RESUMO
The present study aiming to determine the nutritional quality of oat (Avena sativa L.) IPR 126 in order to produce forage for ruminants. Four periods between harvests were used: 14, 21, 28 and 35 days, distributed in randomized blocks with four replicates for each treatment. The variables evaluated were: the concentration of dry matter (DM), neutral detergent insoluble fiber (NDF), crude potein (CP), lignin, dry matter (DM), ash, ether extract (EE) and in vitro digestibility of dry matter obtained by measurement of gas production. The profile of gas production was adjusted to the logistic bicompartimental mathematical model. The variables and the parameters of the adjusted gas production curves were analyzed as repeated measurements through the PROC MIXED of SAS (version 9.0) and the restricted maximum likelihood (REML) as the method of estimation of parameters. Regression analysis was performed for the variables: DM, CP, EE, NDF, ash, and for the parameter k2 of the bicompartimental model. DM and NDF concentrations increased linearly, CP, ash, and the estimations of the parameter k2 reduced linearly and the EE concentration showed a cubic behavior in function of the age of harvest. Lignin and other parameters of the Schofield model were not influenced by the age of harvest. The harvest interval influenced some chemical components and degradation rate of fiber carbohydrates, but do not interfere in lignin concentration. The forage with 21 days of cutting interval has the high nutritional value.
O objetivo com este estudo foi determinar a qualidade nutricional da aveia (Avena sativa L.) IPR 126, a fim de produzir forragem para ruminantes. Foram utilizados quatro períodos entre os cortes: 14, 21, 28 e 35 dias, distribuídos em blocos casualizados com quatro repetições para cada tratamento. As variáveis avaliadas foram: a concentração de fibra insolúvel em detergente neutro (FDN), lignina, matéria seca (MS), cinzas, extrato etéreo (EE) e digestibilidade in vitro da matéria seca obtida pela medição da produção de gás. O perfil de produção de gás foi ajustado ao modelo logístico bicompartimental. As variáveis e os parâmetros ajustados às curvas de produção de gás foram analisados como medidas repetidas através do PROC MIXED do SAS (versão 9.0) e a máxima verossimilhança restrita (REML) como o método de estimação de parâmetros. A análise de regressão foi realizada para as variáveis: MS, PB, EE, FDN, cinzas, e para o parâmetro k2 do modelo bicompartimental. As concentrações de MS e FDN aumentaram linearmente, PB, cinza, e as estimativas do parâmetro k2 diminuíram de forma linear e a concentração de EE apresentou comportamento cúbico em função da idade de colheita. A lignina e outros parâmetros do modelo de Schofield não foram influenciados pela idade de colheita. O intervalo de colheita influenciou alguns componentes químicos e taxa de degradação de carboidratos de fibra, mas não na concentração de lignina. A forragem com 21 dias de intervalo de corte apresentou maior valor nutricional.
Assuntos
Animais , Avena/citologia , Avena/enzimologia , Avena/química , Biodegradação Ambiental , Ruminantes/fisiologia , Valor NutritivoRESUMO
The present study aiming to determine the nutritional quality of oat (Avena sativa L.) IPR 126 in order to produce forage for ruminants. Four periods between harvests were used: 14, 21, 28 and 35 days, distributed in randomized blocks with four replicates for each treatment. The variables evaluated were: the concentration of dry matter (DM), neutral detergent insoluble fiber (NDF), crude potein (CP), lignin, dry matter (DM), ash, ether extract (EE) and in vitro digestibility of dry matter obtained by measurement of gas production. The profile of gas production was adjusted to the logistic bicompartimental mathematical model. The variables and the parameters of the adjusted gas production curves were analyzed as repeated measurements through the PROC MIXED of SAS (version 9.0) and the restricted maximum likelihood (REML) as the method of estimation of parameters. Regression analysis was performed for the variables: DM, CP, EE, NDF, ash, and for the parameter k2 of the bicompartimental model. DM and NDF concentrations increased linearly, CP, ash, and the estimations of the parameter k2 reduced linearly and the EE concentration showed a cubic behavior in function of the age of harvest. Lignin and other parameters of the Schofield model were not influenced by the age of harvest. The harvest interval influenced some chemical components and degradation rate of fiber carbohydrates, but do not interfere in lignin concentration. The forage with 21 days of cutting interval has the high nutritional value.(AU)
