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Plant defensins constitute the innate immune response against pathogens such as fungi and bacteria. Typical plant defensins are small, basic peptides that possess a characteristic three-dimensional fold stabilized by three or four disulfide bridges. In addition to known defensin genes, the Arabidopsis genome comprises > 300 defensin-like genes coding for small cysteine-rich peptides. One of such genes encodes for AtPDFL2.1, a putative antifungal peptide of 55 amino acids, with six cysteine residues in its primary sequence. To understand the functional role of AtPDFL2.1, we carried out antifungal activity assays and determined its high-resolution three-dimensional structure using multidimensional solution NMR spectroscopy. We found that AtPDFL2.1 displays a strong inhibitory effect against Fusarium graminearum (IC 50 ≈ 4 μM). This peptide folds in the canonical cysteine-stabilized αβ (CSαβ) motif, consisting of one α-helix and one triple-stranded antiparallel β-sheet stabilized by three disulfide bridges and a hydrophobic cluster of residues within its core where the α-helix packs tightly against the β-sheets. Nuclear spin relaxation measurements show that the structure of AtPDFL2.1 is essentially rigid, with the L3 loop located between β-strands 2 and 3 being more flexible and displaying conformational exchange. Interestingly, the dynamic features of loop L3 are conserved among defensins and are probably correlated to the antifungal and receptor binding activities.

In this paper, we review our past and current efforts toward the elucidation of the biological chemistry of organotin compounds. In particular, we cover two prominent aspects of organotin compounds: their reactivity toward biological dithiols, and their degradation (or metabolization) mechanism using a combination of experimental and computational techniques.

The complete assignment of the 1H and 13C NMR spectra of bendaline (BNDL) was performed by mono-dimensional and homo- and hetero-correlated two-dimensional NMR experiments. The interaction between bendaline and albumin was also studied by the analysis of the motional parameters spin-lattice relaxation times, allowing the motional state of the BNDL free and bound with albumin to be defined. In absence of albumin the indazolacetic and benzylic moieties are characterized by roughly the same mobility and by positive sigma (cross-relaxation rates) values. In the presence of the macromolecule, the indazolacetic and benzylic moieties and the lysine change their motional behaviour to different extents, as indicated by correlation times. Data obtained in absence and in presence of the protein show that the molecular moiety of the bendaline most involved in the binding with albumin is the fragment H-4 H-5. The binding constant was evaluated at 2.4x10(3)M(-1).

The kinetics of the reaction catalyzed by human erythrocyte Acetylcholinesterase (AChE) is studied in the presence of its inhibitor Tacrine (1,2,3,4-tetrahydro-9-acridinamine), and of two newly synthesized compounds, 6-metoxy-Tacrine and N -eptyl-Tacrine. The proposed kinetic model describes the rate of the enzymatic reaction in terms of competitive and uncompetitive mixed inhibition of the three different inhibitors. The kinetic parameters describing the rate of the reaction are obtained. The competitive and uncompetitive inhibition constants of the different inhibitors are reported and the mixed competitive–uncompetitive inhibition is discussed.

This work presents the realization of enzymatic bioelectrodes, suitable for the determination of γ-aminobutyric acid (GABA). The biosensors are based on the catalytic activity of the enzymes γ-aminobutyric glutamic transaminase (GABA-T), succinic semialdehyde dehydrogenase (SSDH) and horseradish peroxidase (HPO). The first two enzymes are usually indicated by the general term “GABASE”. All the biosensors presented in this work are realized by immobilizing the enzyme HPO on the tip of an amperometric oxygen selective electrode: the resulting NADPH-sensitive biosensor is used in combination with GABASE to determine the concentration of GABA in aqueous samples. Since SSDH depends on the NADP+/NADPH equilibrium, it follows that, in the presence of HPO, the NADPH formed is oxidized to NADP, and the decrease in the concentration of dissolved oxygen is proportional to the concentration of NADPH and, in turn, to that of GABA. The experiments were performed either with GABASE free in solution or co-immobilized with HPO on the surface of the oxygen electrode. In the latter case, the immobilization of the three enzymes has been performed either on a single membrane or on two separated membranes. In both cases there is an almost perfect linearity between the electrode signal and GABA concentration in the range 5.0 × 10−5–1.2 × 10−3M, with a lower detection limit of 2.0 × 10−5M; but the single-membrane biosensor showed a better overall performance, especially in terms of repeatability of measurements and lifetime of operation.

