A new competitive fluorescence assay for the detection of patulin toxin.

Patulin is a toxic secondary metabolite of a number of fungal species belonging to the genera Penicillum and Aspergillus. It has been mainly isolated from apples and apple products contaminated with the common storage-rot fungus of apples, Penicillum expansum, but it has also been extracted from rotten fruits, moldy feeds, and stored cheese. Human exposure to patulin can lead to serious health problems, and according to a long-term investigation in rats, the World Health Organization has set a tolerable weekly intake of 7 ppb body weight. The content of patulin in foods has been restricted to 50 ppb in many countries. Conventional analytical detection methods involve chromatographic analyses, such as HPLC, GC, and, more recently, techniques such as LC/MS and GC/MS. However, extensive protocols of sample cleanup are required prior to the analysis, and to accomplish it, expensive analytical instrumentation is necessary. An immunochemical analytical method, based on highly specific antigen-antibody interactions, would be desirable, offering several advantages compared to conventional techniques, i.e., low cost per sample, high selectivity, high sensitivity, and high throughput. In this paper, the synthesis of two new derivatives of patulin is described, along with their conjugation to the bovine serum albumin for the production of polyclonal antibodies. Finally, a fluorescence competitive immunoassay was developed for the on-line detection of patulin.

Proteins from extremophiles as stable tools for advanced biotechnological applications of high social interest.

Extremophiles are micro-organisms adapted to survive in ecological niches defined as 'extreme' for humans and characterized by the presence of adverse environmental conditions, such as high or low temperatures, extreme values of pH, high salt concentrations or high pressure. Biomolecules isolated from extremophiles possess extraordinary properties and, in particular, proteins isolated from extremophiles represent unique biomolecules that function under severe conditions, comparable to those prevailing in various industrial processes. In this article, we will review some examples of recent applications of thermophilic proteins for the development of a new class of fluorescence non-consuming substrate biosensors for monitoring the levels of two analytes of high social interest, such as glucose and sodium.

Pressure effect on the stability and the conformational dynamics of the D-Galactose/D-Glucose-binding protein from Escherichia coli.

The effect of the pressure on the structure and stability of the D-Galactose/D-Glucose binding protein (GGBP) from Escherichia coli was studied by steady-state and time-resolved fluorescence spectroscopy, and the ability of glucose ligand to stabilize the GGBP structure was also investigated. Steady-state fluorescence experiments showed a marked quenching of fluorescence emission of GGBP in the absence of glucose. Instead, the presence of glucose seems to stabilize the structure of GGBP at low and moderate pressure values. Time-resolved fluorescence measurements showed that the GGBP taumean in the absence of glucose varies significantly up to 600 bar, while in the presence of the ligand it is almost unaffected by pressure increase up to 600 bar. The effect of the pressure on GGBP was also studied by molecular dynamics simulations. The simulation data support the spectroscopic results and confirm that the presence of glucose is able to contrast the negative effects of pressure on the protein structure. Taken together, the spectroscopic and computer simulation studies suggest that at pressure values up to 2000 bar the structure of GGBP in the absence of glucose remains folded, but a significant perturbation of the protein secondary structures can be detected. The binding of glucose reduces the negative effect of pressure on protein structure and confers protection from perturbation especially at moderate pressure values.

Glycomimetics as decorating motifs for oligonucleotides: solid-phase synthesis, stability, and hybridization properties of carbopeptoid-oligonucleotide conjugates.

The online solid-phase synthesis of oligonucleotides conjugated at the 3' end with [1-6]-linked oligosaccharide mimics having the O-glycosidic linkages replaced by amide bonds is here described. The assembly of the carbohydrate domain has been carried out by exploiting classical solid phase peptide synthetic protocols, starting from solid supports functionalized with 1-azido sugars, in association with suitably protected 1-azido uronic acids of glucose and lactose, chosen as model addition monomers. After the insertion of a flexible linker, elongation of the oligodeoxyribonucleotide (ODN) chain was performed by standard automated phosphoramidite protocols. 3'-Glycoconjugated 18-mers exhibited an increased enzymatic stability with respect to the same unmodified ODN sequence. UV thermal denaturation experiments showed that the presence of the oligosaccharide tail at the 3' end of the oligonucleotides did not negatively interfere with their duplex formation abilities.

The role of calcium in the conformational dynamics and thermal stability of the D-galactose/D-glucose-binding protein from Escherichia coli.

