RESUMEN
Changes to water conditions due to eutrophication and climate change have resulted in the proliferation of algae blooms in freshwater and marine environments globally, including in Canadian lakes. We developed and administered an online survey to evaluate the awareness of these blooms and the perceptions of health risks in a sample of New Brunswick waterfront cottage and homeowners. The survey was distributed to lake and cottage associations in New Brunswick and was completed by 186 eligible respondents (18 years of age or older). Participants were asked about the water quality of their lake, awareness about algae blooms, sociodemographic and cottage characteristics, and to complete a self-rated measure of physical and mental health. While approximately 73% of participants reported that the quality of their lake water was good or very good, 41% indicated a concern about algae blooms. We found no differences in self-reported physical or mental health between those who were aware of algae blooms at their cottage and those who were not (p > 0.05). Participants expressed concerns about the impacts of algae blooms on the health of their pets, and wildlife. While climate change was the most frequently identified cause of algae blooms, there was substantial heterogeneity in the responses. In addition, the reporting of the presence and frequency of algae bloom varied between respondents who lived on the same lake. Taken together, the findings from our survey suggest that cottage owners in New Brunswick are aware and concerned about the impacts of algae blooms, however, there is a need to provide additional information to them about the occurrence and causes of these blooms.
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Eutrofización , Lagos , Adolescente , Adulto , Humanos , Nuevo Brunswick , Calidad del AguaRESUMEN
BACKGROUND: Rates of lower respiratory tract infection (LRTI) among First Nations (FN) children living in Canada are elevated. We aimed to quantify indoor environmental quality (IEQ) in the homes of FN children in isolated communities and evaluate any associations with respiratory morbidity. METHODS: We performed a cross-sectional evaluation of 98 FN children (81 with complete data) aged 3 years or younger, living in 4 FN communities in the Sioux Lookout region of Northern Ontario. We performed medical chart reviews and administered questionnaires. We performed a housing inspection, including quantifying the interior surface area of mould (SAM). We monitored air quality for 5 days in each home and quantified the contaminant loading of settled floor dust, including endotoxin. We analyzed associations between IEQ variables and respiratory conditions using univariable and multivariable analyses. RESULTS: Participants had a mean age of 1.6 years and 21% had been admitted to hospital for respiratory infections before age 2 years. Houses were generally crowded (mean occupancy 6.6 [standard deviation 2.6, range 3-17] people per house). Serious housing concerns were frequent, including a lack of functioning controlled ventilation. The mean SAM in the occupied space was 0.2 m2. In multivariable modelling, there was evidence of an association of LRTI with log endotoxin (p = 0.07) and age (p = 0.02), and for upper respiratory tract infections, with SAM (p = 0.07) and age (p = 0.03). Wheeze with colds was associated with log endotoxin (p = 0.03) and age (p = 0.04). INTERPRETATION: We observed poor housing conditions and an association between endotoxin and wheezing in young FN children living in Northern Ontario.
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Contaminación del Aire Interior , Calidad de la Vivienda , Indígena Canadiense , Infecciones del Sistema Respiratorio/etnología , Infecciones del Sistema Respiratorio/epidemiología , Población Rural/estadística & datos numéricos , Preescolar , Estudios Transversales , Polvo , Endotoxinas/efectos adversos , Femenino , Hongos , Humanos , Masculino , Ontario/epidemiología , VentilaciónRESUMEN
Exposure to biocontaminants, such as dust mites, animal dander, bacteria, and mold, is associated with a range of health effects. This study identified household characteristics associated with indoor biocontaminant loadings in four Canadian cities. Floor dust was collected in 290 Canadian homes in Edmonton, Halifax, Montreal, and Windsor. The dust samples were analyzed for house dust mite allergens (Der f 1 and Der p 1), cat allergen (Fel d 1), cockroach allergen (Bla g 1), beta-(1,3)-D-glucan, and endotoxin. Household information was obtained through questionnaires and home inspections. We performed univariate and multivariate analyses to identify household determinants of biocontaminant loadings and mold odor presence. We observed large regional variations for all biocontaminants, except for cockroach allergen. The ranges of the contaminants measured in loadings and concentrations were similar to that of previous Canadian studies. Household characteristics including presence of carpeting, low floor cleaning frequency, older home age, presence of pets, and indoor relative humidity above 45% were positively associated with the presence of multiple indoor biocontaminants. High floor cleaning frequency and use of dehumidifiers were negatively associated with the presence of multiple indoor biocontaminants. Mold odor was positively associated with older home age, past water damage, and visible mold growth.
