Avaliação das propriedades farmacológicas de polissacarídeos do fungo Scleroderma nitidum

Several pharmacological properties have been attributed to isolated compounds from mushroom. Recently, have these compounds, especially the polysaccharides derived from mushrooms, modulate the immune system, and its antitumor, antiviral, antibiotic and antiinflammatory activities. This study assesses the possible pharmacological properties of the polysaccharides from Scleroderma nitidum mushroom. The centesimal composition of the tissue showed that this fungus is composed mainly of fibers (35.61%), ash (33.69%) and carbohydrates (25.31%). The chemical analysis of the polysaccharide fraction showed high levels of carbohydrates (94.71%) and low content of protein (5.29%). These polysaccharides are composed of glucose, galactose, mannose and fucose in the following molar ratios 0.156, 0.044, 0.025, 0.066 and the infrared analysis showed a possible polysaccharide-protein complex. The polysaccharides from Scleroderma nitidum showed antioxidant potential with concentration-dependent antioxidant activity compared to ascorbic acid. The analysis scavenging of superoxide radical and inhibition of lipid peroxidation showed that the polysaccharides from S. nitidum have an IC50 of 12.70 mg/ml and EC50 10.4 μg/ml, respectively. The antioxidant activity was confirmed by the presence of reducing potential of these polysaccharides. The effect of these polymers on the inflammatory process was tested using the carrageenan or histamine-induced paw edema model and the sodium thioglycolate or zymosan-induced model. The polysaccharides were effective in reducing edema (73% at 50 mg/kg) and cell infiltrate (37% at 10 mg/kg) in both inflammation models tested. Nitric oxide, a mediator in the inflammatory process, showed a reduction of around 26% at 10 mg/kg of body weight. Analysis of pro- and anti-inflammatory cytokines showed that in the groups treated with polysaccharides from S. nitidum there was an increase in cytokines such as IL-1ra, IL-10, and MIP-1β concomitant with the decrease in INF-γ (75%) and IL-2 (22%). We observed the influence of polysaccharides on the modulation of the expression of nuclear factor κB. Thus, polysaccharides from S. nitidum reduced the expression of NF-κB by up to 64%. The results obtained suggest that NF-κB modulation is one of the possible mechanisms that explain the anti-inflammatory effect of polysaccharides from the fungus S. nitidum.

Análises Estruturais e atividades biológicas de exopolissacarídeo extraído do fungo comestível pleurotus Sajor-Caju e de seu derivado sulfatado quimicamente.

The exopolysaccharides are extracellular compounds produced by some species of fungi and bacteria. It is suggested that these molecules, even when in the form of complex polysaccharide-peptide, are the main bioactive molecules of many fungus. Some of the biological activities displayed by these compounds can be accentuated and others may arise when you add chemically polar or nonpolar groups to polysaccharides. The fruiting body of Pleurotus sajor-caju produces a heteropolysaccharide with antineoplastic and antimicrobial activity, but other biological activities of this polymer have not been evaluated. In this work the exopolysaccharide of Pleurotus sajor-caju was sulfated chemically and structurally characterized. We also evaluated the antiproliferative, antioxidant and anticoagulant activities from native exopolysaccharide (PN) and its sulfated derivated (PS). Polyacrylamide gel electrophoresis, infrared spectroscopy and nuclear magnetic resonance (¹³C) proved successful in sulfation of PN to obtain PS. Analysis by gas chromatography-mass spectroscopy showed that PN and PS are composed of mannose, galactose, 3-O-methyl-galactose and glucose in proportion percentage of 44,9:16,3:19,8:19 and 49, 7:14,4:17,7:18,2, respectively. The percentage of sulfate found in PS was 22.5%. Antioxidants assays revealed that the sulfation procedure affects differently the activities of exopolysaccharides, while the total antioxidant capacity, the scavenging activity of superoxide radical and ferric chelating were not affected by sulfation, on the other hand the chemical modification of PN enhanced the scavenging activity of hydroxyl radical and reducing power. PS also showed anticoagulant activity in a dose-dependent manner and clotting time was 3.0 times higher than the baseline value in APTT at 2 mg/mL. The exopolysaccharide not presented antiproliferative activity against HeLa tumor cells, but PS affects the cellular proliferation in a time-dependent manner. After 72 h, the inhibition rate of PS (2.0 mg/mL) on HeLa cells was about 60%. The results showed that PN sulfation increase some of their activities.

