant differences between the cultivars where the fruits fresh matter were 68,6g ('CP-9553 CYN') and 141,35g ('Cascata 953'); the fruits longitudinal diameter were 50,32mm ('Marli') and 72,12mm ('Conserva 693') and the fruits transversal diameter were 50,50mm ('CP-9553 CYN') and 62,90mm ('Cascata 953'). The post-harvest characteristics also had significant differences. The texture had changes at 517,00gf ('Marli') and 168,47gf ('Precocinho'); the soluble solids were 9,73 degrees Brix on Marli cultivar. The pH was about 3,36 on 'Diamante Mejorado' until 4,48 on CP-9553 CYN cultivar and the acidity was 0,96g of citric acid 100g(-1) fruits juice on Tropic Beauty cultivar. The crop period of the cultivars evaluated were the end of September month until 15(th) November month, being the earliest cultivars Tropic Beauty, CP-951 and CP-951 CYN and the latest cultivar were Marli. It was observed that the news cultivars, until in observations, with Cascata 953, Cascata 797 and Cascata 587, showed crops potential at Botucatu/SP region.

Produção e qualidade dos frutos de pessegueiro e nectarineira em clima subtropical do estado de São Paulo

Tendo em vista que o conhecimento do comportamento de uma cultura, em determinada região, determina o sucesso da produção, a qualidade do produto final e a garantia de aceitação pelo mercado consumidor, o presente trabalho teve como objetivo avaliar a produção e as características físicas e físico-químicas de frutos de cultivares de pessegueiro e nectarineira, cultivados ou não tradicionalmente em São Paulo, mas que possam vir a apresentar uma opção de diversificação aos fruticultores do município de Botucatu-SP, e regiões climáticas semelhantes. O experimento foi realizado durante três ciclos agrícolas: 2006/2007, 2007/2008 e 2008/2009, na Faculdade de Ciências Agronômicas, UNESP, Botucatu-SP. O clima da região é do tipo mesotérmico, Cwa, ou seja, subtropical úmido com chuvas no verão e seca no inverno. As cultivares de pessegueiro avaliadas foram: Turmalina, Cascata 968, Cascata 848, Cascata 587, Conserva 693, Precocinho, Diamante Mejorado, Oro Azteca, CP-9553CYN e Tropic Beauty. A cultivar de nectarineira avaliada foi 'Sun Blaze'. Foram avaliadas características de produção e qualidade dos frutos. Os resultados obtidos permitiram concluir que a cultivar Turmalina foi a mais produtiva. As cultivares Cascata 968 e Diamante Mejorado apresentaram o maior teor de sólidos solúveis, e as cultivares Cascata 587 e CP 9553 CYN, a maior relação sólidos solúveis/acidez titulável.