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Contaminación del Aire Interior , Cucarachas , Contaminación del Aire Interior/análisis , Alérgenos/análisis , Animales , Antígenos Dermatofagoides , Canadá , Polvo/análisis , ViviendaRESUMEN
Endophytes of healthy needles were collected from Picea rubens (red spruce) and P. mariana (black spruce) in a survey of southeastern New Brunswick, Canada. Four endophyte strains were selected for further investigation based on the production of biologically active extracts from culture filtrates during screening as well as phylogenetic relationship to species known to produce natural products or taxonomic novelty. A novel endophyte within the family Rhytismataceae produced two new dihydropyrones (1 and 2) as major metabolites together with phthalides (3 and 4), isocoumarins (5 and 6), and tyrosol (7). Lachnum cf. pygmaeum synthesized a new chlorinated para-quinone, chloromycorrhizinone A (8), and the nematicidal compounds (1'Z)-dechloromycorrhizin A (9), mycorrhizin A (10), and chloromycorrhizin A (11). A new isocoumarin (12) and four related structures (13-16) were isolated from an undescribed taxon in the Mycosphaerellaceae. The known antifungal metabolites cryptosporiopsin (17), 5-hydroxycryptosporiopsin (18), (+)-cryptosporiopsinol (19), and mellein (20) were produced by Pezicula sporulosa. Phylogenetically diverse conifer endophytes from the Acadian forest continue to be a productive source of new biologically active natural products.
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Antifúngicos/aislamiento & purificación , Antifúngicos/farmacología , Antinematodos/aislamiento & purificación , Antinematodos/farmacología , Productos Biológicos/aislamiento & purificación , Productos Biológicos/farmacología , Endófitos/química , Isocumarinas/aislamiento & purificación , Isocumarinas/farmacología , Picea/química , Pironas/aislamiento & purificación , Pironas/farmacología , Quinonas/aislamiento & purificación , Quinonas/farmacología , Antifúngicos/química , Antinematodos/química , Ascomicetos/química , Ascomicetos/clasificación , Productos Biológicos/química , Canadá , Bosques , Isocumarinas/química , Estructura Molecular , Filogenia , Pironas/química , Quinonas/químicaRESUMEN
Buildings that have been flooded often have high concentrations of Trichoderma spores in the air while drying. Inhaled spores and spore and mycelial fragments contain large amounts of fungal glucan and natural products that contribute to the symptoms associated with indoor mould exposures. In this study, we considered both small molecules and peptaibol profiles of T. atroviride, T. koningiopsis, T. citrinoviride, and T. harzianum strains obtained from damp buildings in eastern Canada. Twenty-residue peptaibols and sorbicillin-derived metabolites (1-6) including a new structure, (R)-vertinolide (1), were characterized from T. citrinoviride. Trichoderma koningiopsis produced several koninginins (7-10), trikoningin KA V, and the 11-residue lipopeptaibols trikoningin KB I and trikoningin KB II. Trichoderma atroviride biosynthesized a mixture of 19-residue trichorzianine-like peptaibols, whereas T. harzianum produced 18-residue trichokindin-like peptaibols and the 11-residue harzianin HB I that was subsequently identified from the studied T. citrinoviride strain. Two α-pyrones, 6-pentyl-pyran-2-one (11) and an oxidized analog (12), were produced by both T. atroviride and T. harzianum. Aside from exposure to low molecular weight natural products, inhalation of Trichoderma spores and mycelial fragments may result in exposure to membrane-disrupting peptaibols. This investigation contributes to a more comprehensive understanding of the biologically active natural products produced by fungi commonly found in damp buildings.