This work presents a new method for the simple and inexpensive determination of atrazine. The method is based on the use of a novel, partially disposable, plant tissue bioelectrode, which is sensitive to a variety of mono- and polyphenols. The biosensor is obtained by coupling a thin slice of potato ( Solanum tuberosum ) tissue, which contains high levels of the enzyme polyphenoloxidase (PPO), to a commercial O 2 -selective Clark electrode. The concentration of atrazine in aqueous samples can be determined thanks to its inhibitory power toward the catalytic activity of PPO. The low cost of this device and its good analytical performance suggest its application in the field of environmental analysis, especially in the continuous monitoring of atrazine in risk areas.

A novel analytical system for the in-flow quantitative determination of acetylcholine (ACh) and acetylcholinesterase (AChE) inhibitors is presented. Reliable and reproducible values in the monitoring of ACh, of carbamic acid derivatives and of organophosphorous compounds have been obtained thanks to a device realized by coupling an AChE-based whole plant tissue column bioreactor with a traditional choline oxidase (ChO) biosensor. AChE was extracted from the inner part of a grapefuit shell (Albedum pomi citreum), coupled to chitin by glutaraldehyde and immobilized into a glass column bioreactor, whose outlet was connected to a thermostated flow-through cell incorporating the ChO biosensor. A sharp improvement in the sensitivity limits was obtained by recycling the sample solution into the bioreactor. The efficacy of the analytical system here presented was checked on standard solutions of ACh and on three of the most commonly used inhibitors of AChE (aldicarb, malathion and paraoxon), widely employed mainly as the active components of pesticides and insecticides.

An amperometric biosensor for the direct determination of L-glutamate was developed by chemical bonding of L-glutamate oxidase (GAO) on a carboxylic Nylon membrane with polyazetidine prepolymer (PAP), and using a hydrogen peroxide electrode as indicating sensor. The biosensor is specific for L-glutamate and the peculiar analytical properties (linearity range, reproducibility, accuracy) were experimentally determined. Furthermore, the same basic biosensor was also modified to be used and characterized for the direct determination of L-glutamine. This L-glutamine biosensor was obtained by coimmobilizing, on two separate membranes, glutamic acid oxidase and glutaminase (GMN) on the same biosensor. The two sensors were then used for the determination of glutamate and L-glutamine contained in pharmaceutical formulations and the results were compared with those obtained by other analytical methods.

The present work deals with the quantitative determination of catechol and other biologically active polyphenols (catecholamines) by means of plant tissue electrodes built up by a whole tissue containing an oxidizing enzyme (polyphenol oxidase, PPO) coupled with an O2 gas-selective electrode. Several tissues have been employed to prepare the biocatalytic layer of the biosensors. The analytical characteristics of the plant tissue electrodes here proposed, tested in catechol standard solutions, are comparable to those of traditional PPO-based enzyme electrodes. Moreover, the whole-tissue biocatalytic membranes are more stable than those prepared by using purified enzymes, thus ensuring longer life times of application. The biosensors presented here are also suitable for the quantitative determination of other catecholamines (epinephrine, norepinephrine, dopamine, l-DOPA).

We have realized an enzyme-modified FET device (ENFET) in which the enzyme has been chemically linked to the FET surface by means of a layer of polymerized [3-(2-aminoethyl)aminopropyl]trimethoxysilane (AEAPS). The enzyme has been bound to the amino functional groups of the polysiloxane surface together with bovine serum albumin by using cross-linking agents, such as glutaraldehyde, and the obtained enzymatic membrane has been characterized by FT-IR directly onto the gate surface. The polysiloxane layer has proved to be a good interface between the enzyme and the device surface allowing a biosensor with the characteristics of good stability and reproducibility to be obtained. In this paper two examples of the fabrication and the performances of this kind of biosensor for urea determination in biomedical applications and pesticide detection in environmental control are shown, employing immobilized urease or acetylcholinesterase ENFET devices, respectively.

An IMMUNOFET device in which an antibody is immobilized onto the gate surface by means of an immunochemical membrane containing Protein A has been realized. Such a membrane is chemically linked to the device surface by using a layer of polyaminosiloxane. The antigen-antibody interaction is detected by an immunoenzymatic assay which allows the response of the immunoreaction to be amplified using as marker an enzyme (e.g., glucose oxidase) that is able to produce, during the catalysed reaction, a pH variation detected by the ISFET device. The possibility of employing this immunosensor in biomedical applications and environmental monitoring is shown using as a model system human IgG (hIgG) and the herbicide atrazine respectively.