O objetivo com este estudo foi determinar a qualidade nutricional da aveia (Avena sativa L.) IPR 126, a fim de produzir forragem para ruminantes. Foram utilizados quatro períodos entre os cortes: 14, 21, 28 e 35 dias, distribuídos em blocos casualizados com quatro repetições para cada tratamento. As variáveis avaliadas foram: a concentração de fibra insolúvel em detergente neutro (FDN), lignina, matéria seca (MS), cinzas, extrato etéreo (EE) e digestibilidade in vitro da matéria seca obtida pela medição da produção de gás. O perfil de produção de gás foi ajustado ao modelo logístico bicompartimental. As variáveis e os parâmetros ajustados às curvas de produção de gás foram analisados como medidas repetidas através do PROC MIXED do SAS (versão 9.0) e a máxima verossimilhança restrita (REML) como o método de estimação de parâmetros. A análise de regressão foi realizada para as variáveis: MS, PB, EE, FDN, cinzas, e para o parâmetro k2 do modelo bicompartimental. As concentrações de MS e FDN aumentaram linearmente, PB, cinza, e as estimativas do parâmetro k2 diminuíram de forma linear e a concentração de EE apresentou comportamento cúbico em função da idade de colheita. A lignina e outros parâmetros do modelo de Schofield não foram influenciados pela idade de colheita. O intervalo de colheita influenciou alguns componentes químicos e taxa de degradação de carboidratos de fibra, mas não na concentração de lignina. A forragem com 21 dias de intervalo de corte apresentou maior valor nutricional.(AU)
Assuntos
Animais , Avena/química , Avena/citologia , Avena/enzimologia , Biodegradação Ambiental , Valor Nutritivo , Ruminantes/fisiologiaRESUMO
Trypanosoma cruzi epimastigotes are auxotrophic for polyamines because they are unable to synthesize putrescine de novo. This deficiency is due to the absence of ornithine and arginine decarboxylase genes in the parasite genome. We have been able to obtain transgenic T. cruzi expressing heterologous genes coding for these enzymes. Since arginine decarboxylase normal expression in oat requires a post-translational proteolytic cleavage of an enzyme precursor, we have investigated whether a similar processing occurs inside the transformed protozoa expressing oat arginine decarboxylase or the same enzyme attached to a C-terminal (his)(6)-tag. We were able to demonstrate that the post-translational processing also takes place inside the transgenic parasites. This cleavage is probably the result of a general proteolytic activity of T. cruzi acting on a protease-sensitive region of the protein. Interestingly, the (his)(6)-tagged enzyme expressed in the transformed parasites showed considerably increased metabolic stability and catalytic efficiency.
Assuntos
Carboxiliases/metabolismo , Regulação Enzimológica da Expressão Gênica , Processamento de Proteína Pós-Traducional , Trypanosoma cruzi/enzimologia , Sequência de Aminoácidos , Animais , Avena/enzimologia , Carboxiliases/genética , Carboxiliases/isolamento & purificação , Cinética , Dados de Sequência Molecular , Plasmídeos , Alinhamento de Sequência , Transcrição Gênica , Trypanosoma cruzi/genéticaRESUMO
Polyamine oxidase from Avena sativa L. cv. Cristal seedlings was purified to homogeneity using a simple four-step purification protocol including an infiltration washing technique. The enzyme had a high affinity for spermidine and spermine (K(m) approximately 5.5 and 1.2 microM, respectively), and also oxidized norspermidine (K(m) approximately 64.0 microM). Natural and synthetic diamines, cyclohexylamine, the putrescine analogue 1-aminooxy-3-aminopropane, and several polyamine analogues had inhibitory effects on polyamine oxidase activity and none were substrates. No inhibitory effect was observed on spermidine oxidation when the reaction product 1,3-diaminopropane was added. By contrast, 1-aminooxy-3-aminopropane showed mixed inhibition kinetics and a K(i) value of 0.113 mM. In addition, in vitro enzymatic activity assays showed that the oligoamine [3,8,13,18,23,28,33,38,43,48-deca-aza-(trans-25)-pentacontene], the tetramine 1,14-bis-[ethylamino]-5,10-diazatetradecane, and the pentamine 1,19-bis-[ethylamino]-5,10,15-triazanonadecane, displayed potent competitive inhibitory activities against polyamine oxidase with K(i) values of 5.8, 110.0 and 7.6 nM, respectively, where cyclohexylamine was a weak competitive inhibitor with a K(i) value of 0.5 mM. These analogues did not inhibit mycelial growth of the fungus Sclerotinia sclerotiorum (Lib.) De Bary and the bacterium Pseudomonas viridiflava (Burkholder) Dowson in vitro. On the contrary, with concentrations similar to those used for polyamine analogues, guazatine (a well-known fungicide and at the same time, a polyamine oxidase inhibitor) inhibited ( approximately 85%) S. sclerotiorum mycelial growth on Czapek-Dox medium. Finally, the analogue 1,19-bis-ethylamino-5,10,15-triazanonadecane inhibited polyamine oxidase activity observed in segments of maize leaves in vivo. The results obtained provide insights into research on the influence of polyamine oxidase activity on plant biotic and abiotic stresses.
