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- A novel colorimetric assay of beta-D glucans in basidiomycete strains by alcian blue dye in a 96-well microtiter platePublication . Semedo, Madga Sofia Cardoso Nobre; Karmali, Amin; Fonseca, LuísBasidiomycete strains synthesize several types of beta-D-glucans, which play a major role in the medicinal properties of mushrooms. Therefore, the specific quantification of these beta-D-glucans in mushroom strains is of great biochemical importance. Because published assay methods for these beta-D-glucans present some disadvantages, a novel colorimetric assay method for beta-D-glucan with alcian blue dye was developed. The complex formation was detected by following the decrease in absorbance in the range of 620 nm and by hypsochromic shift from 620 to 606 nm (similar to 14 nm) in UV-Vis spectrophotometer. Analysis of variance was used for optimization of the slope of the calibration curve by using the assay mixture containing 0.017% (w/v) alcian blue in 2% (v/v) acetic acid at pH 3.0. The high-throughput colorimetric assay method on microtiter plates was used for quantification of beta-D-glucans in the range of 0-0.8 mu g, with a slope of 44.15 x 10(-2) and a limit of detection of 0.017 mu g/well. Recovery experiments were carried out by using a sample of Hericium erinaceus, which exhibited a recovery of 95.8% for beta-1,3-D-glucan. The present assay method exhibited a 10-fold higher sensitivity and a 59-fold lower limit of detection compared with the published method with congo red beta-D-glucans of several mushrooms strains were isolated from fruiting bodies and mycelia, and they were quantified by this assay method. This assay method is fast, specific, simple, and it can be used to quantify beta-D-glucans from other biological sources. (C) 2015 American Institute of Chemical Engineers
- Intrinsic non-stomatal resilience to drought of the photosynthetic apparatus in Coffea spp. is strengthened by elevated air [CO2]Publication . JN; Rodrigues, Ana P.; Lidon, Fernando C.; P. Pais, Isabel; Marques, Isabel; Gouveia, Duarte; ARMENGAUD, Jean; Silva, Maria J.; Martins, Sónia; Semedo, Magda C.; Dubberstein, Danielly; Partelli, Fábio Luiz; Reboredo, Fernando H.; Scotti-Campos, Paula; Ribeiro-Barros, Ana I; DaMatta, Fabio; Ramalho, José C.Growing water restrictions associated with climate changes constitute daunting challenges to crop performance. This study unveils the impacts of moderate (MWD) or severe (SWD) water deficit, and their interaction with air [CO2], on the photosynthetic apparatus of Coffea canephora Pierre ex A. Froehner cv. Conilon Clone 153 (CL153) and Coffea arabica L. cv. Icatu. Seven year-old potted plants grown under 380 (aCO(2)) or 700 mu l l (-1) (eCO(2)) [CO2] gradually reached predawn water potentials between -1.6 and -2.1 MPa (MWD), and below -3.5 MPa (SWD). Under drought, stomata closure was chiefly related to abscisic acid (ABA) rise. Increasing drought severity progressively affected gas exchange and fluorescence parameters in both genotypes, with non-stomatal limitations becoming gradually dominating, especially regarding the photochemical and biochemical components of CL153 SWD plants. In contrast, Icatu plants were highly tolerant to SWD, with minor, if any, negative impacts on the potential photosynthetic functioning and components (e.g., A(max), F-v/F-m, electron carriers, photosystems (PSs) and ribulose-1,5-bisphosphate carboxylase oxygenase (RuBisCO) activities). Besides, drought-stressed Icatu plants displayed increased abundance of a large set of proteins associated with the photosynthetic apparatus (PSs, light-harvesting complexes, cyclic electron flow, RuBisCO activase) regardless of [CO2]. Single eCO(2) did not promote stomatal and photosynthetic down-regulation in both genotypes. Instead, eCO(2) increased photosynthetic performance, moderately reinforced photochemical (PSs activity, electron carriers) and biochemical (RuBisCO, ribulose-5-phosphate kinase) components, whereas photoprotective mechanisms and protein abundance remained mostly unaffected. In both genotypes, under MWD, eCO(2) superimposition delayed stress severity and promoted photosynthetic functioning with lower energy dissipation and PSII impacts, whereas stomatal closure was decoupled from increases in ABA. In SWD plants, most impacts on the photosynthetic performance were reduced by eCO(2), especially in the moderately drought affected CL153 genotype, although maintaining RuBisCO as the most sensitive component, deserving special breeder's attention to improve coffee sustainability under future climate scenarios.
