RESUMO
Most antivenoms are produced by techniques developed over 50 years ago, with minor modifications. Herein we revise the core of traditional antivenom production processes aiming to optimize key determinants for both consistent antivenom production and the best balance between F(ab')2 quality and recovery. Factorial design analysis revealed that pepsin digestion of 1:3 saline diluted equine plasma for 60 min under pH: 3.20, 37 °C temperature and a 1:15 pepsin to protein ratio conditions, allowed to achieve maximal IgG to F(ab')2 conversion with minimal protein aggregate formation. Further downstream processing by salting out with ammonium sulfate was also studied by factorial analysis. The influence of ammonium sulfate (AS) concentration, temperature (T) and the albumin to total plasma protein ratio plasma (Alb:P) were assayed, revealing that both AS, T and their interaction have a significant impact in F(ab')2 quality and recovery. Taking into account the existing compromise between F(ab')2 monomer recovery and quality two alternative conditions were selected: 14 g/dl AS at 56 °C and, alternatively 16 g/dl AS at 30 °C. Reasonable yields (42%) and product quality (2.5% of aggregates) without significant changes in production cost of traditional methodologies was achieved under the optimized conditions found.
Assuntos
Antivenenos/imunologia , Cavalos/imunologia , Fragmentos Fab das Imunoglobulinas/imunologia , Pepsina A/metabolismo , Mordeduras de Serpentes/imunologia , Venenos de Serpentes/imunologia , Sulfato de Amônio/química , Sulfato de Amônio/metabolismo , Animais , Antivenenos/sangue , Antivenenos/metabolismo , Proteínas Sanguíneas/metabolismo , Caprilatos/química , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Cavalos/sangue , Humanos , Fragmentos Fab das Imunoglobulinas/sangue , Fragmentos Fab das Imunoglobulinas/metabolismo , Papaína/metabolismo , Albumina Sérica/metabolismo , Mordeduras de Serpentes/prevenção & controleRESUMO
Plants colonised by dark septate endophytic (DSE) fungi show increased uptake of nutrients available in the environment. The objective of the present study was to evaluate the impact of DSE fungi on the activity of proton pumps, nitrogen (N) recovery from ammonium sulphate, and nutrient accumulation in rice plants. Treatments consisted of non-inoculated plants and plants inoculated with two isolates of DSE fungi, A101 and A103. To determine N recovery from the soil, ammonium sulphate enriched with 15N was added to a non-sterile substrate while parameters associated with the activity of proton pumps and with NO3- uptake were determined in a sterile environment. The A101 and A103 fungal isolates colonised the roots of rice plants, promoting 15N uptake, growth, and accumulation of nutrients as compared with the mock control. A103 induced the expression of the plasma membrane H+-ATPase (PM H+-ATPase) isoforms OsA5 and OsA8, the activity of the PM H+-ATPase and H+-pyrophosphatase. Our results suggest that the inoculation of rice plants with DSE fungi represents a strategy to improve the N recovery from ammonium sulphate and rice plant growth through the induction of OsA5 and OsA8 isoforms and stimulation of the PM H+-ATPase and H+-pyrophosphatase.
