RESUMO
Due to the great significance of amino acids, a substantial number of research studies has been directed toward the development of effective and reliable platforms for their evaluation, detection, and identification. In order to support these studies, a new electrochemical platform based on PANI/ZnO nanowires' modified carbon inks screen-printed electrodes was developed for qualitative analysis of electroactive amino acids, with emphasis on tyrosine (Tyr) and tryptophan (Trp). A comparative investigation of the carbon ink before and after modification with the PANI/ZnO was performed by scanning electron microscopy and by Raman spectroscopy, confirming the presence of PANI and ZnO nanowires. Electrochemical investigations by cyclic voltammetry and electrochemical impedance spectroscopy have shown a higher charge-transfer rate constant, which is reflected into lower charge-transfer resistance and higher capacitance values for the PANI/ZnO modified ink when compared to the simple carbon screen-printed electrode. In order to demonstrate the electrochemical performances of the PANI/ZnO nanowires' modified carbon inks screen-printed electrodes for amino acids analysis, differential pulse voltammograms were obtained in individual and mixed solutions of electroactive amino acids. It has been shown that the PANI/ZnO nanowires' modified carbon inks screen-printed electrodes allowed for tyrosine and tryptophan a peak separation of more than 100 mV, enabling their screening and identification in mixed solutions, which is essential for the electrochemical analysis of proteins within the proteomics research field.
Assuntos
Carbono , Óxido de Zinco , Carbono/química , Aminoácidos , Tinta , Triptofano , Óxido de Zinco/química , Tirosina , Eletrodos , Técnicas Eletroquímicas/métodosRESUMO
An understanding of the mechanisms underlying ammonium (NH(4)(+)) toxicity in plants requires prior knowledge of the metabolic uses for nitrogen (N) and carbon (C). We have recently shown that pea plants grown at high NH(4)(+) concentrations suffer an energy deficiency associated with a disruption of ionic homeostasis. Furthermore, these plants are unable to adequately regulate internal NH4(+) levels and the cell-charge balance associated with cation uptake. Herein we show a role for an extra-C application in the regulation of C-N metabolism in NH(4)(+) -fed plants. Thus, pea plants (Pisum sativum) were grown at a range of NH(4)(+) concentrations as sole N source, and two light intensities were applied to vary the C supply to the plants. Control plants grown at high NH(4)(+) concentration triggered a toxicity response with the characteristic pattern of C-starvation conditions. This toxicity response resulted in the redistribution of N from amino acids, mostly asparagine, and lower C/N ratios. The C/N imbalance at high NH(4)(+) concentration under control conditions induced a strong activation of root C metabolism and the upregulation of anaplerotic enzymes to provide C intermediates for the tricarboxylic acid cycle. A high light intensity partially reverted these C-starvation symptoms by providing higher C availability to the plants. The extra-C contributed to a lower C4/C5 amino acid ratio while maintaining the relative contents of some minor amino acids involved in key pathways regulating the C/N status of the plants unchanged. C availability can therefore be considered to be a determinant factor in the tolerance/sensitivity mechanisms to NH(4)(+) nutrition in plants.