- Adsorption of myoglobin on calixarenes and biocatalysis in organic mediaPublication . Semedo, Magda C.; Karmali, Amin; Barata, Patrícia; Prata, José V.Derivatives of p-tert-butylcalix[4,6,8]arene carboxylic acids were used for selective adsorption of myoglobin.Amixtureofmyoglobin,laccaseandperoxidase wasusedforextractionwithcalixarenesandonlymyoglobin was selectively extracted to organic media. Myoglobin and Mb c–calixarene exhibited pseudoactivity of peroxidase in aqueous and organic media. This protein-calixarene complex exhibited the highest specific activity of 1.37 × 10−1 U.mg protein−1 at initial pH 6.5 of myoglobin aqueous solution. Apparent kinetic parameters (V max, K m, k cat and k cat/K m) for the pseudoperoxidase activity were determined in organic media for different initial pH values of myoglobin aqueous solution by Michaelis-Menten plot. The stability of this complex was studied for different initial pH values and t1/2 values were obtained in the range of 3.5–5.2 days. The extracted Mb c in organic media was recovered into fresh aqueous solutions at alkaline pH with a recovery of pseudoperoxidase activity of over 100%.
- Resilient and sensitive key points of the photosynthetic machinery of coffea spp. to the single and superimposed exposure to severe drought and heat stressesPublication . Dubberstein, Danielly; Lidon, Fernando C.; Rodrigues, Ana P.; Semedo, José N.; Marques, Isabel; Rodrigues, Weverton P.; Gouveia, Duarte; ARMENGAUD, Jean; Semedo, Magda C.; Martins, Sónia; Simoes-Costa, Maria C.; Moura, Isabel; Pais, Isabel P.; Scotti-Campos, Paula; Partelli, Fábio Luiz; E, Campostrini; Ribeiro-Barros, Ana I.; DaMatta, Fabio; Ramalho, José C.This study unveils the single and combined drought and heat impacts on the photosynthetic performance ofCoffea arabicacv. Icatu andC. canephoracv. Conilon Clone 153 (CL153). Well-watered (WW) potted plants were gradually submitted to severe water deficit (SWD) along 20 days under adequate temperature (25/20 degrees C, day/night), and thereafter exposed to a gradual temperature rise up to 42/30 degrees C, followed by a 14-day water and temperature recovery. Single drought affected all gas exchanges (includingA(max)) and most fluorescence parameters in both genotypes. However, Icatu maintainedF(v)/F(m)and RuBisCO activity, and reinforced electron transport rates, carrier contents, and proton gradient regulation (PGR5) and chloroplast NADH dehydrogenase-like (NDH) complex proteins abundance. This suggested negligible non-stomatal limitations of photosynthesis that were accompanied by a triggering of protective cyclic electron transport (CEF) involving both photosystems (PSs). These findings contrasted with declines in RuBisCO and PSs activities, and cytochromes (b(559),f,b(563)) contents in CL153. Remarkable heat tolerance in potential photosynthetic functioning was detected in WW plants of both genotypes (up to 37/28 degrees C or 39/30 degrees C), likely associated with CEF in Icatu. Yet, at 42/30 degrees C the tolerance limit was exceeded. ReducedA(max)and increasedC(i)values reflected non-stomatal limitations of photosynthesis, agreeing with impairments in energy capture (F(0)rise), PSII photochemical efficiency, and RuBisCO and Ru5PK activities. In contrast to PSs activities and electron carrier contents, enzyme activities were highly heat sensitive. Until 37/28 degrees C, stresses interaction was largely absent, and drought played the major role in constraining photosynthesis functioning. Harsher conditions (SWD, 42/30 degrees C) exacerbated impairments to PSs, enzymes, and electron carriers, but uncontrolled energy dissipation was mitigated by photoprotective mechanisms. Most parameters recovered fully between 4 and 14 days after stress relief in both genotypes, although some aftereffects persisted in SWD plants. Icatu was more drought tolerant, with WW and SWD plants usually showing a faster and/or greater recovery than CL153. Heat affected both genotypes mostly at 42/30 degrees C, especially in SWD and Icatu plants. Overall, photochemical components were highly tolerant to heat and to stress interaction in contrast to enzymes that deserve special attention by breeding programs to increase coffee sustainability in climate change scenarios.
