Delineation of cellular stages and identification of key proteins for reduction and biotransformation of Se(IV) by Stenotrophomonas bentonitica BII-R7.

The widespread use of selenium (Se) in technological applications (e.g., solar cells and electronic devices) has led to an accumulation of this metalloid in the environment to toxic levels. The newly described bacterial strain Stenotrophomonas bentonitica BII-R7 has been demonstrated to reduce mobile Se(IV) to Se(0)-nanoparticles (Se(0)NPs) and volatile species. Amorphous Se-nanospheres are reported to aggregate to form crystalline nanostructures and trigonal selenium. We investigated the molecular mechanisms underlying the biotransformation of Se(IV) to less toxic forms using differential shotgun proteomics analysis of S. bentonitica BII-R7 grown with or without sodium selenite for three different time-points. Results showed an increase in the abundance of several proteins involved in Se(IV) reduction and stabilization of Se(0)NPs, such as glutathione reductase, in bacteria grown with Se(IV), in addition to many proteins with transport functions, including RND (resistance-nodulation-division) systems, possibly facilitating Se uptake. Notably proteins involved in oxidative stress defense (e.g., catalase/peroxidase HPI) were also induced by Se exposure. Electron microscopy analyses confirmed the biotransformation of amorphous nanospheres to trigonal Se. Overall, our results highlight the potential of S. bentonitica in reducing the bioavailability of Se, which provides a basis both for the development of bioremediation strategies and the eco-friendly synthesis of biotechnological nanomaterials.

A homogeneous caspase-3 activity assay using HTRF technology.

Caspases are cysteine proteases presenting a conserved active site that cleaves protein substrates at a highly specific position. They are involved in different aspects of the active cell death pathway. Most of them act through proteolytic degradations of cellular components. This paper describes the assay development, assay validation, and screening for inhibitors of this enzyme, which could be potential drug candidates. The assay uses homogeneous time-resolved fluorescence based on energy transfer from europium cryptate as donor to cross-linked allophycocyanin as acceptor (XL665). A double-tagged substrate, biotinyl-epsilon-aminocaproyl-L-aspartyl-L-glutamyl-L-valyl-Laspartyl-L-alanyl-L-propyl-N(epsilon)-(2,4-dinitrophenyl)-L-lysine-amide (biotin-X-DEVDAPK(dnp)-NH(2)), is conjugated with streptavidin cryptate and anti-dnp-XL665 monoclonal antibody. The close proximity between donor and acceptor induces a specific time-resolved fluorescence signal. In the presence of enzyme activity, the substrate cleavage induces an unlinking of the two fluorescent probes and, subsequently, the disappearance of the specific signal as a result of loss of proximity. Experiments to optimize the reagent concentration, incubation times, precision, reproducibility, and robustness are discussed in comparison with a fluorometric method.

Europium cryptate labeled deoxyuridine-triphosphate analog: synthesis and enzymatic incorporation.

The synthesis of an europium tris-bipyridine cryptate labeled 2'-deoxyuridine-5 '-triphosphate analog (K-11-dUTP) is described. This labeled triphosphate was incorporated into DNA through enzymatic reactions with terminal transferase and DNA polymerases. The enzymatic reactions were monitored by TRACE (Time Resolved Amplification of Cryptate Emission), a homogeneous method using Fluorescence Resonance Energy Transfer (FRET) from an europium cryptate as donor to a modified allophycocyanine as acceptor.

Europium cryptate-tethered ribonucleotide for the labeling of RNA and its detection by time-resolved amplification of cryptate emission.

TRACE (time-resolved amplification of cryptate emission), also called HTRF for pharmaceutical applications, is a homogeneous time-resolved fluorescence technique well adapted for the study of molecular interactions. It is based on fluorescence resonance energy transfer (FRET) between europium trisbipyridine cryptate (TBPEu(3+)) as energy donor and cross-linked allophycocyanin, symbolized by XL665, as acceptor, leading to a long-lived FRET signal. TBPEu(3+)-labeled uridine triphosphate (UTP), referred to as K-11-UTP in the text, was obtained by coupling TBPEu(3+) moiety to a C-5 functionalized UTP analog. K-11-UTP can be directly incorporated in RNA strands during enzymatic synthesis. This was demonstrated in an in vitro transcription reaction promoted by T(7) RNA polymerase. The reaction was performed in the presence of K-11-UTP and biotin-labeled cytidine triphosphate (biotin-16-CTP) in admixture with natural ribonucleotides. After the addition of streptavidin-XL665 conjugate (SA-XL665), which binds on biotinylated cytidine residues, a long-lived FRET signal was obtained. This proved that both europium cryptate and biotin were incorporated into the same RNA strand and are close enough to generate a FRET signal. The study of this FRET detection assay format showed that such doubly labeled RNA can be easily detected even when a very low percentage of K-11-UTP is used (less than 1% of total UTP concentration). Europium-cryptate-labeled RNA can also be monitored using a homogeneous hybridization assay format involving a biotinylated probe. After the addition of SA-XL665, the FRET signal generated demonstrates the formation of RNA:DNA hybrids. Europium-cryptate-labeled nucleotide thus gives access to a new type of RNA nonisotopic labeling and homogeneous detection assays.

Analysis of BCR/ABL transcripts in chronic myeloid patients by nested PCR using an immunoassay-like detection format.

We describe an 'immunoassay-like' detection format, based on the AMPLICIS technique for the routine detection of BCR/ABL transcripts in leukaemic patients. The AMPLICIS technique is characterized as a nested PCR coupled to solid phase isotopic or non-isotopic detection of PCR products. Comparison between this assay and a conventional PCR technique using Southern-blot analysis provided a good correlation between the two procedures. The assay, easier and faster than the conventional one, gives access to automation and therefore appears well suited for the routine screening of BCR/ABL chimaeric mRNAs.

Fast solid support detection of human papillomavirus in in vitro amplified DNA using a DNP-anti DNP monoclonal antibody couple.

In some anogenital lesions the detection of certain types of human papilloma virus, especially oncogenic types, is of interest. In a first step during a prospective study, we compared two methods for the detection of human papillomavirus (HPV) DNA in clinical samples: Southern blotting followed by hybridization with a cloned radioactive genomic probe and a classical polymerase chain reaction (PCR) followed by hybridization with a 32P-labelled oligonucleotide probe. 118 biopsies and swabs were examined for HPV 6/11, 16, 18 and 33, 67 positive reactions were found by both methods, 5 positives only by PCR and 2 positives only by Southern blot for unidentified HPV. Patients with anogenital condylomas, dysplasias and carcinomas or asymptomatic patients were studied. Most high grade (II and III) dysplasias were associated with HPV 16 and HPV 18. Condylomata lesions and low grade dysplasia (grade I) were associated mostly with HPV 6/11, mixed type of HPV, less frequently with HPV 16 or HPV 18. As a second step a nested PCR coupled to solid support detection method was used as described by Sauvaigo et al. (1990) Nucleic Acids Res. 18, 3175-3183) to study a panel of 30 previously qualified different HPV DNA extracts. In this procedure the second round of PCR amplification involves biotinylated and dinitrophenylated labelled primers allowing the capture of PCR amplified HPV DNA sequences on streptavidin coated tubes and its revelation. We describe an improvement of HPV DNA detection by means of single-step immunoenzymatic revelation involving anti-DNP monoclonal antibodies conjugated to horseradish peroxidase enzyme. A perfect correlation with the previous results was obtained. This solid support method allows a faster and easier HPV typing compared to methods using membrane transfer.