The widespread use of NO(3)(-) fertilization has had a major ecological impact. NH(4)(+) nutrition may help to reduce this impact, although high NH(4)(+) concentrations are toxic for most plants. The underlying tolerance mechanisms are not yet fully understood, although they are thought to include the limitation of C, the disruption of ion homeostasis, and a wasteful NH(4)(+) influx/efflux cycle that carries an extra energetic cost for root cells. In this study, high irradiance (HI) was found to induce a notable tolerance to NH(4)(+) in the range 2.5-10mM in pea plants by inducing higher C availability, as shown by carbohydrate content. This capacity was accompanied by a general lower relative N content, indicating that tolerance is not achieved through higher net N assimilation on C-skeletons, and it was also not attributable to increased GS content or activity in roots or leaves. Moreover, HI plants showed higher ATP content and respiration rates. This extra energy availability is related to the internal NH(4)(+) content regulation (probably NH(4)(+) influx/efflux) and to an improvement of the cell ionic balance. The limited C availability at lower irradiance (LI) and high NH(4)(+) resulted in a series of metabolic imbalances, as reflected in a much higher organic acid content, thereby suggesting that the origin of the toxicity in plants cultured at high NH(4)(+) and LI is related to their inability to avoid large-scale accumulation of the NH(4)(+) ion.

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.

An understanding of the mechanisms underlying ammonium (NH4+) 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 NH4+ 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 NH4+‐fed plants. Thus, pea plants (Pisum sativum) were grown at a range of NH4+ concentrations as sole N source, and two light intensities were applied to vary the C supply to the plants. Control plants grown at high NH4+ 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 NH4+ 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 NH4+ nutrition in plants.

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 ϳ24 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.

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The cytosolic enzymes asymmetrical diadenosine tetraphosphate hydrolase (EC 3.6.1.17, Ap R Aase) and diadenosine triphosphate hydrolase (EC 3.6.1.29, Ap Q Aase) are inhibited competitively by suramin. Ap R Aase and Ap Q Aase were assayed in cytosolic rat brain extracts using fluorogenic analogues of the respective substrates diadenosine tetraphosphate (Ap R A) and diadenosine triphosphate (Ap Q A). K i values for suramin as inhibitor of Ap R Aase and Ap Q Aase were 5U10 3T M and 3U10 3U M, respectively. Results indicate that suramin or suramin-like derivatives may be useful tools to investigate diadenosine polyphosphate cleaving enzymes and that the intracellular diadenosine polyphosphate metabolism may be a pharmacological target of suramin with biological and clinical implications.z 1998 Federation of European Biochemical Societies.

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