[Show abstract][Hide abstract] ABSTRACT: This study evaluated the reactivity of membrane antigens of Leishmania (Viannia) braziliensis for the diagnosis of ACL by enzyme immunoassay (EIA). Promastigotes of L. (V.) braziliensis were grown in medium 199 and lysed in a sonicator. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting showed that specific proteins of L. (V.) braziliensis (apparent molecular weights 36 kDa and 48–56 kDa) were recognized by sera from ACL patients. These proteins were eluted from the SDS-PAGE and tested in EIA-IgG with sera from ACL patients, healthy individuals, patients with toxoplasmosis, paracoccidioidomycosis, syphilis, tuberculosis, leprosy, and Chagas disease. The EIA-IgG with membrane antigens allowed us to distinguish patients with ACL from healthy individuals and patients with other diseases (P < 0.0001), and showed a sensitivity of 93.3% and specificity of 90.8%, not including Chagas disease patients. 2D-SDS-PAGE followed by Western blotting was performed to improve the characterization of the antigens, and showed a component with isoelectric points near the acid pH side and apparent molecular weights of 48–56 kDa. The results showed good sensitivity and specificity of EIA-IgG with membrane antigens, indicating their potential use for diagnosis of ACL, as well as seroepidemiological surveys and follow-up of clinically cured patients.
[Show abstract][Hide abstract] ABSTRACT: Identification of the nucleotide consensus sequence in mammalian replication origins is a difficult and controversial problem. The hypothesis that local DNA topology could be involved in recognition by replication proteins is an exciting possibility. Secondary DNA structures, including intrinsically bent DNA, can be easily detected, and they may indicate a specific pattern in or near mammalian replication origins. This work presents the entire mapping of the intrinsically bent DNA sites (IBDSs), using in silico analysis and a circular permutation assay, of the DNA replication origins oriGNAI3, oriC, oriB, and oriA in the mammalian amplified AMPD2 gene domain. The results show that each origin presents an IBDS that flanks the straight core of these DNA replication sites. In addition, the in silico prediction of the nucleosome positioning reveals a strong indication that the center of an IBDS is localized in a nucleosome-free region (NFR). The structure of each of these curved sites is presented together with their helical parameters and topology. Together, the data that we present here indicate that the oriGNAI3 origin where preferential firing to the replication initiation events in the amplified AMPD2 domain occurs is the only origin that presents a straight, narrow region that is flanked on both sides by two intrinsically bent DNA sites within a short distance (~300 bp); however, all of the origins present at least one IBDS, which is localized in the NFR region. These results indicate that structural features could be implicated in the mammalian DNA replication origin and support the possibility of detecting and characterizing these segments.
[Show abstract][Hide abstract] ABSTRACT: The glucans of basidiomycetes are an important class of polysaccharides with potential biological activities. In this work, the β-glucans were isolated from the fruiting bodies of edible mushrooms, Pleurotus eryngii and Pleurotus ostreatoroseus, via extraction with hot water, and then fractionation by freeze-thawing. The insoluble glucans gave similar 13C NMR spectra, monosaccharide composition and methylation analyses, and P. eryngii was selected for further controlled Smith degradation, and DEPT and 1H (obs.), 13C HMQC spectroscopy. It was a branched β-glucan, with a main chain of (1 → 3)-linked-Glcp residues, substituted at O-6 by single-unit β-Glcp side-chains, on average to every third residue of the backbone, as in scleroglucan.
[Show abstract][Hide abstract] ABSTRACT: The two main water-soluble extracellular polysaccharides produced by the basidiomycete fungus Pleurotus ostreatoroseus Sing were isolated and purified. They were characterized using 13C, 1H, and 1H,13C HMQC NMR spectroscopy, methylation analysis, and Smith degradation. One was a mannan having a main chain of (1→6)-linked -d-mannopyranosyl residues, almost all of which were branched at O-2 with side chains of different lengths, containing 2-O- and 3-O-linked mannopyranosyl units. The other was a partially 3-O-methylated (1→4)-linked -d-galactopyranan, a structure that has not been previously described.
[Show abstract][Hide abstract] ABSTRACT: The two main polysaccharides from the basidiomycetous fungus Laetiporus sulphureus were isolated, purified and characterized. The structural assignments were carried out using (13)C, (1)H, and (1)H,(13) HSQC nuclear magnetic resonance spectroscopy, methylation analysis, and Smith degradation. One was a linear beta-glucan having a (1-->3)-linked main chain, namely laminaran. The other was a fucomannogalactan, which consisted of a main chain of (1-->6)-linked alpha-D-galactopyranosyl residues, a part of them being substituted at O-2 by 3-O-D-mannopyranosyl-L-fucopyranosyl, alpha-D-mannopyranosyl and in a minor proportion, alpha-L-fucopyranosyl groups. This heteropolysaccharide is related to those of other Basidiomycetes heterogalactans, although it differs distinctly in its side-chain structures. Whereas part of the single-unit L-fucopyranosyl and/or 3-O-alpha-mannopyranosyl-L-fucopyranosyl residues are present as side chains of the other heterogalactans, additional alpha-D-mannopyranosyl units are present in our fucomannogalactan of L. sulphureus.