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Peptaiboles/metabolismo , Trichoderma/metabolismo , Secuencia de Aminoácidos , Materiales de Construcción/microbiología , Microbiología Ambiental , Nueva Escocia , Ontario , Peptaiboles/química , Peptaiboles/aislamiento & purificación , Quebec , Trichoderma/química , Trichoderma/aislamiento & purificaciónRESUMEN
Seven new secondary metabolites, four isochromans (1-4) and three α-pyrones (5-7), were isolated from Penicillium corylophilum DAOM 242293 collected from a damp building in Halifax, Canada. Their structures were elucidated by HRESIMS, 1D and 2D NMR, chemical derivatization, ORD, UV, and comparison to the literature. Related isochromans have previously been reported from other Penicillium species; however, to our knowledge this is the first report of α-pyrones from P. corylophilum. Compounds 1-4 demonstrated antifungal activity against Saccharomyces cerevisiae at 100 µg mL(-1).
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Antifúngicos/aislamiento & purificación , Cromanos/aislamiento & purificación , Penicillium/química , Pironas/aislamiento & purificación , Antifúngicos/química , Antifúngicos/farmacología , Canadá , Cromanos/química , Cromanos/farmacología , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Pironas/química , Pironas/farmacología , Saccharomyces cerevisiae/efectos de los fármacosRESUMEN
From the epidemiological studies conducted on the effect of mould and dampness on health a decade ago, the role of toxin-producing fungi in damp and mouldy buildings involved opinion more than evidence. Very little was known about the metabolites that were produced by the fungi that grew on damp building materials, and almost nothing had been reported on their occurrence in buildings. As a consequence, the focus was on speculations involving the fungal toxins that occur in agriculture. Over the past decade, particularly in the last 5 years, considerable progress has been made concerning the relevant toxins from fungi that grow on damp building materials. This paper summarizes the available data on the low-molecular-weight toxins reliably known from fungi common on damp building materials, the toxins that have been measured on mouldy building materials and the new understanding of the role that they play in the documented health effects of individuals living and working in damp and mouldy buildings.
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Contaminantes Atmosféricos/aislamiento & purificación , Contaminación del Aire Interior , Hongos/metabolismo , Micotoxinas/metabolismo , Metabolismo SecundarioRESUMEN
Indoor exposure to the spores and mycelial fragments of fungi that grow on damp building materials can result in increased non-atopic asthma and upper respiratory disease. The mechanism appears to involve exposure to low doses of fungal metabolites. Penicillium corylophilum is surprisingly common in damp buildings in USA, Canada and western Europe. We examined isolates of P. corylophilum geographically distributed across Canada in the first comprehensive study of secondary metabolites of this fungus. The sesquiterpene phomenone, the meroterpenoids citreohybridonol and andrastin A, koninginin A, E and G, three new alpha pyrones and four new isochromans were identified from extracts of culture filtrates. This is the first report of koninginins, meroterpenoids and alpha pyrones from P. corylophilum. These secondary metabolite data support the removal of P. corylophilum from Penicillium section Citrina and suggest that further taxonomic studies are required on this species.
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Contaminación del Aire Interior/efectos adversos , Asma/microbiología , Micotoxinas/aislamiento & purificación , Penicillium/química , Infecciones del Sistema Respiratorio/microbiología , Androstadienos/química , Androstadienos/aislamiento & purificación , Canadá , Cromanos/química , Cromanos/aislamiento & purificación , Exposición a Riesgos Ambientales , Compuestos Heterocíclicos con 3 Anillos/química , Compuestos Heterocíclicos con 3 Anillos/aislamiento & purificación , Vivienda , Humedad , Micotoxinas/química , Naftoles/química , Naftoles/aislamiento & purificación , Penicillium/clasificación , Penicillium/aislamiento & purificación , Penicillium/metabolismo , Pironas/química , Pironas/aislamiento & purificación , Metabolismo Secundario , Sesquiterpenos/química , Sesquiterpenos/aislamiento & purificación , Terpenos/química , Terpenos/aislamiento & purificaciónRESUMEN
This parameter was developed by the Joint Task Force on Practice Parameters, representing the American Academy of Allergy, Asthma & Immunology (AAAAI); the American College of Allergy, Asthma & Immunology (ACAAI); and the Joint Council of Allergy, Asthma & Immunology. The AAAAI and the ACAAI have jointly accepted responsibility for establishing "Environmental assessment and remediation: a practice parameter." This is a complete and comprehensive document at the current time. The medical environment is a changing environment, and not all recommendations will be appropriate for all patients. Because this document incorporated the efforts of many participants, no single person, including those who served on the Joint Task Force, is authorized to provide an official AAAAI or ACAAI interpretation of these practice parameters. Any request for information about or an interpretation of these practice parameters by the AAAAI or ACAAI should be directed to the Executive Offices of the AAAAI, the ACAAI, and the Joint Council of Allergy, Asthma & Immunology. These parameters are not designed for use by pharmaceutical companies in drug promotion. The findings and conclusions in this manuscript are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention (CDC).