We have characterized stability and conformational dynamics of the calcium depleted D-galactose/D-glucose-binding protein (GGBP) from Escherichia coli. The structural stability of the protein was investigated by steady state and time resolved fluorescence, and far-UV circular dichroism in the temperature range from 20 degrees C to 70 degrees C. We have found that the absence of the Ca(2+) ion results in a significant destabilization of the C-terminal domain of the protein. In particular, the melting temperature decreases by about 10 degrees C with the simultaneous loss of the melting cooperativity. Time resolved fluorescence quenching revealed significant loosening of the protein when highly shielded Trp residue(s) became accessible to acrylamide at higher temperatures. We have documented a significant stabilizing effect of glucose that mostly reverts the effect of calcium, that is, the thermal stability of the protein increases by about 10 degrees C and the melting cooperativity is restored. Moreover, the protein structure remains compact with low amplitude of the segmental mobility up to high temperatures. We have used molecular dynamics to identify the structural feature responsible for changes in the temperature stability. Disintegration of the Ca(2+)-binding loop seems to be responsible for the loss of the stability in the absence of calcium. The new insights on the structural properties and temperature stability of the calcium depleted GGBP contribute to better understanding of the protein function and constitute important information for the development of new biotechnological applications of this class of proteins.

Improved synthesis of daunomycin conjugates with triplex-forming oligonucleotides. The polypurine tract of HIV-1 as a target.

Triple helix-forming oligonucleotides (TFOs) are promising agents for the control of gene expression, as they can selectively bind to a chosen oligopyrimidine.oligopurine region of a gene of interest thus interfering with its expression. The stability of the triplex formed by the TFO and the duplex is often too poor for successful applications of TFOs in vivo and the conjugation of a DNA intercalating moiety to the TFO is a common way to enhance the TFO affinity for its target. In a previous work, we investigated the properties of daunomycin conjugated TFO (dauno-TFO) and found that this class of compounds showed a higher degree of affinity than native oligonucleotides for an oligopyrimidine.oligopurine duplex target and that the presence of the amino sugar increases such stability. Here, we report a significantly improved synthetic procedure for the preparation of the conjugates, based on the protection of the daunosamine moiety by N-trifluoroacetylation. This protecting group is removed as a final step from the conjugation product by mild basic hydrolysis to give the desired dauno-TFO. Compared to the previous synthetic procedure, the improvement is important. The synthesis is now more reproducible and no side products are formed. Moreover, the thus protected daunomycin derivative is very stable, up to at least one year. Two dauno-TFOs, differing by the length of the oligonucleotide moiety, were prepared to target the polypurine tract (PPT) of HIV-1. Triplex formation by these compounds with model duplexes was studied by UV spectroscopy, thermal gradient gel electrophoresis (TGGE) and gel electrophoretic mobility shift. The experimental results demonstrate that dauno-TFOs bind to the PPT of HIV-1 more strongly than the unconjugated TFOs.

Excess electron transfer in G-quadruplex.

The excess electron transfer in a G-quadruplex is successfully probed by using the reaction of hydrated electrons with quadruplex complex of pentamers and the 8-bromoguanine moieties as the detection system.

Antibiotics in the environment: occurrence in Italian STPs, fate, and preliminary assessment on algal toxicity of amoxicillin.

Amoxicillin is a widely used penicillin-like antibiotic, and due to its presence in several effluents of Italian STPs, its environmental fate along with its toxicity toward simple organisms have been investigated in model conditions. The present study shows that under abiotic conditions both hydrolysis and direct photolysis could be responsible for the transformation and removal of amoxicillin in aquatic environment, especially in slightly basic media. Quantum yields for the solar direct photolysis have been calculated along with kinetic constants and half-life times. Indirect photolysis experiments in the presence of natural photosensitizers such as nitrate ions and humic acids indicate that nitrate ions have no influence on the photodegradation rate of amoxicillin, while humic acids are able to enhance it. Standard batch experiments have been also performed under biotic conditions. The results indicated that also biodegradation on activated sludge is an effective pathway through which amoxicillin can be removed from the aquatic environment. Rate constants for biodegradation and adsorption have been calculated by applying simple pseudo-first-order kinetic models. Algal bioassays indicate that, in the range of concentrations from 50 ng/L to 50 mg/L, amoxicillin is not toxic toward eucariotic organisms such as the Chlorophyceae Pseudokirkneriella subcapitata and Closterium ehrenbergii and the Bacillariophyceae Cyclotella meneghiniana, but it shows a marked toxicity toward the Cyanophyta Synechococcus leopolensis.

Cyclic uridine diphosphate glucose: a new pyrimidine analog of cyclic ADP ribose.

Novel compound 1, as the first example of cyclic ADP-ribose analogs containing a pyrimidine residue, was synthesized by a chemical strategy employing a Mitsunobu reaction for the condensation of the glucosyl moiety on protected uridine, and a Matsuda procedure for the cyclization step.

New solid supports linking nucleoside scaffolds.

An easy and efficient strategy to obtain new nucleoside based solid supports in which the nucleoside moieties have been anchored to the solid support through the nucleobase is here proposed. A simple and efficient solid-phase synthesis of 5' and 3'-derivatized uridine analogues has so been developed, following methodologies well established in organic chemistry.