[Show abstract][Hide abstract] ABSTRACT: The species of the genus Pleurotus produce large amounts of biomass and exopolysaccharide (EPS) in submerged cultures, which may be used for biotechnological purposes. In the present work two Brazilian autochthonous strains of edible Pleurotus (P. ostratoroseus Sing. and P. ostreatus "florida") were used. The fungi grown in liquid Potato Dextrose medium (PD) were used as inocula to cultivate those microorganisms in the POL culture medium. After a 9-day incubation, the optimal growth time for biomass production, P. ostreatus "florida" presented higher biomass production (22.8 g d.w./l) than P. ostreatoroseus (16.8 g d.w./l). After a 7-day incubation, the optimal growth time for EPS production, P. ostreatoroseus produced higher amounts of crude EPS (5.8 g d.w./l) than P. ostreatus "florida" (1.4 g d.w./l). Relative carbohydrate composition for P. ostreatoroseus and P. ostreatus "florida" EPS were: glucose (95.5-87.7), galactose (traces - 4.9), mannose (traces - 3.1), xylose (1.3-2.5), and arabinose (3.2-1.8). Lower ammonium sulfate concentration in the POL culture medium increased the exopolysaccharides production by P. ostreatoroseus.
[Show abstract][Hide abstract] ABSTRACT: The partially 3-O-methylated mannogalactans were isolated from the fruiting bodies of edible basidiomycetes Pleurotus ostreatus 'florida' Berk. and Pleurotus ostreatoroseus Sing. They were obtained via successive aqueous extraction, freeze thawing, and precipitation with Fehling solution and then investigated using (13)C- and (1)H-nuclear magnetic resonance spectroscopy (including COSY, TOCSY and HMQC techniques), methylation analysis and Smith degradation. The main chain consisted of (1-->6)-linked alpha-D-galactopyranosyl residues containing 3-O-Me-alpha-D-galactopyranoses, a part of these units being substituted in the position O-2 with beta-D-mannopyranose residues. The heteropolysaccharides found were similar with differences only in the levels of the 3-O-Me-alpha-D-galactopyranoses residues. The presence of partially 3-O-methylated mannogalactan appears to be typical of Pleurotus spp.
[Show abstract][Hide abstract] ABSTRACT: The two main water-soluble extracellular polysaccharides produced by the basidiomycete fungus Pleurotus ostreatoroseus Sing were isolated and purified. They were characterized using 13C, 1H, and 1H, 13C HMQC NMR spectroscopy, methylation analysis, and Smith degradation. One was a mannan having a main chain of (1-->6)-linked alpha-D-mannopyranosyl residues, almost all of which were branched at O-2 with side chains of different lengths, containing 2-O- and 3-O-linked mannopyranosyl units. The other was a partially 3-O-methylated (1-->4)-linked alpha-D-galactopyranan, a structure that has not been previously described.
[Show abstract][Hide abstract] ABSTRACT: Efficiency of solid and liquid inocula and their use for spawn production were compared so that improved cultivation conditions for the edible mushroom Pleurotus ostreatoroseus could be tested. Solid and liquid inocula were prepared respectively with Potato Dextrose Agar (PDA) and Liquid Potato Dextrose (LPD). Wheat grains and cotton residues were used as substrates for spawn preparation. Inoculum types did not affect the development of P. ostreatoroseus, and LPD spawns were cheaper, more homogenous, less contaminated. Decomposition activity of mushroom growth, as a percentage of organic matter loss (OML), was higher in the wheat grain spawn and was not influenced by the inoculum type. Advantages in the use of cotton residue for spawn production were longer storage time, lower contamination and reduced costs. The cotton residue substrate may be also used for the production of mushroom fruiting bodies.
[Show abstract][Hide abstract] ABSTRACT: RESUMO: Os cogumelos comestíveis e as plantas medicinais estão ganhando importância na atualidade pelos seus valores nutritivos e terapêuticos. O fungo Basidiomicete Pleurotus ostreatus é considerado de alta importância, porque, além da produção de exopolissacarídeos e biomassa, possui substâncias terapêuticas ativas. A planta medicinal Ginkgo biloba é oriunda da China e possui constituintes farmacológicos ativos presentes nas folhas, sementes e raízes que vêm sendo usados para a prevenção e tratamento de doenças. Foi avaliada a produção de biomassa de Pleurotus ostreatus em cultura submersa, o fungo foi inoculado em meio de cultura BD líquida em diferentes concentrações do extrato aquoso de Ginkgo biloba. Estes inóculos que contêm o fungo foram transferidos para erlenmyers e submetidos à agitação a 150 rpm por 14 dias. Após determinação de biomassa, houve crescimento micelial e o extrato desta planta pode ser uma alternativa para estimular o crescimento do fungo e aplicá-lo na indústria Nutricêutica e em Processos Biotecnológicos. ABSTRACT: The importance of comestible mushrooms and medicinal herbs is currently on the increase owing to their nutrition and therapeutic properties. The basidiomycete fungus Pleurotus ostreatus is highly important due to its active therapeutic compounds besides its production of exopolysaccharides and biomass. Hailing from China, the medicinal plant Ginkgo biloba has active pharmaceutical properties in leaves, seeds and roots which are used for the prevention and treatment of several diseases. Production of Pleurotus ostreatus products in a submerged culture has been evaluated. Fungus was inoculated in liquid BD medium at different concentrations of Ginkgo biloba aqueous extract. Inocula with fungus were transferred to Erlenmeyer flasks and stirred for 14 days at 150 rpm. Posterior to biomass determination, mycelial growth occurred and the plant extract may be an alternative to stimulate fungus growth. Its application in nutritional and biotechnological productions is highly appreciated.