Vicilinas de sementes de leguminosas selvagens:purificação, caracterização, efeito de deletério e mecanismo de ação para bruquídeos e para fungos fitopatogêncios

Globulins fractions of legume seeds of Crotalaria pallida, Erytrina veluntina and Enterolobium contortisiliquum were isolated and submitted to assays against serine, cysteine and aspartic proteinases, as also amylase present in midgut of C. maculatus and Z. subfasciatus. Hemagglutination assays indicated presence of a lectin in E. veluntina globulin fractions. This lectin had affinity to human erythrocytes type A, B and O. Vicilins were purified by chromatography on Sephacryl S-300 followed of a chromatography on Sephacryl S-200, which was calibrated using protein markers. Vicilins from C. pallida (CpV) and E. veluntina (EvV) seeds had a molecular mass of 124.6 kDa and E. contortisiliquum a molecular mass of 151kDa. Eletrophoresis in presence of SDS showed that CpV was constituted by four subunities with apparent molecular mass of 66, 63, 57 and 45 kDa, EvV with three subunities with apparent molecular mass of 45kDa and EcV four subunities, two with 37.1 kDa and two with 25.8 kDa. Non denaturantig eletrophoresis displayed single bands with high homogeneity, where CpV had lower acidic behavior. All vicilins are glycoproteins with carbohydrate contents at 1 to1.5%. Bioassays were done to detect deleterious effects of vicilins against C. maculatus and Z. subfasciatus larvae. CpV, EvV and EcV exhibited a WD50 of 0.28, 0.19 and 1.03%; LD50 0.2, 0.26, and 1.11% respectively to C. maculatus. The dose responses of CpV, EvV and EcV to Z. subfasciatus were: WD50 of 0.12, 0.14, 0.65% and LD50 of 0.09, 0.1, and 0.43% respectively. The mechanism of action of these proteins to bruchids should be based on their properties of bind to chitin present in mid gut of larvae associated with the low digestibility of vicilin. In assays against phytopatogenous fungus, only EcV was capable of inhibit F. solani growth at concentrations of 10 and 20 µg and its action mechanism should be also based in the affinity of EcV to chitin present in the fungi wall

Produção e caracterização de quitooligossacarídeos produzidos pelo fungo Metarhizium anisopliae e avaliação da citotoxicidade em células tumorais

Chitosan is a natural polymer, biodegradable, nontoxic, high molecular weight derived from marine animals, insects and microorganisms. Oligomers of glucosamine (GlcN) and N-acetylglucosamine (GlcNAc) have interesting biological activities, including antitumor effects, antimicrobial activity, antioxidant and others. The alternative proposed by this work was to study the viability of producing chitooligosaccharides using a crude enzymes extract produced by the fungus Metarhizium anisopliae. Hydrolysis of chitosan was carried out at different times, from 10 to 60 minutes to produce chitooligosaccharides with detection and quantification performed by High Performace Liquid Chromatography (HPLC). The evaluation of cytotoxicity of chitosan oligomers was carried out in tumor cells (HepG2 and HeLa) and non-tumor (3T3). The cells were treated for 72 hours with the oligomers and cell viability investigated using the method of MTT. The production of chitosan oligomers was higher for 10 minutes of hydrolysis, with pentamers concentration of 0.15 mg/mL, but the hexamers, the molecules showing greater interest in biological properties, were observed only with 30 minutes of hydrolysis with a concentration of 0.004 mg/mL. A study to evaluate the biological activities of COS including cytotoxicity in tumor and normal cells and various tests in vitro antioxidant activity of pure chitosan oligomers and the mixture of oligomers produced by the crude enzyme was performed. Moreover, the compound with the highest cytotoxicity among the oligomers was pure glucosamine, with IC50 values of 0.30; 0.49; 0.44 mg/mL for HepG2 cells, HeLa and 3T3, respectively. Superoxide anion scavenging was the mainly antioxidant activity showed by the COS and oligomers. This activity was also depending on the oligomer composition in the chitosan hydrolysates. The oligomers produced by hydrolysis for 20 minutes was analyzed for the ability to inhibit tumor cells showing inhibition of proliferation only in HeLa cells, did not show any effect in HepG2 cells and fibroblast cells (3T3)