Assuntos
Compostos de Amônio/farmacologia , Aspartato-Amônia Ligase/metabolismo , Carbono/metabolismo , Metabolismo Energético/efeitos dos fármacos , Nitrogênio/metabolismo , Pisum sativum/enzimologia , Asparagina/biossíntese , Metabolismo Energético/efeitos da radiação , Ácido Glutâmico/metabolismo , Glutamina/metabolismo , Luz , Pisum sativum/efeitos dos fármacos , Pisum sativum/efeitos da radiação , Poliaminas/metabolismo , Ácido gama-Aminobutírico/metabolismoRESUMO
The iron superoxide dismutase (FeSOD) is a first barrier to defend photosynthetic organisms from superoxide radicals. Although it is broadly present in plants and bacteria, FeSODs are absent in animals. They belong to the same phylogenic family as Mn-containing SODs, which are also highly efficient at detoxifying superoxide radicals. In addition, SODs can react with peroxynitrite, and FeSOD enzyme has already been used to evaluate the anti-nitrative capacity of plant antioxidants. Gold nanoparticles (AuNPs) have been shown to significantly improve the functionality and the efficiency of ligands, providing they are properly assembled. In this work, the characteristics of the recombinant cowpea (Vigna unguiculata) FeSOD (rVuFeSOD) immobilized onto AuNPs were investigated as a function of (1) NP surface chemistry and (2) biofunctionalization methods, either physical adsorption or covalent bonding. The NP surface chemistry was studied by varying the concentration of the ligand molecule 11-mercaptoundecanoic acid (MUA) on the NP surface. The coverage and activity of the protein on AuNPs was determined and correlated to the surface chemistry and the two biofunctionalization methods. rVuFeSOD-AuNPs conjugate stability was monitored through absorption measurements, agarose gel electrophoresis and DLS, enzymatic activity by a colorimetric assay and by in-gel activity assay, and coverage was measured by colorimetric assay. When using physical adsorption, the NP is the most perturbing agent for the activity of the enzyme. In contrast, only the NP coverage was affected by MUA ligand concentration. However, during covalent attachment, both the NP and the concentration of MUA on the surface influenced the enzyme activity, while the coverage of the NP remained constant. The results evidence the importance of the biomolecule and AuNP interaction for the functionality of the hybrid. These strategies can be used to develop electrochemical biosensors for O2â¢- and for peroxynitrite in biomedical applications.
RESUMO
Two phylogenetically unrelated superoxide dismutase (SOD) families, i.e., CuZnSOD (copper and zinc SOD) and FeMn-CamSOD (iron, manganese, or cambialistic SOD), eliminate superoxide radicals in different locations within the plant cell. CuZnSOD are located within the cytosol and plastids, while the second family of SOD, which are considered to be of bacterial origin, are usually located within organelles, such as mitochondria. We have used the reactive oxygen species-producer methylviologen (MV) to study SOD isozymes in the indeterminate nodules on pea (Pisum sativum). MV caused severe effects on nodule physiology and structure and also resulted in an increase in SOD activity. Purification and N-terminal analysis identified CamSOD from the Rhizobium leguminosarum endosymbiont as one of the most active SOD in response to the oxidative stress. Fractionation of cell extracts and immunogold labeling confirmed that the CamSOD was present in both the bacteroids and the cytosol (including the nuclei, plastids, and mitochondria) of the N-fixing cells, and also within the uninfected cortical and interstitial cells. These findings, together with previous reports of the occurrence of FeSOD in determinate nodules, indicate that FeMnCamSOD have specific functions in legumes, some of which may be related to signaling between plant and bacterial symbionts, but the occurrence of one or more particular isozymes depends upon the nodule type.
Assuntos
Pisum sativum/enzimologia , Pisum sativum/genética , Rhizobium/enzimologia , Rhizobium/genética , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , DNA Bacteriano/genética , Genes Bacterianos , Genes de Plantas , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/fisiologia , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Fixação de Nitrogênio/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Paraquat/farmacologia , Pisum sativum/microbiologia , Pisum sativum/ultraestrutura , Frações Subcelulares/enzimologiaRESUMO
The genome of Spodoptera frugiperda multiple nucleopolyhedrovirus (NPV) was inserted into a bacmid (Sfbac) and used to produce a mutant lacking open reading frame 29 (Sf29null). Sf29null bacmid DNA was able to generate an infection in S. frugiperda. Approximately six times less DNA was present in occlusion bodies (OBs) produced by the Sf29null bacmid in comparison to viruses containing this gene. This reduction in DNA content was consistent with fewer virus particles being packaged within Sf29null bacmid OBs, as determined by fractionation of dissolved polyhedra and comparison of occlusion-derived virus (ODV) infectivity in cell culture. DNA from Sfbac, Sf29null, or Sf29null-repair, in which the gene deletion had been repaired, were equally infectious when used to transfect S. frugiperda. All three viruses produced similar numbers of OBs, although those from Sf29null were 10-fold less infectious than viruses with the gene. Insects infected with Sf29null bacmid died approximately 24 h later than positive controls, consistent with the reduced virus particle content of Sf29null OBs. Transcripts from Sf29 were detected in infected insects 12 h prior to those from the polyhedrin gene. Homologs to Sf29 were present in other group II NPVs, and similar sequences were present in entomopoxviruses. Analysis of the Sf29 predicted protein sequence revealed signal peptide and transmembrane domains, but the presence of 12 potential N-glycosylation sites suggest that it is not an ODV envelope protein. Other motifs, including zinc-binding and threonine-rich regions, suggest degradation and adhesion functions. We conclude that Sf29 is a viral factor that determines the number of ODVs occluded in each OB.