Assuntos
Sulfato de Amônio/metabolismo , Endófitos/isolamento & purificação , Fungos/isolamento & purificação , Micronutrientes/metabolismo , Oryza/metabolismo , Oryza/microbiologia , Proteínas de Plantas/metabolismo , Bombas de Próton/metabolismo , Transporte Biológico , Endófitos/classificação , Endófitos/genética , Fungos/classificação , Fungos/genética , Oryza/genética , Oryza/crescimento & desenvolvimento , Proteínas de Plantas/genética , Bombas de Próton/genéticaRESUMO
Analyzed by means of simulation, the effect of managements of taming primiparous during antepartum and higher body weight at calving profitability of dairy farming of milk production system. During the rainy season, in the summer season, cows were kept on pastures and, during the dry season, were confined with use of cane sugar in natura enriched with urea and ammonium sulfate. The reference zootechnical was composed herd of F1 Holstein x Gir cows of the Experimental Farm Felixlândia (FEFX) of the Agricultural Research Corporation of Minas Gerais (EPAMIG), located in the municipality of Felixlândia-MG, central region of the state. The inventory and expenditure, revenue and other data were registered on COST BOVINE MILK 1.0 software, to obtain profitability analysis. The use of managements taming primiparous during antepartum and higher body weight at calving improves the profitability of a system of milk production with F1 Holstein x Gir cows.(AU)
Analisou-se, por meio de simulação, o efeito do uso dos manejos de amansamento de primíparas no pré-parto e do maior peso vivo ao parto na rentabilidade da atividade leiteira de um sistema de produção de leite. Durante o período chuvoso, na época do verão, as vacas foram mantidas em pastos e, durante o período da seca, foram confinadas com o uso da cana-de-açúcar in natura enriquecida com ureia e sulfato de amônio. A referência zootécnica foi do rebanho composto por vacas F1 Holandês x Gir da Fazenda Experimental de Felixlândia (FEFX) da Empresa de Pesquisa Agropecuária de Minas Gerais (EPAMIG), localizada no município de FelixlândiaMG, região central do estado. O inventário bem como as despesas, as receitas e os demais dados foram cadastrados no software CUSTO BOVINO LEITE 1.0, visando obter a análise de rentabilidade. O uso dos manejos de amansamento de primíparas no pré-parto e do maior peso vivo ao parto melhora a rentabilidade de um sistema de produção de leite com vacas F1 Holandês x Gir.(AU)
Assuntos
Animais , Feminino , Gravidez , Bovinos , Saccharum/metabolismo , Sulfato de Amônio/metabolismo , Leite/estatística & dados numéricos , Análise Custo-Benefício , Criação de Animais Domésticos/métodosRESUMO
Lignocellulose is the most abundant environmental component and a renewable organic resource in soil. There are some filamentous fungi which developed the ability to break down and use cellulose, hemicellulose and lignin as an energy source. The objective of this research was to analyze the effect of three nitrogen resources (ammonium sulfate, saltpetre, soybean) in the holocellulolitic activity of Lentinula edodes EF 50 using as substrate sawdust E. benthamii. An experimental design mixture was applied with repetition in the central point consisting of seven treatments (T) of equal concentrations of nitrogen in ammonium sulfate, potassium nitrate and soybean. The enzymatic activity of avicelase, carboxymetilcellulase, ß-glucosidase, xylanases and manganese peroxidase was determined. The humidity, pH, water activity (aw) and qualitative analysis of mycelial growth in 8 times of cultivation were evaluated. The results showed negative effect on enzyme production in treatments with maximum concentration of ammonium sulfate and potassium nitrate. The treatments with cooked soybean flour expressed higher enzymatic activities in times of 3, 6 and 9 days of culture, except in the activity of manganese peroxidase. The highest production was observed in the treatment with ammonium sulfate, and soybean (83.86 UI.L-1) at 20 days of cultivation.
Assuntos
Biomassa , Lignina/farmacologia , Nitrogênio/metabolismo , Cogumelos Shiitake/enzimologia , Cogumelos Shiitake/crescimento & desenvolvimento , Sulfato de Amônio/metabolismo , Eucalyptus/química , Nitratos/metabolismo , Compostos de Potássio/metabolismo , Glycine max/química , Madeira/análiseRESUMO
Abstract Lignocellulose is the most abundant environmental component and a renewable organic resource in soil. There are some filamentous fungi which developed the ability to break down and use cellulose, hemicellulose and lignin as an energy source. The objective of this research was to analyze the effect of three nitrogen resources (ammonium sulfate, saltpetre, soybean) in the holocellulolitic activity of Lentinula edodes EF 50 using as substrate sawdust E. benthamii. An experimental design mixture was applied with repetition in the central point consisting of seven treatments (T) of equal concentrations of nitrogen in ammonium sulfate, potassium nitrate and soybean. The enzymatic activity of avicelase, carboxymetilcellulase, β-glucosidase, xylanases and manganese peroxidase was determined. The humidity, pH, water activity (aw) and qualitative analysis of mycelial growth in 8 times of cultivation were evaluated. The results showed negative effect on enzyme production in treatments with maximum concentration of ammonium sulfate and potassium nitrate. The treatments with cooked soybean flour expressed higher enzymatic activities in times of 3, 6 and 9 days of culture, except in the activity of manganese peroxidase. The highest production was observed in the treatment with ammonium sulfate, and soybean (83.86 UI.L–1) at 20 days of cultivation.
