Fluorescent siderophore-based chemosensors: iron(III) quantitative determinations

 A highly sensitive and selective method is described for a rapid and easy determination of iron(III). This procedure is based on fluorimetric detection combined with the attractive properties of siderophores and biomimetic ligands, which are strong and selective ferric chelators. Azotobactin δ, a bacterial fluorescent siderophore, three fluorescent derivatives of desferriferrioxamine B with a linear structure (NBD-, MA-, NCP-desferriferrioxamine B) and one tripodal biomimetic ligand of desferriferrichrome carrying an anthracenyl fluorescent probe were examined. A very efficient static quenching mechanism by iron was observed for all the ligands considered in this work. Our results identify azotobactin δ as the most promising chemosensor of ferric traces in water, more sensitive than the NBD-desferriferrioxamine B fluorescent ligand. Under more lipophilic conditions, the anthryl-desferriferrichrome biomimetic analogue showed similar analytical potential and was found to be more sensitive than the lipophilic MA- and NCP-desferriferrioxamine B. Their detection limits were respectively 0.5 ng mL^–1 for azotobactin δ and 0.6 ng mL^–1 for the anthryl tripodal chelator. The calibration curves were linear over the range 0–95 ng mL^–1 and 0–180 ng mL^–1. Various foreign cations have been examined and only copper(II) and aluminium(III) were shown to interfere when present in similar concentrations as iron(III). The developed procedure using fluorescent siderophores or biomimetic ligands of iron(III) may be applied (1) to monitor iron(III)-dependent biological systems and (2) to determine iron(III) quantitatively in natural waters and in biological systems. Received: 12 September 1998 · Accepted: 19 January 1999     

The Use of Kinetic and Dynamic Data in Risk Assessment of Drugs

The risk assessment process for non-carcinogens must incorporate all available scientific information, including toxicokinetic and toxicodynamic data. The framework for exposure limit setting proposed by Renwick and the International Programme on Chemical Safety (IPCS) subdivides traditional 10X uncertainty factors (UFs) into separate partial-log default values based on kinetic and dynamic considerations and allows for incorporation of compound-specific data when available. In this investigation, an extensive literature search was conducted on nine pharmaceuticals in order to incorporate information on kinetics and dynamics to allow extrapolation across species and among susceptible humans. The drugs are diazepam, oxazepam, midazolam, buspirone, fluoxetine, venlafaxine, amlodipine, felodipine, and nifedipine. The composite factors were calculated using the highest ratio or the average ratio for appropriate parameters and default subfactor. For the drugs examined, most of the subfactors for kinetics and dynamics were less than the proposed values by Renwick and IPCS, and the composite factors were far less than 100. From this study, it was concluded that relevant compound-specific kinetic and dynamic data can reduce uncertainties associated with interspecies differences and interindividual variability.     

Kinetic Behavior of Some Polyphosphate-Accumulating Bacteria Isolates in the Presence of Nitrate and Oxygen

This paper studies the phosphate uptake by pure cultures of Aeromonas hydrophila, Klebsiella oxytoca, Agrobacterium tumefaciens, and Aquaspirillum dispar in the presence of both nitrate and oxygen. It is shown that species were able to respire both electron acceptors for phosphate accumulation. A. tumefaciens and A. dispar accumulated overall phosphate both in oxic and anoxic culture conditions, whereas A. hydrophila and K. oxytoca eliminated overall phosphate only in oxic conditions. A. dispar was able to remove phosphate by reducing oxygen and nitrate simultaneously with the production of dinitrogen gas. The anoxic denitrification observed in the cultures of adapted and nonadapted cells to nitrate showed that only A. dispar have a denitrification rate superior when the cells were adapted to nitrate. Received: 15 December 1998 / Accepted: 21 January 1999     

The CRL4DCAF1 cullin‐RING ubiquitin ligase is activated following a switch in oligomerization state

The cullin‐4‐based RING‐type (CRL4) family of E3 ubiquitin ligases functions together with dedicated substrate receptors. Out of the ˜29 CRL4 substrate receptors reported, the DDB1‐ and CUL4‐associated factor 1 (DCAF1) is essential for cellular survival and growth, and its deregulation has been implicated in tumorigenesis. We carried out biochemical and structural studies to examine the structure and mechanism of the CRL4^DCAF1 ligase. In the 8.4 Å cryo‐EM map of CRL4^DCAF1, four CUL4‐RBX1‐DDB1‐DCAF1 protomers are organized into two dimeric sub‐assemblies. In this arrangement, the WD40 domain of DCAF1 mediates binding with the cullin C‐terminal domain (CTD) and the RBX1 subunit of a neighboring CRL4^DCAF1 protomer. This renders RBX1, the catalytic subunit of the ligase, inaccessible to the E2 ubiquitin‐conjugating enzymes. Upon CRL4^DCAF1 activation by neddylation, the interaction between the cullin CTD and the neighboring DCAF1 protomer is broken, and the complex assumes an active dimeric conformation. Accordingly, a tetramerization‐deficient CRL4^DCAF1 mutant has higher ubiquitin ligase activity compared to the wild‐type. This study identifies a novel mechanism by which unneddylated and substrate‐free CUL4 ligases can be maintained in an inactive state.     

An ingenious technique based on the usage of fluorone-based dye; pyrosin B in prucalopride assay in different matrices through an “on–off” dye native fluorescence probe

Prucalopride (PCD), is a modern medication approved by the United States in 2018 to alleviate constipation caused by motility issues. PCD demonstrates a strong affinity and selectivity toward the 5-HT4 receptor. The study here introduces a feasible, direct, non-extractive, and affordable pathway for PCD analytical tracking. The fluorimetric study is based on the on–off effect on the emission amplitude of fluorone-based dye (pyrosin B). In a one-pot experiment, the complex between PCD and pyrosin B is formed instantly in an acidic medium. Correlation between decreased pyrosin B emission and PCD concentrations provides a linear calibration plot from 50 to 900 ng/mL. PCD–dye complex system affecting variables were meticulously tuned. The values of the estimated limit of quantitation and limit of detection for the current methodology were 47.5 and 15.7 ng/mL, respectively. Conformity of the strategy validity was achieved by a comprehensive study of the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use criteria. The method was convincingly applied for PCD assay in tablets and content uniformity investigation. Furthermore, PCD tracking in the spiked biological fluid was applied. Finally, the method uses distilled water as dispersing medium which rise accommodation with the green chemistry principle.     

Surface-exposed phenylalanines in the RNP1/RNP2 motif stabilize the cold-shock protein CspB from Bacillus subtilis

In the cold-shock protein CspB from Bacillus subtilis three exposed Phe residues (F15, F17, and F27) are essential for its function in binding to single-stranded nucleic acids. Usually, the hydrophobic Phe side chains are buried in folded proteins. We asked here whether the exposition of the essential Phe residues could be a cause for the very low conformational stability of CspB. Urea-induced and heat-induced equilibrium unfolding transitions were measured for three mutants of CspB, where Phe 15, Phe 17, and Phe 27 were individually replaced by alanine. Unexpectedly, all three mutations strongly destabilized CspB. The aromatic side chains of Phe 15, Phe 17, and Phe 27 in the active site are thus important for both binding to nucleic acids and conformational stability. There is no compromise between function and stability in the active site. Model calculations indicate that, although they are partially exposed to solvent, all three Phe residues nevertheless lose accessible surface upon folding, and this should favor the native state. A different result is obtained with the F38A variant. Phe 38 is hyperexposed in native CspB, and its substitution by Ala is in fact stabilizing. Proteins 30:401–406, 1998. © 1998 Wiley-Liss, Inc.