Assuntos
Regulação Viral da Expressão Gênica , Nucleocapsídeo/genética , Nucleopoliedrovírus/metabolismo , Vírion , Animais , Células Cultivadas , DNA Viral/metabolismo , Deleção de Genes , Genes Virais , Glicosilação , Humanos , Insetos , Nucleocapsídeo/metabolismo , Fases de Leitura Aberta , Sinais Direcionadores de Proteínas , Vírion/químicaRESUMO
Synaptosomes and plasma membranes obtained from rat brain display ectoenzymatic hydrolytic activity responsible for hydrolysis of the neurotransmitter/neuroregulatory nucleotides diadenosine polyphosphates. Intact synaptosomes and plasma and synaptic membranes isolated by sucrose-gradient ultracentrifugation from several brain regions (hypothalamus, hippocampus, temporal cortex, frontal cortex striatum and cerebellum) degraded the fluorogenic substrates diethenoadenosine polyphosphates up to ethenoadenosine as by-product. Purified ectoenzyme cleaved substrates always releasing the mononucleotide moieties ethenoadenosine 5'-monophosphate and the corresponding ethenoadenosine (n-1) 5'-phosphate. Ectoenzymatic hydrolysis reached maximal activity at pH 9.0 (pH range 6.5-9.0) and was activated by Ca(2+) and Mg(2+) ions, with maximal effects around 2.0 mM cation. EDTA drastically reduced activity and Zn(2+) was required for enzyme reactivation. Hydrolysis of substrates followed hyperbolic kinetics with K(m) values in the 3-10 microM range. Diadenosine polyphosphates and heparin behaved as competitive inhibitors in the enzymatic hydrolysis of diethenoadenosine polyphosphates and AMP, ATP, alpha,beta-methyleneADP, ADPbetaS ATPgammaS, beta,gamma-methyleneATP, suramin and diethyl pyrocarbonate were also inhibitors. Ectoenzymatic activity shared the typical characteristics of members of the ecto-nucleotide pyrophosphatase/phosphodiesterase (E-NPP) family and inhibition data suggest that NPP1 ectoenzyme is involved in the cleavage of extracellular diadenosine polyphosphates in brain. Synaptic membranes from cerebellum, hypothalamus and hippocampus presented the highest activities and no activity differences were observed between young and aged animals. However, plasma membranes showed a more homogeneous distribution of ectoenzymatic activity but a general increase was detected in aged animals. Enhancement of ectoenzymatic diadenosine polyphosphate cleaving activity found in plasma membranes from old animals could play a deleterious role in aged brain by limiting neuroprotective effects reported for extracellular diadenosine tetraphosphate.