Termoterapia de banana 'Prata-Anã' no controle de podridões em pós-colheita

O objetivo desse estudo foi determinar a tolerância de banana (Musa spp.) 'Prata-Anã' (AAB) e do fungo Colletotrichum musae à termoterapia no controle de podridões em pós-colheita. Experimentos in vivo e in vitro foram instalados em delineamento inteiramente casualizado, seguindo um esquema fatorial 4x5 (temperatura x tempo). Os tratamentos consistiram na imersão dos frutos (buquês) e do fungo (esporos e micélio) em água aquecida a 47, 50, 53 e 56 ºC, durante 0, 3, 6, 9 e 12 min. A exposição dos frutos a 56 ºC durante 9 min causou escurecimento da casca nas extremidades dos frutos, porém, as características físicas e químicas dos frutos não foram alteradas pelos tratamentos. Frutos inoculados e tratados a 56 ºC durante 6 min não apresentaram podridões nem escurecimento da casca, enquanto aqueles não tratados apresentaram 64% da área lesionada / fruto. A partir das combinações 53 ºC / 9 mi. e 56 ºC / 3 min a germinação de esporos foi reduzida para 4% e 0%, respectivamente. A combinação 56 ºC / 12 min reduziu, mas não paralisou o crescimento micelial. O tratamento 56 ºC / 6 min retardou mas não paralisou o crescimento micelial in vitro, porém foi efetivo no controle completo das podridões in vivo. Esse tratamento evitou a manifestação de podridões no inverno (maio), mas não no verão (novembro), mostrando-se influenciado pelas condições climáticas próximas à colheita dos cachos. A termoterapia pode ser recomendada para controle de podridão em pós-colheita de banana devendo ser ajustada para diferentes estações do ano.

Incidência de fungos em pós-colheita de banana 'Prata anã'(Musa AAB)

Este trabalho teve por objetivo determinar a ocorrência e a freqüência de fungos em banana 'Prata anã' e elucidar o agente causal das podridões em pós-colheita de frutos provenientes do norte de Minas Gerais. Dois métodos de isolamento foram adotados: diluição em placas, a partir da lavagem de frutos verdes, e direto de frutos maduros. Os fungos Colletotrichum musae, Trichoderma harzianum, Fusarium equisetii, Penicillium sp. Aspergillus parasiticus, Trichothecium roseum, Colletotrichum acutatum, Alternaria sp., Cladosporium musae e Curvularia lunata foram os mais freqüentemente associados aos frutos. A patogenicidade desses fungos foi testada pela substituição de discos da casca de frutos verdes por discos de micélio. Colletotrichum musae apresentou área média lesionada em torno do ponto de inoculação igual a 5,8 cm², enquanto para os demais fungos testados não passou de 1,50 cm². Os resultados mostraram que C. musae é o agente primário das podridões dos frutos examinados com 100 % de incidência e os demais fungos limitaram-se a necrosar os ferimentos em torno do ponto de inoculação. O modo de infecção latente, causada por C. musae, parece favorecer, primeiramente, a colonização interna dos tecidos e, posteriormente, a ação dos fungos oportunistas, que aceleram as podridões nos frutos e na coroa.

Freqüência e etiologia das dermatomicoses em pacientes atendidos no Hospital Giselda Trigueiro, Natal, RN

Dermatomycoses are fungal infections that attack the skin, hair and nails, in addition to the mucosal and cutaneous-mucosal zones. Objective: Observe the frequency of dermatomycoses, identify etiological agents and establish an association between the results and sex. Age, collection site, time and lesion location. Methods: Between February, 2002 and December, 2004, samples were collected from patients at Giselda Trigueiro Hospital in Natal, Brazil, by lesion scraping and hair removal, following 70% alcohol disinfection, and submitted to direct and culture examination. Results: Of the 817 lesions collected, 325 (39.8%) were fungus positive, with the hair collection site yielding the highest number of positive results (65.8%) and the scalp and hair representing the most frequent lesion sites (65.9%). Negative results occurred mainly in the lower limbs (78.6%). Of the species identified, 55.9% were yeasts, 41.6% dermatophytes and 2.5% Fusarium spp. Non-albicans Candida was the most isolated yeast (43.3%), mainly in females (61.7%) over the age of 40 years (56.4%). T. rubrum was the most isolated dermatophyte (67.9%),notably in males (59.2%) in the 0-20 age group (44.7%). With respect to collection site, 73.9% of the dermatophytes were present in the skin and 61.1% of the yeasts in the nails. When assessing the collection site, the inguinocrural regional was 22.6% positive for dermatophytes, and the nails and hands, 41.8% for yeasts. Conclusions: The results obtained verified that: most of the positive lesions were found in the hair, whereas skin and nail lesions yielded more negative results; T. rubrum was the most isolated dermatophyte and non-albicans candida the most commonly found yeast; positivity was greater in males in the 0-20 year age group at the skin site and in the inguinocrural region, while yeasts were more frequent in females in the over-40 age group at the nail sites

Triterpene benzoates from the bark of Picramnia teapensis (Simaroubaceae)

Two new benzoic acid esters of triterpene alcohols [lup-20 (29)-en-28-oic acid 3 alpha, 7 beta -dibenzoate and 3 alpha -hydroxy-lup-20(29)-en-28-ic acid 7 beta -benzoate] were isolated from the stem bark of Picramnia teapensis Tul. The structures of these compounds were established on the basis of spectral analyses. Other known compounds, beta -sitosterol, estigmasterol, lupeol and epilupeol, were identified in mixture by GC-MS. The triterpene esters have not shown in-vitro inhibitory effect on the growth of Leucoagaricus gongylophorus (Fisher), referred also as Leucocoprinus gongylophorus (Heim), syn Rozites gongylophora (Moller), the symbiotic fungus cultivated by the leaf-cutting ant Atta sexdens L.