- Next-generation proteomics reveals a greater antioxidative response to drought in Coffea arabica than in Coffea canephoraPublication . Marques, Isabel; Gouveia, Duarte; Gaillard, Jean-Charles; Martins, Sónia; Semedo, Magda C.; Lidon, Fernando C.; DaMatta, Fabio; Ribeiro, Ana; ARMENGAUD, Jean; Ramalho, José C.Drought is a major threat to coffee, compromising the quality and quantity of its production. We have analyzed the core proteome of 18 Coffea canephora cv. Conilon Clone 153 and C. arabica cv. Icatu plants and assessed their responses to moderate (MWD) and severe (SWD) water deficits. Label-free quantitative shotgun proteomics identified 3000 proteins in both genotypes, but less than 0.8% contributed to ca. 20% of proteome biomass. Proteomic changes were dependent on the severity of drought, being stronger under SWD and with an enrolment of different proteins, functions, and pathways than under MWD. The two genotypes displayed stress-responsive proteins under SWD, but only C. arabica showed a higher abundance of proteins involved in antioxidant detoxification activities. Overall, the impact of MWD was minor in the two genotypes, contrary to previous studies. In contrast, an extensive proteomic response was found under SWD, with C. arabica having a greater potential for acclimation/resilience than C. canephora. This is likely supported by a wider antioxidative response and an ability to repair photosynthetic structures, being crucial to develop new elite genotypes that assure coffee supply under water scarcity levels.
- Extraction of hemoglobin with calixarenes and biocatalysis in organic media of the complex with pseudoactivity of peroxidasePublication . Semedo, Madga Sofia Cardoso Nobre; Karmali, Amin; Barata, Patrícia; Prata, José VirgílioThe present work involves the use of p-tert-butylcalix[4,6,8]arene carboxylic acid derivatives ((t)Butyl[4,6,8]CH2COOH) for selective extraction of hemoglobin. All three calixarenes extracted hemoglobin into the organic phase, exhibiting extraction parameters higher than 0.90. Evaluation of the solvent accessible positively charged amino acid side chains of hemoglobin (PDB entry 1XZ2) revealed that there are 8 arginine, 44 lysine and 30 histidine residues on the protein surface which may be involved in the interactions with the calixarene molecules. The hemoglobin-(t)Butyl[6]CH2COOH complex had pseudoperoxidase activity which catalysed the oxidation of syringaldazine in the presence of hydrogen peroxide in organic medium containing chloroform. The effect of pH, protein and substrate concentrations on biocatalysis was investigated using the hemoglobin-(t)Butyl[6]CH2COOH complex. This complex exhibited the highest specific activity of 9.92 x 10(-2) U mg protein(-1) at an initial pH of 7.5 in organic medium. Apparent kinetic parameters (V'(max), K'(m), k'(cat) and k'(cat)/K'(m)) for the pseudoperoxidase activity were determined in organic media for different pH values from a Michaelis-Menten plot. Furthermore, the stability of the protein-calixarene complex was investigated for different initial pH values and half-life (t(1/2)) values were obtained in the range of 1.96 and 2.64 days. Hemoglobin-calixarene complex present in organic medium was recovered in fresh aqueous solutions at alkaline pH, with a recovery of pseudoperoxidase activity of over 100%. These results strongly suggest that the use of calixarene derivatives is an alternative technique for protein extraction and solubilisation in organic media for biocatalysis.