Assuntos
Membrana Celular/enzimologia , Sistema Nervoso Central/enzimologia , Fosfatos de Dinucleosídeos/metabolismo , Líquido Extracelular/enzimologia , Neurônios/enzimologia , Diester Fosfórico Hidrolases/metabolismo , Pirofosfatases/metabolismo , Fatores Etários , Envelhecimento/metabolismo , Animais , Membrana Celular/ultraestrutura , Sistema Nervoso Central/ultraestrutura , Ativação Enzimática/fisiologia , Hidrólise , Cinética , Masculino , Neurônios/ultraestrutura , Fármacos Neuroprotetores/metabolismo , Ratos , Ratos Wistar , Membranas Sinápticas/enzimologia , Membranas Sinápticas/ultraestrutura , Sinaptossomos/enzimologia , Sinaptossomos/ultraestruturaRESUMO
The enzyme-linked immunosorbent assay (ELISA) technique is based on the specific recognition ability of the molecular structure of an antigen (epitope) by an antibody and is likely the most important diagnostic technique used today in bioscience. With this methodology, it is possible to diagnose illness, allergies, alimentary fraud, and even to detect small molecules such as toxins, pesticides, heavy metals, etc. For this reason, any procedures that improve the detection limit, sensitivity or reduce the analysis time could have an important impact in several fields. In this respect, many methods have been developed for improving the technique, ranging from fluorescence substrates to methods for increasing the number of enzyme molecules involved in the detection such as the biotin-streptavidin method. In this context, nanotechnology has offered a significant number of proposed solutions, mainly based on the functionalization of nanoparticles from gold to carbon which could be used as antibody carriers as well as reporter enzymes like peroxidase. However, few works have focused on the study of best practices for nanoparticle functionalization for ELISA enhancement. In this work, we use 20 nm gold nanoparticles (AuNPs) as a vehicle for secondary antibodies and peroxidase (HRP). The design of experiments technique (DOE) and four different methods for biomolecule loading were compared using a rabbit IgG/goat anti-rabbit IgG ELISA model (adsorption, directional, covalent and a combination thereof). As a result, AuNP probes prepared by direct adsorption were the most effective method. AuNPs probes were then used to detect gliadin, one of the main components of wheat gluten, the protein composite that causes celiac disease. With this optimized approach, our data showed a sensitivity increase of at least five times and a lower detection limit with respect to a standard ELISA of at least three times. Additionally, the assay time was remarkably decreased.
RESUMO
We describe here the purification and characterisation of the human enzyme diadenosine triphosphatase isolated from human platelets and leukocytes, offering biochemical and immunochemical evidence to identify this enzyme with the novel tumour suppressor Fhit protein, a homodimer composed of approximately 17 kDa monomers. It catalyses the Mg(2+)-dependent hydrolysis of diadenosine triphosphate, Ap(3)A, to AMP+ADP. The fluorogenic substrate di-ethenoadenosine triphosphate, epsilon-(Ap(3)A), and Fhit antibodies were used for enzymatic and immunochemical characterisations, respectively. Human Ap(3)Aase presents a native molecular mass of approximately 32 kDa and no significant differences were found in K(m) values (2 microM), activating effects by Mg(2+), Ca(2+), and Mn(2+), optimum pH (7.0-7.2) or inhibition by Zn(2+) and diethyl pyrocarbonate between the human enzyme and the recombinant Fhit protein. Suramin is a very potent competitive inhibitor of both human Ap(3)Aase and Fhit protein with K(i) values in the range 20-30 nM. Both human and rat Ap(3)Aase activity co-purifies with Fhit immunoreactivity under gel filtration, ion-exchange and affinity chromatography. Homogeneous human Ap(3)Aase preparations analysed by SDS-PAGE and Western blot analysis with Fhit antibodies elicit immunochemical responses corresponding to a approximately 17 kDa polypeptide, indicating a dimeric structure for the enzyme Ap(3)Aase. The strong inhibition of Fhit enzyme by the drug suramin, supports the need to investigate the therapeutic potential of Fhit-Ap(3)Aase mediated by its interaction with suramin or related drugs.