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Cucarachas/inmunología , Exposición a Riesgos Ambientales/prevención & control , Hipersensibilidad Inmediata/prevención & control , Alérgenos/efectos adversos , Alérgenos/inmunología , Animales , Cucarachas/fisiología , Humanos , Hipersensibilidad Inmediata/diagnóstico , Hipersensibilidad Inmediata/etiología , Hipersensibilidad Inmediata/inmunologíaRESUMEN
Mycotoxin emergence and co-occurrence trends in Canadian grains are dynamic and evolving in response to changing weather patterns within each growing season. The mycotoxins deoxynivalenol and zearalenone are the dominant mycotoxins detected in grains grown in Eastern Canada. Two potential emerging mycotoxins of concern are sterigmatocystin, produced by Aspergillus versicolor, and diacetoxyscirpenol, a type A trichothecene produced by a number of Fusarium species. In response to a call from the 83rd Joint Expert Committee on Food Additives and Contaminants, we conducted a comprehensive survey of samples from cereal production areas in Ontario, Canada. Some 159 wheat and 160 corn samples were collected from farms over a three-year period. Samples were extracted and analyzed by LC-MS/MS for 33 mycotoxins and secondary metabolites. Ergosterol was analyzed as an estimate of the overall fungal biomass in the samples. In wheat, the ratio of DON to its glucoside, deoxynivalenol-3-glucoside (DON-3G), exhibited high variability, likely attributable to differences among cultivars. In corn, the ratio was more consistent across the samples. Sterigmatocystin was detected in some wheat that had higher concentrations of ergosterol. Diacetoxyscirpenol was not detected in either corn or wheat over the three years, demonstrating a low risk to Ontario grain. Overall, there was some change to the mycotoxin profiles over the three years for wheat and corn. Ongoing surveys are required to reassess trends and ensure the safety of the food value chain, especially for emerging mycotoxins.
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Contaminación de Alimentos , Micotoxinas , Triticum , Zea mays , Zea mays/microbiología , Zea mays/química , Triticum/microbiología , Triticum/química , Ontario , Micotoxinas/análisis , Contaminación de Alimentos/análisis , Tricotecenos/análisis , Espectrometría de Masas en Tándem , Ergosterol/análisisRESUMEN
We are interested in identifying human fungal allergens and antigens from species common on water-damaged or damp building materials for use as marker proteins and diagnostic tests. The cellulolytic fungus Chaetomium globosum is common on damp materials in the building environment worldwide. ELISA and immunoblotting tests identified two related proteins of molecular weights 45 and 47 kDa which were identified as fungal antigens found on spore surfaces and in culture filtrate. The sequences were determined by liquid chromatography tandem mass spectrometry (LC-MS/MS), which indicated that the two proteins were chitosanases, confirmed by enzyme assay. The 47 kDa protein was not glycosylated and had an acidic pI of 4.5. These proteins have not been reported from other fungi and similar antigens were not seen in other fungi common in buildings. The production of polyclonal antibodies in rabbits showed the antigenicity of the target proteins and confirmed they were not artifacts of the isolation process. The proteins isolated are useful biomarkers for the detection of C. globosum in the building environment.