- Novel polyol-responsive monoclonal antibodies against extracellular b-D-Glucans from pleurotus ostreatusPublication . Semedo, Magda; Karmali, Amin; Martins, Sónia; Fonseca, Luís P.b-D-glucans from mushroom strains play a major role as biological response modifiers in several clinical disorders. Therefore, a specific assay method is of critical importance to find useful and novel sources of b-D-glucans with anti-tumor activity. Hybridoma technology was used to raise monoclonal antibodies (Mabs) against extracellular b-D-glucans (EBG) from Pleurotus ostreatus. Two of these hybridoma clones (3F8_3H7 and 1E6_1E8_B3) secreting Mabs against EBG from P. ostreatus were selected and 3F8_3H7 was used to investigate if they are polyol-responsive Mabs (PR-Mabs) by using ELlSA-elution assay. This hybridoma cell line secreted Mab of IgM class, which was purified in a single step by gel filtration chro-matography on Sephacryl S-300HR, which revealed a protein band on native PAGE with Mr of 917 kDa. Specificity studies of Mab 3F8_3H7 revealed that it recognized a common epitope on several b-D-glucans from different basidiomycete strains as determined by indirect EllSA and Western blotting under native conditions. This Mab exhibited high apparent affinity con-stant (KApp) for b-D-glucans from several mushroom strains. However, it revealed differential reactivity to some heat-treated b-D-glucans compared with the native forms suggesting that it binds to a conformation-sensitive epitope on b-D-glucan molecule. Epitope analysis of Mab 3F8_3H7 and 1E6_1E8_B3 was investigated by additivity index parameter, which revealed that they bound to the same epitope on some b-D-glucans and to different epitopes in other antigens. Therefore, these Mab can be used to assay for b-D-glucans as well as to act as powerful probes to detect conformational changes in these biopolymers.
- Generation of high-affinity monoclonal antibodies of IgG class against native β-d-glucans from basidiomycete mushroomsPublication . Semedo, Madga Sofia Cardoso Nobre; Karmali, Amin; Martins, Sónia; Fonseca, Luísβ-d-glucans from basidiomycete strains are powerful immunomodulatory agents in several clinical conditions. Therefore, their assay, purification and characterization are of great interest to understand their structure-function relationship. Hybridoma cell fusion was used to raise monoclonal antibodies (Mabs) against extracellular β-d-glucans (EBGs) from Pleurotus ostreatus. Two of the hybridoma clones (1E6-1E8-B5 and 3E8-3B4) secreting Mabs against EBGs were selected. This hybridoma cell line secreted Mabs of the IgG class which were then purified by hydroxyapatite chromatography to apparent homogeneity on native and SDS-PAGE. Mabs secreted by 1E6-1E8-B5 clone were found to recognize a common epitope on several β-d-glucans from different basidiomycete strains. This Mab exhibited high affinity constant (KA) for β-d-glucans from several mushroom strains in the range of 3.20 × 109 ± 3.32 × 103-1.51 × 1013 ± 3.58 × 107 L/mol. Moreover, they reacted to some heat-treated β-d-glucans in a different mode when compared with the native forms; these data suggest that this Mab binds to a conformational epitope on the β-d-glucan molecule. The epitope-binding studies of Mabs obtained from 1E6-1E8-B5 and 3E8-3B4 revealed that the Mabs bind to the same epitope on some β-d-glucans and to different epitopes in other antigen molecules. Therefore, these Mabs can be used to assay for β-d-glucan from basidiomycete mushrooms. © 2015 Elsevier Ltd. All rights reserved.
- A high throughput colorimetric assay of beta-1,3-D-glucans by Congo red dyePublication . Semedo, Madga Sofia Cardoso Nobre; Karmali, Amin; Fonseca, LuísMushroom strains contain complex nutritional biomolecules with a wide spectrum of therapeutic and prophylactic properties. Among these compounds, β-d-glucans play an important role in immuno-modulating and anti-tumor activities. The present work involves a novel colorimetric assay method for β-1,3-d-glucans with a triple helix tertiary structure by using Congo red. The specific interaction that occurs between Congo red and β-1,3-d-glucan was detected by bathochromic shift from 488 to 516 nm (> 20 nm) in UV–Vis spectrophotometer. A micro- and high throughput method based on a 96-well microtiter plate was devised which presents several advantages over the published methods since it requires only 1.51 μg of polysaccharides in samples, greater sensitivity, speed, assay of many samples and very cheap. β-d-Glucans of several mushrooms (i.e., Coriolus versicolor, Ganoderma lucidum, Pleurotus ostreatus, Ganoderma carnosum, Hericium erinaceus, Lentinula edodes, Inonotus obliquus, Auricularia auricular, Polyporus umbellatus, Cordyseps sinensis, Agaricus blazei, Poria cocos) were isolated by using a sequence of several extractions with cold and boiling water, acidic and alkaline conditions and quantified by this microtiter plate method. FTIR spectroscopy was used to study the structural features of β-1,3-d-glucans in these mushroom samples as well as the specific interaction of these polysaccharides with Congo red. The effect of NaOH on triple helix conformation of β-1,3-d-glucans was investigated in several mushroom species.