RESUMO
Aleurites moluccana L. is grown as a roadside tree in southern China and the oil content of its seed is higher than other oil plants, such as Jatropha curcas and Camellia oleifera. A. moluccana is considered a promising energy plant because its seed oil could be used to produce biodiesel and bio-jet fuel. In addition, the bark, leaves, and kernels of A. moluccana have various medical and commercial uses. Here, a novel gene coding the biotin carboxyl carrier protein subunit (BCCP) was cloned from A. moluccana L. using the homology cloning method combined with rapid amplification of cDNA end (RACE) technology. The isolated full-length cDNA sequence (designated AM-accB) was 1188 bp, containing a 795-bp open reading frame coding for 265 amino acids. The deduced amino acid sequence of AM-accB contained a biotinylated domain located between amino acids 190 and 263. A. moluccana BCCP shows high identity at the amino acid level to its homologues in other higher plants, such as Vernicia fordii, J. curcas, and Ricinus communis (86, 77, and 70%, respectively), which all contain conserved domains for ACCase activity. The expression of the AM-accB gene during the middle stage of development and maturation in A. moluccana seeds was higher than that in early and later stages. The expression pattern of the AM-accB gene is very similar to that of the oil accumulation rate.
Assuntos
Acetil-CoA Carboxilase/genética , Aleurites/genética , Clonagem Molecular , Expressão Gênica , Subunidades Proteicas/genética , Acetil-CoA Carboxilase/química , Aleurites/metabolismo , Sequência de Aminoácidos , DNA Complementar/genética , Ácido Graxo Sintase Tipo II/química , Ácido Graxo Sintase Tipo II/genética , Dados de Sequência Molecular , Óleos de Plantas/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Fatores de TempoRESUMO
Herbicides that inhibit acetyl coenzyme A carboxylase (ACCase) are commonly used in Mexico to control weedy grasses such as little seed canarygrass (Phalaris minor). These herbicides are classified into three major families (ariloxyphenoxypropionates (APP), cyclohexanodiones (CHD), and, recently, phenylpyrazolines (PPZ)). In this work, the resistance to ACCase (APP, CHD, and PPZ) inhibiting herbicides was studied in a biotype of Phalaris minor (P. minor) from Mexico, by carrying out bioassays at the whole-plant level and investigating the mechanism behind this resistance. Dose-response and ACCase in vitro activity assays showed cross-resistance to all ACCase herbicides used. There was no difference in the absorption, translocation, and metabolism of the (14)C-diclofop-methyl between the R and S biotypes. The PCR generated CT domain fragments of ACCase from the R biotype and an S reference were sequenced and compared. The Ile-1781-Leu and Asp-2078-Gly point mutations were identified. These mutations could explain the loss of affinity for ACCase by the ACCase-inhibing herbicides. This is the ï¬rst report showing that this substitution confers resistance to APP, CHD, and PPZ herbicides in P. minor from Mexico. The mutations have been described previously only in a few cases; however, this is the first study reporting on a pattern of cross-resistance with these mutations in P. minor. The findings could be useful for better management of resistant biotypes carrying similar mutations.
Assuntos
Acetil-CoA Carboxilase/genética , Substituição de Aminoácidos , Códon , Resistência a Herbicidas/genética , Herbicidas/farmacologia , Mutação , Phalaris/efeitos dos fármacos , Phalaris/genética , Acetil-CoA Carboxilase/química , Acetil-CoA Carboxilase/metabolismo , Sequência de Aminoácidos , Relação Dose-Resposta a Droga , Ativação Enzimática , Éteres Difenil Halogenados/metabolismo , México , Dados de Sequência Molecular , Phalaris/metabolismo , Alinhamento de SequênciaRESUMO
A stochastic simulation of adsorption processes was developed to simulate the coverage of an atomic force microscope (AFM) tip with enzymes represented as rigid polyhedrons. From geometric considerations of the enzyme structure and AFM tip, we could estimate the average number of active sites available to interact with substrate molecules in the bulk. The procedure was exploited to determine the interaction force between acetyl-CoA carboxylase enzyme (ACC enzyme) and its substrate diclofop, for which steered molecular dynamics (SMD) was used. The theoretical force of (1.6±0.5) nN per enzyme led to a total force in remarkable agreement with the experimentally measured force with AFM, thus demonstrating the usefulness of the procedure proposed here to assist in the interpretation of nanobiosensors experiments.