Assuntos
Hidrolases Anidrido Ácido/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Hidrolases Anidrido Ácido/sangue , Hidrolases Anidrido Ácido/isolamento & purificação , Animais , Sítios de Ligação , Plaquetas/enzimologia , Encéfalo/enzimologia , Catálise/efeitos dos fármacos , Cromatografia em Gel/métodos , Cromatografia Líquida de Alta Pressão/métodos , Fosfatos de Dinucleosídeos/metabolismo , Eletroforese em Gel de Poliacrilamida , Fluorometria , Humanos , Imuno-Histoquímica , Cinética , Leucócitos/enzimologia , Masculino , Proteínas de Neoplasias/sangue , Proteínas de Neoplasias/isolamento & purificação , Ratos , Ratos Wistar , Especificidade por Substrato , Suramina/farmacologia , Proteínas Supressoras de Tumor/sangue , Proteínas Supressoras de Tumor/isolamento & purificaçãoRESUMO
The VirPphA/AvrPtoB family of type III effector proteins from the phytopathogenic bacterium Pseudomonas syringae is one of the models providing insights into the molecular mechanisms conferring plant disease resistance and pathogenesis. In this review we summarize recent advances concerning the VirPphA/AvrPtoB family of effectors involved in the elicitation and suppression of plant defense responses.
Assuntos
Proteínas de Bactérias/fisiologia , Doenças das Plantas/microbiologia , Pseudomonas syringae/fisiologia , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Dados de Sequência Molecular , Plantas/microbiologia , Pseudomonas syringae/genética , Pseudomonas syringae/metabolismo , Pseudomonas syringae/patogenicidade , Alinhamento de Sequência , VirulênciaRESUMO
Nitric oxide cytotoxicity arises from its rapid conversion to peroxynitrite (ONOO(-)) in the presence of superoxide, provoking functional changes in proteins by nitration of tyrosine residues. The physiological significance of this post-translational modification is associated to tissue injury in animals, but has not been yet clarified in plants. The objective of this study was to establish new approaches that could help to understand ONOO(-) reactivity in plants. A recombinant Fe-superoxide dismutase from cowpea (Vigna unguiculata (L.) Walp.), rVuFeSOD, was the target of the ONOO(-)-generator SIN-1, and the anti-nitrative effect of plant antioxidants and haemoglobins was tested in vitro. Nitration on rVuFeSOD was evaluated immunochemically or as the loss of its enzymatic activity. This assay proved to be useful to test a variety of plant compounds for anti-nitrative capacity. Experimental data confirmed that rice (Oryza sativa L.) haemoglobin-1 (rOsHbI) and cowpea leghaemoglobin-2 exerted a protective function against ONOO(-) by diminishing nitration on rVuFeSOD. Both plant haemoglobins were nitrated by SIN-1. The chelator desferrioxamine suppressed nitration in rOsHbI, indicating that Fe plays a key role in the reaction. The removal of the haem moiety in rOsHbI importantly suppressed nitration, evidencing that this reaction may be self-catalyzed. Among small antioxidants, ascorbate remarkably decreased nitration in all tests. The phenolic compounds caffeic acid, gallic acid, pyrogallol, 4-hydroxybenzoic acid and the flavonoid gossypin also diminished tyrosine nitration and protected rVuFeSOD to different extents. It is concluded that small plant antioxidants, especially ascorbate, and haemoglobins may well play key roles in ONOO(-) homeostasis in vivo.
Assuntos
Antioxidantes/química , Fabaceae/enzimologia , Ácido Peroxinitroso/química , Proteínas de Plantas/química , Superóxido Dismutase/química , Proteínas Recombinantes/químicaRESUMO
Superoxide dismutase enzymes (SODs) are an essential part of the first line of cellular defense system against free radicals species. They catalyze the dismutation of superoxide radicals into oxygen and hydrogen peroxide. Although several studies have examined the attachment of superoxide dismutases to nanoparticles and nanostructures, never has been used a member of the Fe/MnSOD family. In this study, the behavior of plant origin FeSOD enzyme on three different nanopatterned surfaces was investigated as a function of covalent and electrostatic binding. Fluorescence microscopy was used to demonstrate that the protein is attached only to the gold layer. We also examined the activity of SOD by a colorimetric assay, and we have shown that the enzyme remains active after attachment to the three different surfaces under both kind of binding (electrostatic and covalent). This methodology could be useful for those who want to functionalize nanostructures with a SOD enzyme and test the activity. This process could be of great interest for the development of peroxynitrite and superoxide biosensors.