Resumo Lignocelulose é o componente mais abundante do meio ambiente e recurso orgânico renovável no solo. Alguns fungos filamentosos têm desenvolvido a habilidade de degradar e utilizar celulose, hemicelulose e lignina como fonte de energia. O objetivo deste trabalho foi analisar o efeito de três fontes de nitrogênio (sulfato de amônio, nitrato de potássio e farelo de soja) na atividade enzimática de Lentinula edodes EF 50 utilizando como substrato serragem de E. benthamii. Foi aplicado um planejamento experimental de mistura com três repetições no ponto central constituído de sete tratamentos (T) de iguais concentrações em nitrogênio de sulfato de amônia, nitrato de potássio e farinha de soja cozida. Foram determinadas a atividade enzimática da avicelase, carboximetilcelulase, β-glicosidase, xilanases e manganês peroxidase. Foram avaliados o teor de umidade, pH, atividade de água (aw) e análise qualitativa do crescimento micelial em 8 tempos de cultivo. Os resultados mostraram efeito negativo na produção das enzimas nos tratamentos com máxima concentração de sulfato de amônia e nitrato de potássio. Os tratamentos com farinha de soja cozida expressaram maiores atividades enzimáticas, nos tempos de 3, 6 e 9 dias de cultivo exceto na atividade do manganês peroxidase. A maior produção foi observada no tratamento com sulfato de amônia e farinha de soja cozida (83.86 UI.L–1) em 20 dias de cultivo.
Assuntos
Biomassa , Lignina/farmacologia , Nitrogênio/metabolismo , Cogumelos Shiitake/enzimologia , Cogumelos Shiitake/crescimento & desenvolvimento , Sulfato de Amônio/metabolismo , Eucalyptus/química , Nitratos/metabolismo , Compostos de Potássio/metabolismo , Glycine max/química , Madeira/análiseRESUMO
The effects of aging on the specific growth rate of Kluyveromyces lactis cultures, as a function of (NH4)2SO4 concentration, were evaluated. The growth kinetic parameters maximum specific growth rate and saturation constant for (NH4)2SO4 were calculated to be 0.44 h(-1) and 0.15 mmol·L(-1), respectively. Batch cultures were allowed to age for 16 days without influence of cell density or starvation. The specific growth rates of these cultures were determined each day and decreased as the population aged at different nitrogen concentrations. Aging signals (N-acetylglucosamine content of the cell wall, cell dimensions, and apoptosis markers) were measured. Apoptosis markers were detected after 5 days at limiting (NH4)2SO4 concentrations (0.57, 3.80, and 7.60 mmol·L(-1)) but only after 8 days at a nonlimiting (NH4)2SO4 concentration (38.0 mmol·L(-1)). Similarly, continuous cultures of K. lactis performed under nitrogen limitation and, at lower dilution rates, accumulated cells exhibiting aging signals. The results demonstrate that aging affects growth rate and raise the question of whether nitrogen limitation accelerates aging. Because aging is correlated with growth rate, and each dilution rate of the continuous cultures tends to select and accumulate cells with a respective age, cultures growing at lower growth rates can be useful to investigate yeast physiological responses, including aging.
Assuntos
Sulfato de Amônio/metabolismo , Kluyveromyces/crescimento & desenvolvimento , Acetilglucosamina/metabolismo , Apoptose , Parede Celular/metabolismo , Meios de Cultura , Cinética , Kluyveromyces/citologia , Kluyveromyces/fisiologiaRESUMO
Neutralizing monoclonal antibodies against three major toxic components of Bothrops atrox venom were produced and tested. The mAbs against phospholipase A2, hemorrhagic metalloprotease, and thrombin-like enzymes were produced in large amounts and purified with caprylic acid followed by ammonium sulfate precipitation. Purified mAbs were analyzed by SDS-PAGE and their ability to neutralize the respective toxins was tested. Five Swiss mice were injected i.p. with 13.5 mg of pooled mAbs and challenged via s.c. route with venom. Survival rate was recorded for the next 48 h. All mice treated and challenged with venom survived, whereas only one mouse in the control group survived. Bleeding time in mice treated with mAbs was similar to that observed in control mice. Our results show that monoclonal antibodies neutralized the lethal toxicity of Bothrops venom and indicate that there is a reasonable possibility of developing antivenoms based on humanized mAbs to treat victims of venomous animals in the future.