Assuntos
Enzimas Imobilizadas/química , Acetil-CoA Carboxilase/antagonistas & inibidores , Acetil-CoA Carboxilase/química , Técnicas Biossensoriais , Domínio Catalítico , Microscopia de Força Atômica , Simulação de Dinâmica Molecular , Éteres Fenílicos/química , Propionatos/química , Ligação Proteica , Estrutura Quaternária de Proteína , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/química , Processos Estocásticos , TermodinâmicaRESUMO
OBJECTIVE: The present study investigates the level of Sterol-regulatory element-binding proteins (SREBP-1c) and related proteins in obese mice (DIO) treated with SREBP-1c antisense oligonucleotide (ASO) to observe a reversal of steatosis. MATERIALS AND METHODS: Swiss mice were fed on chow containing 61 kJ% saturated fat for 8 weeks to develop obesity. After this period, one group of animals was used to assess the molecular effects of SREBP-1c antisense oligonucleotide treatment by immunoblot analysis in a dose-response curve (0; 1.0; 2.0; 3.0; 4.0 nmol/day). After the dose (3.0 nmol/day) was determined, another group was treated for 14 days. After a period of 24 h following the last injection mice were killed and plasma and hepatic tissue were obtained to evaluate plasma triglycerides and total liver fat. Western blot was performed to evaluate SREBP-1c, FAS, SCD-1, PPARγ and CPT1 expression and AMPK[Thr172] and ACC[Ser79] phosphorylation. Livers were stained using the hematoxylin and eosin method for histological analysis. RESULTS: Body weight, epididymal fat and glucose levels were not affected by one daily dose of ASO. However, total plasma triglycerides and total liver fat were significantly reduced. Also, this treatment inhibited SREBP-1c and reduced protein levels of a series of proteins involved in lipogenesis, including ACC, FAS and SCD-1. Moreover, mice treated with ASO presented a significant reduction in macroscopic and microscopic features of hepatic steatosis. CONCLUSION: Our results demonstrate that the inhibition of SREBP-1c decreased the expression of lipogenic enzymes, reducing the accumulation of triglycerides and, finally, reversing hepatic steatosis in mice.
Assuntos
Fígado Gorduroso/tratamento farmacológico , Fígado Gorduroso/enzimologia , Oligonucleotídeos Antissenso/farmacologia , Proteína de Ligação a Elemento Regulador de Esterol 1/efeitos dos fármacos , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Proteínas Quinases Ativadas por AMP/química , Acetil-CoA Carboxilase/química , Adiposidade , Animais , Ácido Graxo Sintases/metabolismo , Fígado Gorduroso/patologia , Camundongos , Camundongos Obesos , Hepatopatia Gordurosa não Alcoólica , Oligonucleotídeos Antissenso/uso terapêutico , PPAR gama/metabolismo , Fosforilação , Estearoil-CoA Dessaturase/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Triglicerídeos/sangueRESUMO
Nanobiosensors can be built via functionalization of atomic force microscopy (AFM) tips with biomolecules capable of interacting with the analyte on a substrate, and the detection being performed by measuring the force between the immobilized biomolecule and the analyte. The optimization of such sensors may require multiple experiments to determine suitable experimental conditions for the immobilization and detection. In this study we employ molecular modeling techniques to assist in the design of nanobiosensors to detect herbicides. As a proof of principle, the properties of acetyl co-enzyme A carboxylase (ACC) were obtained with molecular dynamics simulations, from which the dimeric form in an aqueous solution was found to be more suitable for immobilization owing to a smaller structural fluctuation than the monomeric form. Upon solving the nonlinear Poisson-Boltzmann equation using a finite-difference procedure, we found that the active sites of ACC exhibited a positive surface potential while the remainder of the ACC surface was negatively charged. Therefore, optimized biosensors should be prepared with electrostatic adsorption of ACC onto an AFM tip functionalized with positively charged groups, leaving the active sites exposed to the analyte. The preferential orientation for the herbicides diclofop and atrazine with the ACC active site was determined by molecular docking calculations which displayed an inhibition coefficient of 0.168 µM for diclofop, and 44.11 µM for atrazine. This binding selectivity for the herbicide family of diclofop was confirmed by semiempirical PM6 quantum chemical calculations which revealed that ACC interacts more strongly with the herbicide diclofop than with atrazine, showing binding energies of -119.04 and +8.40 kcal mol(-1), respectively. The initial measurements of the proposed nanobiosensor validated the theoretical calculations and displayed high selectivity for the family of the diclofop herbicides.
Assuntos
Acetil-CoA Carboxilase/metabolismo , Técnicas Biossensoriais/instrumentação , Nanotecnologia/instrumentação , Acetil-CoA Carboxilase/química , Técnicas Biossensoriais/métodos , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Desenho de Equipamento , Herbicidas/análise , Microscopia de Força Atômica , Modelos Moleculares , Simulação de Dinâmica Molecular , Nanotecnologia/métodos , Teoria Quântica , Propriedades de SuperfícieRESUMO
Regulation of the cytosolic acetyl-coenzyme A carboxylase (ACCase) gene promoter from common bean (Phaseolus vulgaris) was studied in transgenic Arabidopsis (Arabidopsis thaliana) plants using a beta-glucuronidase (GUS) reporter gene fusion (PvACCase::GUS). Under normal growth conditions, GUS was expressed in hydathodes, stipules, trichome bases, flowers, pollen, and embryos. In roots, expression was observed in the tip, elongation zone, hypocotyl-root transition zone, and lateral root primordia. The PvACCase promoter was induced by wounding, Pseudomonas syringae infection, hydrogen peroxide, jasmonic acid (JA), ethylene, or auxin treatment. Analysis of PvACCase::GUS expression in JA and ethylene mutants (coronatine insensitive1-1 [coi1-1], ethylene resistant1-1 [etr1-1], coi1-1/etr1-1) suggests that neither JA nor ethylene perception participates in the activation of this gene in response to wounding, although each of these independent signaling pathways is sufficient for pathogen or hydrogen peroxide-induced PvACCase gene expression. We propose a model involving different pathways of PvACCase gene activation in response to stress.