The hybridization of single-stranded oligonucleotide-derivatized gold nanoparticles (An nanoprobes) with double stranded complementary DNA was directly observed by atomic force microscopy (AFM). This specific interaction is the basis for an An nanoprobe-based homogeneous assay for specific DNA sequence detection, based on salt-induced particle aggregation that is prevented when a complementary target is present. For long DNA targets (linearized plasmid DNA) complicated hybridized target DNA-Au-nanoprobes structures were formed, that were interpreted as the basis for stability of the An nanoprobes against salt-induced aggregation. For shorter DNA targets (PCR amplified fragments) hybridization with the An nanoprobes occurred, in the majority of cases, in the expected location of the DNA target fragment containing the specific sequence. The formation of the observed DNA hybridized structures provides evidence at the molecular level for specific hybridization to the target sequence as the method of binding of the An nanoprobes.

Advances in nanosciences are having a significant impact in many areas of research. The impact of new nanotechnologies has been particularly large in biodiagnostics, where a number of nanoparticle-based assays have been introduced for biomolecules detection. To date, applications of nanoparticles have largely focused on DNA-functionalised gold nanoparticles used as the target-specific probes. These gold nanoparticle-based systems can be used for the detection of specific sequences of DNA (pathogen detection, characterisation of mutation and/or single nucleotide polymorphisms) or RNA (without prior retro-transcription and amplification). Here a rapid and inexpensive nanoparticle-based method for single-base mismatch detection (single nucleotide polymorphism/mutation) in DNA samples is reported. Gold nanoparticles derivatised with thiol modified oligonucleotides complementary to DNA targets - Au-nanoprobes - are used to distinguish fully complementary from mismatched sequences, with a single-base mismatch. The authors have successfully applied this strategy to detect common mutations within the beta-globin gene.

Fast (120 s) and high-throughput (more than six samples at once) in-gel trypsin digestion of proteins using sonoreactor technology has been achieved. Successful protein identification was done by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, MALDI-TOF-MS. Specific identification of the adenylylsulphate reductase alfa subunit from a complex protein mixture from Desulfovibrio desulfuricans ATCC 27774 was done as a proof of the methodology. The new sample treatment is of easy implementation, saves time and money, and can be adapted to online procedures and robotic platforms.

The synthetic flavylium salt 6-hydroxy-4'-(dimethylamino)flavylium hexa. uorophosphate displays a set of pH-driven chemical reactions in aqueous solutions, involving the formation of hemiketal species and chalcones with cis and trans configurations. Such reactions were studied by steady-state and transient UV-Vis spectroscopy and by stopped-flow techniques. A novel and more generalized kinetic scheme is developed, in order to take account of possible acid/base pairs that occur in the network of chemical reactions as the pH is changed. It is found that the formation of the hemiketal species by hydration of the. avylium is slow, and it is not possible to isolate each process that leads to the formation of the cis-chalcone ( hydration and tautomerization reactions). The cis/trans isomerization reaction of cis-chalcone is slow, and the system takes several hours to reach equilibrium after a pH jump at room temperature. In basic conditions, negatively charged trans-chalcones are dominant. Comparison with other. avylium compounds shows that the hydration process is affected mainly by the amino group, while the hydroxyl group influences the tautomerization and isomerization reactions.

In the absence of arabinose, the AraR transcription factor represses the expression of genes involved in the utilization of arabinose, xylose and galactose in Bacillus subtilis. AraR exhibits a chimeric organization: the N-terminal DNA-binding region belongs to the GntR family and the C-terminal effector-binding domain is homologous to the GalR/LacI family. Here, the AraR–DNA-binding interactions were characterized in vivo and in vitro. The effect of residue substitutions in the AraR N-terminal domain and of base-pair exchanges into an AraR–DNA-binding operator site were examined by assaying for AraR-mediated regulatory activity in vivo and DNA-binding activity in vitro. The results showed that residues K4, R45 and Q61, located in or near the winged-helix DNA-binding motif, were the most critical amino acids required for AraR function. In addition, the analysis of the various mutations in an AraR palindromic operator sequence indicated that bases G9, A11 and T16 are crucial for AraR binding. Moreover, an AraR mutant M34T was isolated that partially suppressed the effect of mutations in the regulatory cis-elements. Together, these findings extend the knowledge on the nature of AraR nucleoprotein complexes and provide insight into the mechanism that underlies the mode of action of AraR and its orthologues.