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Antígenos Fúngicos/análisis , Antígenos Fúngicos/inmunología , Chaetomium/enzimología , Chaetomium/inmunología , Glicósido Hidrolasas/análisis , Glicósido Hidrolasas/inmunología , Animales , Antígenos Fúngicos/química , Chaetomium/aislamiento & purificación , Cromatografía Liquida , Microbiología Ambiental , Ensayo de Inmunoadsorción Enzimática , Glicósido Hidrolasas/química , Humanos , Punto Isoeléctrico , Peso Molecular , Conejos , Espectrometría de Masas en TándemAsunto(s)
Diabetes Mellitus Tipo 2 , Ácidos Grasos Omega-3 , Animales , Dieta , Contaminantes Ambientales/análisis , Peces , Ontario , Alimentos MarinosRESUMEN
PURPOSE OF REVIEW: Over the past 40âyears, the circumstances where fungal bioaerosols are major issues have shifted because of changes in the industrial sector from mainly agriculture to operations, including composting, cannabis production and forestry in hot humid conditions. Changes in the design and operation of nonindustrial workplaces meant that mould and dampness became major issues that are just being reduced. This review attempts to frame that history offers a perspective on the current thinking on mechanisms and provide potentially useful sources of information for physicians and their patients. RECENT FINDINGS: The major impact of fungal exposures is not only from their allergens but also from an array of Danger-Associated Molecular Pattern molecules, possibly the most important of which is the type of glucan found in moulds that grow in damp buildings, wood chips and crops, that is beta 1, 3 D glucan in triple helical form. Located in lung epithelia, the dectin receptor is exquisitely sensitive to this compound. Except in some agricultural workplaces, low molecular weight secondary metabolites often mischaracterized as mycotoxins play little, if any, role on population health. SUMMARY: There has been a convergence in thinking between the allergy and industrial hygiene communities as well as government agencies on mould and occupational health. This has led to some useful strategies for better managing these issues as well as increasing consumer awareness.
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Hongos , Exposición Profesional , Humanos , Alérgenos , Pulmón , GlucanosRESUMEN
A recent study of the health of Indigenous children in four First Nations Communities in remote northwestern Ontario found that 21% of children had been admitted to hospital for respiratory infections before age 2 years. Here we report a detailed analysis of the housing conditions in these communities. We employed a variety of statistical methods, including linear regression, mixed models, and logistic regression, to assess the correlations between housing conditions and loadings of biocontaminants (dust mite allergens, fungal glucan, and endotoxin) and indoor concentrations of PM2.5, CO2, benzene, and formaldehyde. The houses (n = 101) were crowded with an average of approximately 7 people. Approximately 27% of the homes had sustained CO2 concentrations above 1500 ppm. Most homes had more than one smoker. Commercial tobacco smoking and the use of non-electric heating (e.g., wood, oil) were associated with increased fine particle concentrations. Over 90% of the homes lacked working Heat Recovery Ventilators (HRVs), which was associated with increased fine particle concentrations and higher CO2. Of the 101 homes, 12 had mold damage sufficient to increase the relative risk of respiratory disease. This resulted from roof leaks, through walls or around the windows due to construction defects or lack of maintenance. A similar percentage had mold resulting from condensation on windows. Endotoxin loadings were much higher than any previous study in Canada. This work provides evidence for the need for more effort to repair existing houses and to ensure the HRVs are properly installed and maintained.
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Contaminación del Aire Interior , Vivienda , Humanos , Contaminación del Aire Interior/efectos adversos , Contaminación del Aire Interior/análisis , Dióxido de Carbono/análisis , Endotoxinas/análisis , Ontario/epidemiología , Indígena CanadienseAsunto(s)
Socorristas , Exposición a Riesgos Ambientales/efectos adversos , Desastres Naturales , Salud Pública/métodos , Accidentes/estadística & datos numéricos , Enfermedades Transmisibles/epidemiología , Desinfectantes/efectos adversos , Hongos , Humanos , Salud Laboral , Saneamiento/métodos , Hipoclorito de Sodio/efectos adversosRESUMEN
Penicillium chrysogenum clade 4, is a common mold on damp building materials. A capture ELISA assay for the major allergen from P. chrysogenum Pch52 has been developed and tested in house dust samples and potential cross-reactivity examined. Minimal cross-reactivity with other relevant indoor fungi was observed for the assay following thorough purification of the monoclonal and polyclonal antibodies. The limit of quantification for ELISA analysis of Pch52 in sieved house dust is comparable to other assays of other fungi. The LOQ for Pch52 was 0.31 ng/mL in solution or 110 ng/g dust. The LOQ for Asp f1 and Alt a1 were 2.2 ng/g and 17 ng/g, respectively. These results indicate this assay is suitable for the quantification of Pch52 in sieved house dust.