Assuntos
Anticorpos Monoclonais/farmacologia , Antivenenos/farmacologia , Bothrops , Venenos de Crotalídeos/antagonistas & inibidores , Sulfato de Amônio/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Antivenenos/imunologia , Coagulação Sanguínea/efeitos dos fármacos , Caprilatos/metabolismo , Venenos de Crotalídeos/imunologia , Venenos de Crotalídeos/toxicidade , Eletroforese em Gel de Poliacrilamida , Camundongos , Testes de NeutralizaçãoRESUMO
Arthrospira platensis was cultivated in tubular photobioreactor using different photosynthetic photon flux densities (PPFD) and protocols of (NH4)2SO4 fed-batch supply. Results were evaluated by variance analysis selecting maximum cell concentration (X(m)), cell productivity (P(x)), nitrogen-to-cell conversion factor (Y(X/N)) and biomass, protein and lipid contents as responses. At PPFD of 120 and 240 µmol-photons/m² s, a parabolic profile of (NH4)2SO4 addition aiming at producing biomass with 7% nitrogen content ensured X(m) values (14.1 and 12.2 g/L, respectively) comparable to those obtained with NaNO3. At PPFD of 240 µmol-photons/m² s, P(x) (1.69 g/Ld) was 36% higher, although the photosynthetic efficiency (3.0%) was less than one-half that at PPFD of 120 µmol-photons/m² s. Biomass was shown to be constituted by about 35% proteins and 10% lipids, without any dependence on PPFD or kind of nitrogen source. These results highlight the possible use of (NH4)2SO4 as alternative, cheap nitrogen source for A. platensis cultivation in tubular photobioreactors.
Assuntos
Sulfato de Amônio/metabolismo , Fotobiorreatores/microbiologia , Spirulina/metabolismo , Análise de Variância , Fotossíntese/fisiologiaRESUMO
The present study aimed at maximizing cellulase production by Penicillium funiculosum using sequential experimental design methodology for optimizing the concentrations of nitrogen sources. Three sequential experimental designs were performed. The first and the second series of experiments consisted of a 2(4) and a 2(3) factorial designs, respectively, and in the third one, a central composite rotational design was used for better visualizing the optimum conditions. The following nitrogen sources were evaluated: urea, ammonium sulfate, peptone, and yeast extract. Peptone and ammonium sulfate were removed from the medium optimization since they did not present significant statistical effect on cellulase production. The optimal concentrations of urea and yeast extract predicted by the model were 0.97 and 0.36 g/L, respectively, which were validated experimentally. By the use of the desirability function, it was possible to maximize the three main enzyme activities simultaneously, which resulted in values for FPase of 227 U/L, for CMCase of 6,917 U/L, and for beta-glucosidase of 1,375 U/L. These values corresponded to increases of 3.3-, 3.2-, and 6.7-folds, respectively, when compared to those obtained in the first experimental design. The results showed that the use of sequential experimental designs associated to the use of the desirability function can be used satisfactorily to maximize cellulase production by P. funiculosum.
Assuntos
Biotecnologia/métodos , Celulase/metabolismo , Meios de Cultura/química , Nitrogênio/metabolismo , Penicillium/metabolismo , Projetos de Pesquisa , Sulfato de Amônio/química , Sulfato de Amônio/metabolismo , Extratos Celulares/química , Celulose/química , Celulose/metabolismo , Fermentação , Lignina/química , Lignina/metabolismo , Penicillium/química , Peptonas/química , Peptonas/metabolismo , Engenharia de Proteínas/métodos , Saccharum , Ureia/química , Ureia/metabolismoRESUMO
Glutathione reductase (E.C.1.8.1.7) was purified from Phaeodactylum tricornutum cells grown axenically in an autotrophic medium. The overall procedure started with preparation of the cell extract and addition of ammonium sulfate to 20% saturation, followed by anion exchange and affinity interaction chromatography (Blue-A- and 2',5'-ADP-Sepharose). Complete purification required native polyacrylamide gel electrophoresis as the final step. The enzyme was purified to homogeneity and functionally characterized. Its native molecular mass was estimated to be 118 kDa; which corresponds to a dimer. The enzyme exhibited a specific activity of 190 U mg(-1) with an optimal activity at pH 8.0 and 32 degrees C. We determined K(m) values of 14 microM and 60 microM for NADPH and oxidized glutathione, respectively. Products inhibited the enzyme according to a hybrid ping-pong reaction mechanism. After MALDI-TOF analysis, the purified enzyme was unambiguously identified as one of the two proteins annotated as glutathione reductases in the genome of the diatom. The properties of the enzyme help to understand redox metabolic scenarios in P. tricornutum.