The simultaneous separation and quantification of two casein peptides {(IPP}, {VPP)} presenting potent inhibitory activity of angiotensin-converting-enzyme {(ACE)} and casein in fermented milks was developed. Gradient elution was carried out at a flow-rate of 1 {mL/min}, using a mixture of two solvents. Solvent A was 0.1% {TFA} in water and solvent B was acetonitrile-water-trifluoracetic acid 95:5:0.1. The effluent was monitored by {UV} detector at 214 nm. Calibration curves were constructed in the interval of 0.01-1.0 {mg/mL} for {VPP}, 0.005-1.0 {mg/mL} for {IPP}, and 0.05-3.0 {mg/mL} for casein. R2 invariably exceeded 0.999. The detection limits were 0.004 for {VPP}, 0.002 {mg/mL} for {IPP}, and 0.02 {mg/mL} for casein. Repeatability of the method was evaluated by six consecutive injections of two standard solutions containing {VPP}, {IPP}, and casein. The {RSD} values for concentration were all below 5.08%. Recovery studies were carried out to determine the accuracy of the method. Recoveries ranged between 88 and 98.2%. The methodology was applied, not only, for the monitorization of {VPP}, {IPP}, and casein in commercial fermented milks labeled as presenting antihypertensive properties, but also, in milk with different degrees of fermentation by L. Helveticus, and in other commercial functional fermented milks, such as, those presenting cholesterol lowering properties.

The simultaneous separation and quantification of two casein peptides (IPP, VPP) presenting potent inhibitory activity of angiotensin-converting-enzyme (ACE) and casein in fermented milks was developed. Gradient elution was carried out at a flow-rate of 1 mL/min, using a mixture of two solvents. Solvent A was 0.1% TFA in water and solvent B was acetonitrile-water-trifluoracetic acid 95:5:0.1. The effluent was monitored by UV detector at 214 nm. Calibration curves were constructed in the interval of 0.01-1.0 mg/mL for VPP, 0.005-1.0 mg/mL for IPP, and 0.05-3.0 mg/mL for casein. R 2 invariably exceeded 0.999. The detection limits were 0.004 for VPP, 0.002 mg/mL for IPP, and 0.02 mg/mL for casein. Repeatability of the method was evaluated by six consecutive injections of two standard solutions containing VPP, IPP, and casein. The RSD values for concentration were all below 5.08%. Recovery studies were carried out to determine the accuracy of the method. Recoveries ranged between 88 and 98.2%. The methodology was applied, not only, for the monitorization of VPP, IPP, and casein in commercial fermented milks labeled as presenting anti hypertensive properties, but also, in milk with different degrees of fermentation by L Helveticus, and in other commercial functional fermented milks, such as, those presenting cholesterol lowering properties.

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Aldehyde oxidoreductase (AOR) activity has been found in a number of sulfate-reducing bacteria. The enzyme that is responsible for the conversion of aldehydes to carboxylic acids is a mononuclear molybdenum enzyme belonging to the xanthine oxidase family. We report here the purification and characterization of AOR isolated from the sulfate-reducing bacterium Desulfovibrio (D.) aminophilus DSM 12254, an aminolytic strain performing thiosulfate dismutation. The enzyme is a homodimer (ca. 200 kDa), containing a molybdenum centre and two [2Fe-2S] clusters per monomer. UV/Visible and electron paramagnetic resonance (EPR) spectra of D. aminophilus AOR recorded in as-prepared and reduced states are similar to those obtained in AORs from Desulfovibrio gigas, Desulfovibrio desulfuricans and Desulfovibrio alaskensis. Despite AOR from D. aminophilus is closely related to other AORs, it presents lower activity towards aldehydes and no activity towards N-heterocyclic compounds, which suggests another possible role for this enzyme in vivo. A comparison of the molecular and EPR properties of AORs from different Desulfovibrio species is also included.