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Alérgenos/análisis , Anticuerpos Monoclonales , Polvo/análisis , Penicillium chrysogenum/inmunología , Alérgenos/aislamiento & purificación , Alternaria/inmunología , Aspergillus fumigatus/inmunología , Reacciones Cruzadas , Ensayo de Inmunoadsorción EnzimáticaRESUMEN
Coffee silverskin is a byproduct of the coffee roasting process contributing to organic waste burdens in urban areas. Silverskin is a potential source of dietary fiber, protein, carbohydrates, caffeine as well as vitamins and minerals. However, phytosterols present in the plant are susceptible to thermal oxidation resulting in the formation of phytosterol oxidation products (POPs) in the silverskin during roasting. In collaboration with a small roastery, the formation of POPs in three coffee varieties with roasting time was monitored by GC-MS. The objective was to evaluate the safety and potential benefits of incorporating coffee silverskin into value-added products. The qualitative profile of POPs in the silverskin from the three varieties was similar. Average total POPs were 0.32 g POPs/kg silverskin. POPs from the dominant plant sterol, sitosterol, were present at the highest concentrations. Caffeine, total antioxidant capacity, and total flavonoids were measured in the silverskin of the three coffees. Average values were 1.3 g caffeine/100 g silverskin, TEAC of 11 mmol Trolox/kg silverskin, and 1.94 to 8.60 mg catechin equivalent (CE)/g silverskin, respectively. An analysis of the impact of consuming teas and baked goods containing silverskin was also performed. Using published formulations, a tea or cookie containing silverskin would contribute approximately 1 and 0.3 mg POP per day, respectively. Consumption of these products would not substantially increase dietary exposure to POPs, while increasing fiber and antioxidants while reducing organic waste. PRACTICAL APPLICATION: Coffee silverskin has been studied as a possible source of fiber, antioxidants, and caffeine when incorporated in snack foods and used to make teas. To assess possible concerns about increasing dietary oxidized phytosterols, the formation of phytosterol oxidation products (POPs) was investigated in the silverskin fraction during the roasting process in three coffee varieties. In addition, caffeine, antioxidant capacity, and total flavonoids were determined. We found that silverskin can be safely used for value-added products including caffeinated teas, cookies, and bars with minimal impact on dietary POP exposures.
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Coffea , Fitosteroles , Antioxidantes , Coffea/química , Café , Oxidación-Reducción , Fitosteroles/química , Extractos VegetalesRESUMEN
NX and its acetylated form 3ANX are two new type A trichothecenes produced by Fusarium graminearum whose toxicity is poorly documented. The aim of this study was to obtain a general view of the intestinal toxicity of these toxins. Deoxynivalenol (DON), which differs from NX by the keto group at C8, served as a benchmark. The viability of human intestinal Caco-2 cells decreased after 24 h of exposure to 3 µM NX (-21.4%), 3 µM DON (-20.2%) or 10 µM 3ANX (-17.4%). Histological observations of porcine jejunal explants exposed for 4 h to 10 µM of the different toxins showed interstitial edema and cellular debris. Explants exposed to NX also displayed cell vacuolization, a broken epithelial barrier and high loss of villi. Whole transcriptome profiling revealed that NX, DON and 3ANX modulated 369, 146 and 55 genes, respectively. Functional analyses indicated that the three toxins regulate the same gene networks and signaling pathways mainly; cell proliferation, differentiation, apoptosis and growth, and particularly immune and pro-inflammatory responses. Greater transcriptional impacts were observed with NX than with DON. In conclusion, our data revealed that the three toxins have similar impacts on the intestine but of different magnitude: NX > DON â« 3ANX. NX and 3ANX should consequently be included in overall risk analysis linked to the presence of trichothecenes in our diet.