Preliminary studies on the effects of d-limonene to workers of the leaf-cutting ant Atta sexdens rubropilosa and its implications for control

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Plant or fungal sequences? An alternative optimized PCR protocol to avoid ITS (nrDNA) misamplification

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

Isolation and maintenance of symbiotic fungi of ants in the tribe Attini (Hymenoptera: Formicidae)

O isolamento e a manutenção de fungos basidiomicetos simbiontes de formigas da tribo Attini tem sido dificultado pela baixa velocidade de crescimento desses fungos, bem como pela presença de muitos microrganismos que vivem na superfície do material que as formigas mantêm no interior nos ninhos como substrato para o crescimento dos seus fungos simbiontes. No presente trabalho nós descrevemos um método que aumenta em mais de sete vezes a eficiência de isolamento desses fungos, quando comparada àquela obtida por procedimentos tradicionais. Ninhos subterrâneos de formigas atíneas dos gêneros Atta, Acromyrmex, Trachymyrmex e Mycetarotes foram localizados e deles foram coletadas amostras contendo fungos simbiontes e formigas, que foram transportadas para o laboratório, onde as formigas foram capazes de limpar a cultura do fungo e estimular o seu crescimento. em seguida, porções dos micélios foram assepticamente coletadas e transferidas para meio Yeast Nitrogen Base contendo glicose e cloranfenicol. Para facilitar a manutenção dos isolados em culturas de laboratório, diferentes nutrientes foram analisados para a elaboração de um meio de cultivo complexo, que possibilitou aumentar a velocidade de crescimento dos fungos e estocá-los por longos períodos. O método foi aplicado com sucesso para os fungos simbiontes de todos os gêneros de formigas estudados, gerando, assim, um procedimento extremamente útil para a formação e manutenção de uma coleção representativa de diferentes fungos simbiontes de formigas da tribo Attini.

Extrações de óleos de sementes de citros e suas atividades sobre a formiga cortadeira Atta sexdens e seu fungo simbionte

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

Low variation in ribosomal DNA and internal transcribed spacers of the symbiotic fungi of leaf-cutting ants (Attini: Formicidae)

Leaf-cutting ants of the genera Atta and Acromyrmex (tribe Attini) are symbiotic with basidiomycete fungi of the genus Leucoagaricus (tribe Leucocoprineae), which they cultivate on vegetable matter inside their nests. We determined the variation of the 28S, 18S, and 5.8S ribosomal DNA (rDNA) gene loci and the rapidly evolving internal transcribed spacers 1 and 2 (ITS1 and ITS2) of 15 sympatric and allopatric fungi associated with colonies of 11 species of leafcutter ants living up to 2,600 km apart in Brazil. We found that the fungal rDNA and ITS sequences from different species of ants were identical (or nearly identical) to each other, whereas 10 GenBank Leucoagaricus species showed higher ITS variation. Our findings suggest that Atta and Acromyrmex leafcutters living in geographic sites that are very distant from each other cultivate a single fungal species made up of closely related lineages of Leucoagaricus gongylophorus. We discuss the strikingly high similarity in the ITS1 and ITS2 regions of the Atta and Acromyrmex symbiotic L. gongylophorus studied by us, in contrast to the lower similarity displayed by their non-symbiotic counterparts. We suggest that the similarity of our L. gongylophorus isolates is an indication of the recent association of the fungus with these ants, and propose that both the intense lateral transmission of fungal material within leafcutter nests and the selection of more adapted fungal strains are involved in the homogenization of the symbiotic fungal stock.

Host specificity of a Brazilian isolate of Alternaria cassiae (Cenargen CG593) under greenhouse conditions

Several Alternaria cassiae isolates were recovered from diseased sicklepod plants (Senna obtusifolia) in the southern regions of Brazil. A representative isolate (Cenargen CG593) was tested for its host range under greenhouse conditions. The fungus promoted symptoms in sicklepod, cassava (Manihot dulce), tomato (Lycopersicon esculentum) and eggplant (Solanum melongena) when tested at a spore concentration of 10(6) spores ml(-1). When the plants were inoculated with a suspension of 10(5) spores ml(-1) and held at a dew period of 12 h (cassava) or 18 h (tomato and eggplant), the plants showed symptoms of the disease, but they recovered and continued their normal vegetative growth. These results show that the fungus A. cassiae is safe to use for the control of S. obtusifolia under Brazilian conditions, because it did not cause excessive damage in the three plants tested.

Crescimento e esporulação de Bipolaris euphorbiae cultivado sob diferentes condições nutricionais

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)