Assuntos
Avena/enzimologia , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/antagonistas & inibidores , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Poliaminas/metabolismo , Antibacterianos/farmacologia , Antifúngicos/farmacologia , Ascomicetos/efeitos dos fármacos , Peróxido de Hidrogênio/metabolismo , Estrutura Molecular , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/isolamento & purificação , Propilaminas/metabolismo , Pseudomonas/efeitos dos fármacos , Estresse Fisiológico , Especificidade por Substrato , Poliamina OxidaseRESUMO
We have previously demonstrated that wild-type Trypanosoma cruzi epimastigotes lack arginine decarboxylase (ADC) enzymatic activity as well as its encoding gene. A foreign ADC has recently been expressed in T. cruzi after transformation with a recombinant plasmid containing the complete coding region of the oat ADC gene. In the present study, upon modulation of exogenous ADC expression, we found that ADC activity was detected early after transfection; subsequently it decreased to negligible levels between 2 and 3 weeks after electroporation and was again detected approximately 4 weeks after electroporation. After this period, the ADC activity increased markedly and became expressed permanently. These changes of enzymatic activity showed a close correlation with the corresponding levels of ADC transcripts. To investigate whether the genome organization of the transgenic T. cruzi underwent any modification related to the expression of the heterologous gene, we performed PCR amplification assays, restriction mapping and pulse-field gel electrophoresis with DNA samples or chromosomes obtained from parasites collected at different time-points after transfection. The results indicated that the transforming plasmid remained as free episomes during the transient expression of the foreign gene. Afterwards, the free plasmid disappeared almost completely for several weeks and, finally, when the expression of the ADC gene became stable, two or more copies of the transforming plasmid arranged in tandem were integrated into a parasite chromosome (1.4 Mbp) bearing a ribosomal RNA locus. The sensitivity of transcription to alpha-amanitin strongly suggests involvement of the protozoan RNA polymerase I in the transcription of the exogenous ADC gene.
Assuntos
Avena/enzimologia , Carboxiliases/genética , Regulação Enzimológica da Expressão Gênica , Genoma , Trypanosoma cruzi/enzimologia , Amanitinas/farmacologia , Animais , Avena/genética , Carboxiliases/efeitos dos fármacos , Carboxiliases/metabolismo , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Organismos Geneticamente Modificados , RNA Polimerase I/antagonistas & inibidores , RNA Polimerase I/metabolismo , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Especificidade da Espécie , Fatores de Tempo , Transcrição Gênica/fisiologia , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/metabolismoRESUMO
In this work, cadmium phytotoxicity and quantitative sensitivity relationships between different hierarchical endpoints in plants cultivated in a contaminated soil were studied. Thus, germination rate, biomass growth and antioxidative enzyme activity (i.e. superoxide dismutase, peroxidase, catalase and glutathione reductase) in three terrestrial plants (Avena sativa L., Brassica campestris L. cv. Chinensis, Lactuca sativa L. cv. hanson) were analyzed. Plant growth tests were carried out according to an International Standard Organization method and the results were analyzed by ANOVA followed by Williams' test. The concentration of Cd2+ that had the smallest observed significant negative effect (LOEC) on plant biomass was 6.25, 12.5 and 50 mg Cd/kg dry soil for lettuce, oat and Chinese cabbage, respectively. Activity of all enzymes studied increased significantly compared to enzyme activity in plant controls. For lettuce, LOEC values (mg Cd/kg dry soil) for enzymic activity ranged from 0.05 (glutathione reductase) to 0.39 (catalase). For oat, LOEC values (mg Cd/kg dry soil) ranged from 0.19 (for superoxide dismutase and glutathione reductase) to 0.39 (for catalase and peroxidase). For Chinese cabbage, LOEC values (mg Cd/kg dry soil) ranged from 0.19 (peroxidase, catalase and glutathione reductase) to 0.39 (superoxide dismutase). Classical (i.e. germination and biomass) and biochemical (i.e. enzyme activity) endpoints were compared to establish a sensitivity ranking, which was: enzyme activity>biomass>germination rate. For cadmium-soil contamination, the determination of quantitative sensitivity relationships (QSR) between classical and antioxidative enzyme biomarkers showed that the most sensitive plant species have, generally, the lowest QSR values.
Assuntos
Avena/efeitos dos fármacos , Brassica/efeitos dos fármacos , Cádmio/toxicidade , Lactuca/efeitos dos fármacos , Poluentes do Solo/toxicidade , Antioxidantes/metabolismo , Avena/enzimologia , Avena/crescimento & desenvolvimento , Biomarcadores , Brassica/enzimologia , Brassica/crescimento & desenvolvimento , Germinação/efeitos dos fármacos , Lactuca/enzimologia , Lactuca/crescimento & desenvolvimento , Oxirredutases/metabolismoRESUMO
Biotechnological uses of plant cell-tissue culture usually rely on constitutive transgene expression. However, such expression of transgenes may not always be desirable. In those cases, the use of an inducible promoter could be an alternative approach. To test this hypothesis, we developed two binary vectors harboring a stress-inducible promoter from Arabidopsis thaliana, driving the beta-glucuronidase reporter gene and the oat arginine decarboxylase. Transgenic hairy roots of Lotus corniculatus were obtained with osmotic- and cold-inducible beta-glucuronidase and arginine decarboxylase activities. The increase in the activity of the latter was accompanied by a significant rise in total free polyamines level. Through an organogenesis process, we obtained L. corniculatus transgenic plants avoiding deleterious phenotypes frequently associated with the constitutive over-expression of arginine decarboxylation and putrescine accumulation.