Assuntos
Enzimas Imobilizadas/química , Nanoestruturas/química , Nanotecnologia/métodos , Superóxido Dismutase/química , Compostos Azo/química , Técnicas Biossensoriais/instrumentação , Enzimas Imobilizadas/metabolismo , Fabaceae/enzimologia , Ouro/química , Peróxido de Hidrogênio/química , Microscopia de Fluorescência , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Ligação Proteica , Dióxido de Silício/química , Eletricidade Estática , Superóxido Dismutase/metabolismo , Propriedades de SuperfícieRESUMO
Three main families of SODs in plants may be distinguished according to the metal in the active center: CuZnSODs, MnSOD, and FeSOD. CuZnSODs have two sub-families localized either in plant cell cytosol or in plastids, the MnSOD family is essentially restricted to mitochondria, and the FeSOD enzyme family has been typically localized into the plastid. Here, we describe, based on a phylogenetic tree and experimental data, the existence of two FeSOD sub-families: a plastidial localized sub-family that is universal to plants, and a cytosolic localized FeSOD sub-family observed in determinate-forming nodule legumes. Anti-cytosolic FeSOD (cyt_FeSOD) antibodies were employed, together with a novel antibody raised against plastidial FeSOD (p_FeSOD). Stress conditions, such as nitrate excess or drought, markedly increased cyt_FeSOD contents in soybean tissues. Also, cyt_FeSOD content and activity increased with age in both soybean and cowpea plants, while the cyt_CuZnSOD isozyme was predominant during early stages. p_FeSOD in leaves decreased with most of the stresses applied, but this isozyme markedly increased with abscisic acid in roots. The great differences observed for p_FeSOD and cyt_FeSOD contents in response to stress and aging in plant tissues reveal distinct functionality and confirm the existence of two immunologically differentiated FeSOD sub-families. The in-gel FeSOD activity patterns showed a good correlation to cyt_FeSOD contents but not to those of p_FeSOD. This indicates that cyt_FeSOD is the main active FeSOD in soybean and cowpea tissues. The diversity of functions associated with the complexity of FeSOD isoenzymes depending of the location is discussed.
Assuntos
Citosol/enzimologia , Fabaceae/enzimologia , Plastídeos/enzimologia , Estresse Fisiológico/fisiologia , Superóxido Dismutase/metabolismo , Anticorpos/análise , Fabaceae/crescimento & desenvolvimento , Isoenzimas , Filogenia , Folhas de Planta/enzimologia , Raízes de Plantas/enzimologia , Glycine max/enzimologia , Glycine max/crescimento & desenvolvimento , Superóxido Dismutase/classificação , Superóxido Dismutase/imunologia , TempoRESUMO
The development of effective vaccines against HIV-1 infection constitutes one of the major challenges in viral immunology. One of the protein candidates in vaccination against this virus is p24, since it is a conserved HIV antigen that has cytotoxic and helper T cell epitopes as well as B cell epitopes that may jointly confer enhanced protection against infection when used in immunization-challenge approaches. In this context, the adjuvant effect of EDA (used as EDAp24 fusion protein) and poly(I:C), as agonists of TLR4 and TLR3, respectively, was assessed in p24 immunizations using a recombinant Listeria monocytogenes HIV-1 Gag proteins (Lm-Gag, where p24 is the major antigen) for challenge in mice. Immunization with EDAp24 fusion protein together with poly(I:C) adjuvant induced a specific p24 IFN-γ production (Th1 profile) as well as protection against a Lm-Gag challenge, suggesting an additive or synergistic effect between both adjuvants. The combination of EDA (as a fusion protein with the antigen, which may favor antigen targeting to dendritic cells through TLR4) and poly(I:C) could thus be a good adjuvant candidate to enhance the immune response against HIV-1 proteins and its use may open new ways in vaccine investigations on this virus.