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Fusarium , Tricotecenos Tipo A , Tricotecenos , Animales , Células CACO-2 , Perfilación de la Expresión Génica , Humanos , Mucosa Intestinal , Porcinos , Tricotecenos/toxicidadRESUMEN
BACKGROUND: SchS21 and SchS34 are proteins from Stachybotrys chartarum sensu latto that are antigenic in goats, mice and humans. Monoclonal antibodies to these proteins react with spores of S. chartarum and S. chlorohalonata but do not cross-react with a diverse taxonomic and ecological array of other fungi. METHODS: Based on partial sequences of the 21- and 34-kDa proteins, obtained from tandem mass spectra and Edman degradation, degenerate primers were designed for touchdown PCR and the resulting amplicons were sequenced. Subsequently, inverse-PCR was used to obtain genomic DNA sequences encoding SchS21 and SchS34. RT-PCR products were sequenced to predict the mature protein sequences of SchS21 and SchS34. Based on the speculation that SchS21 protein was a DNase, the enzymatic properties were investigated. RESULTS: Sequences of 435 and 666 bp in length were obtained from SchS21 and SchS34 cDNAs. The SchS21 open reading frame encodes a mature protein of 144 amino acids, while that of SchS34 is 221 amino acids in length. SchS21 is a secretory, alkaline, Mg-dependent exodeoxyribonuclease, while SchS34 is a secretory protein of unknown function. His-tagged forms of the mature SchS21 and SchS34 proteins were separately overexpressed in Escherichia coli and purified using Ni-NTA columns (0.5 mg/l yield). CONCLUSIONS: Based on Western blots, the expressed proteins were similar in molecular weight and bound to the respective monoclonal antibodies to SchS21 and SchS34 proteins from S. chartarum. Interactions with human sera IgE confirmed the expressed forms of SchS21 and SchS34 as naturally occurring allergens.
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Antígenos Fúngicos/genética , Antígenos Fúngicos/inmunología , Desoxirribonucleasas/genética , Desoxirribonucleasas/inmunología , Proteínas Fúngicas/genética , Proteínas Fúngicas/inmunología , Stachybotrys/química , Stachybotrys/inmunología , Alérgenos/genética , Alérgenos/inmunología , Alérgenos/metabolismo , Secuencia de Aminoácidos , Anticuerpos Monoclonales/inmunología , Antígenos Fúngicos/metabolismo , Secuencia de Bases , Western Blotting , Catálisis , Cationes Bivalentes/química , ADN Complementario/genética , ADN Viral/metabolismo , Desoxirribonucleasas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/inmunología , Exodesoxirribonucleasas/metabolismo , Proteínas Fúngicas/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Inmunoglobulina E/sangre , Inmunoglobulina E/inmunología , Cinética , Datos de Secuencia Molecular , Plásmidos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Análisis de Secuencia de Proteína , Homología de Secuencia de Aminoácido , Stachybotrys/genética , TemperaturaRESUMEN
A DNA aptamer with high affinity and specificity to ochratoxin A (OTA) was conjugated to a coupling gel and used as sorbent for the preparation of solid phase extraction (SPE) columns. The SPE columns packed with 300µl oligosorbent (24nmol DNA) showed a linear (r=0.999) behaviour in the range of 0.4-500ng OTA. After optimisation of the extraction step, SPE columns were used for clean-up of OTA from wheat prior to liquid chromatographic (HPLC) analysis with fluorescence detection (FLD). Average recoveries from wheat samples spiked at levels of 0.5-50ng/g ranged from 74% to 88% (relative standard deviation <6%) with limits of detection and of quantification of 23 and 77pg/g, respectively. The comparative HPLC/FLD analyses of 33 naturally contaminated durum wheat samples cleaned-up on both aptamer-SPE and immunoaffinity (IMA) columns showed a good correlation (r=0.990). Aptamer-SPE columns could be re-used up to five times without any loss of performance.