Organometallic derivatives of estradiol as bioligands: targetted binding of the estradiol receptor

The complexation of estrogens by transitional metal units e.g. (alkyne)Co2(CO)6 and (alkyne)Mo2Cp2(CO)4, at the 17 alpha-position brings about a dramatic change in the chemical behavior of these compounds with respect to that of the free ligands. The 17 beta-OH function becomes particularly labile, even in weakly acidic medium, giving rise to carbenium ion-like species, from which, depending on the metal and the nucleophile, substitution, elimination and rearrangement take place. This situation provides the basis for a new type of active site directed-reagent for estradiol receptor. The hypothesis of vicinal space positioning of an acidic and a nucleophilic group in the estradiol receptor cavity is examined in the light of the amino-acid composition of the steroid binding domain. The requirement of the sulfhydryl group of a cysteine residue is suspected in the first step of the receptor inactivation process.     

pH effects on the N-demethylation and formation of the cytochrome P-450 iron II nitrosoalkane complex for erythromycin derivatives.

The effects of pH on access to the cytochrome P-450 active site, N-demethylation and formation of the cytochrome P-450 Fe(II)-RNO metabolite complex for a series of erythromycin derivatives were examined. Studies were performed with dexamethasone-treated rat liver microsomes containing large amounts of cytochrome P-450 3A isozymes. In addition to factors such as hydrophobicity or hindrance around the dimethyl-amino function, the ionisation state of the N(CH3)2 group played an important role in the recognition and metabolism of the substrate by cytochrome P-450. Esterification of the desosamine in the beta position of the N(CH3)2 group leads to lower pKa values for the R--N+ H(CH3)2 <--> [R--N (CH3)2] + H+ equilibrium. At physiological pH, the amine group is mainly in the unprotonated form. Consequently, easier access to the protein active site and significant formation of cytochrome P-450 Fe(II)-RNO metabolite complex are observed for these derivatives. These results led us to interpret the formation of cytochrome P-450 Fe(II)-RNO metabolite complex as a series of multiple steps equilibria depending on the ionisation state of the N(CH3)2 group, the partition coefficient of the substrate between the microsomal layer and the aqueous media and a series of metabolic reactions leading partially to the final inhibitory nitrosoalkane-cytochrome P-450 Fe(II) complex.     

The role of exopolysaccharides in fouling phenomenon during ultrafiltration of microalgae (Chlorella sp. and Porphyridium purpureum): Advantage of a swirling decaying flow

Ultrafiltration of two microalgae species, Chorellasp. and Porphyridium purpureum, has been performed in two filtration units of different configurations: a module generating a classical tangential-plane flow and another one inducing a swirling decaying flow. The swirling motion is generated by a unique tangential inlet at the bottom of an annulus. Permeation flux obtained in each ultrafiltration unit depends on the filtered species, and specific deposits are associated to each species and module. Enhancement in permeation flux in the swirling configuration compared with that obtained in the plane unit can reach 40% during the ultrafiltration of Porphyridium purpureum and is negligible for the suspensions of Chlorella sp. The experimental data revealed the significant role of the soluble compounds in the medium culture in terms of flux limitation. It seems that the soluble products and their interactions with the microalgal cells are strongly involved in the fouling mechanism.     

Transition metal-carbonyl labeling of biotin and avidin for use in solid-phase carbonyl metallo immunoassay (CMIA)

The preparation of several transition metal-carbonyl tracers of biotin and avidin is described. Multiple labeling of avidin was achieved by acylation of some of its amine-bearing residues with N-succinimidyl 4-pentynoate (dicobalt hexacarbonyl). By varying the initial amount of this complex, protein conjugates with the extent of derivatization of up to 13 were obtained. Biotin was labeled with one (eta5-cyclopentadienyl)manganese tricarbonyl moiety by reaction of biotin hydrazide, whereas multiple labeling was reached by successive conjugation of biotin and N-succinimidyl 4-pentynoate (dicobalt hexacarbonyl) or (eta5-cyclopentadienyl)iron dicarbonyl (eta1-N-maleimidato) to poly-L-lysine or fourth generation Starburst dendrimer. All the conjugates displayed a good to excellent bioaffinity toward their respective counterparts, as measured by competitive enzymatic assays.     

Chaperone-assisted expression,purification, and characterization of recombinant nitrile hydratase NI1 from Comamonas testosteroni

Nitrile hydratases (NHases) are industrially important iron- and cobalt-containing enzymes that are used in the large-scale synthesis of acrylamide. Heterologous expression of NHases has been complicated by the fact that other proteins (activators or metallochaperones) appear to be required to produce NHases in their catalytically active form. We report a novel heterologous system for the expression of catalytically active iron-containing NI1 NHase in Escherichia coli, involving coexpression with the E. coli GroES and GroEL chaperones. The purified recombinant enzyme was found to be highly similar to the enzyme purified from Comamonas testosteroni according to its spectroscopic features, catalytic properties with various substrates, and post-translational modifications. In addition, we report a rapid and convenient spectrophotometric method to monitor the activity of NI1 NHase during purification.     

Recognition and oxidative metabolism of cyclodipeptides by hepatic cytochrome P450

Possible recognition of peptide derivatives by hepatic cytochrome P450 3A has been suggested by binding and metabolism of numerous pseudopeptidic compounds such as ergot derivatives and cyclosporin.Natural linear or cyclic dipeptides containing hydrophobic amino acids produced by microorganisms and present in mammals are able to interact with the P450 active site through either iron-amine interactions (Type II) or hydrophobic Type I interactions. P450 3A from dexamethasone-treated rats or yeast-expressed P450 human 3A4 are the most potent in such interactions, which are particularly strong with peptides containing a histidyl residue.Some cyclodipeptides are rapidly transformed by rat cytochrome P450 3A to mono- or dihydroxylated metabolites, with turnovers around 3 nmoles min(-1) P450(-1). Linear peptides are poorly transformed in these conditions. This metabolism of cyclodipeptides occurs in 8 species including man.Such interactions and metabolism have only minor consequences in terms of P450 3A binding and metabolism of classical P450 3A substrates. These data reinforce the concept that, in addition to their effect on the regulation of P450 neosynthesis, naturally occurring endogenous peptides are also substrates of P450 3A. The physiological activities of these peptides may be modulated by their metabolism.     

Sequence diversity of the OprD protein of environmental Pseudomonas strains

OprD has been widely described for Pseudomonas aeruginosa at both structural and functional levels. Here, we describe the sequence diversity of the OprD proteins from other fluorescent Pseudomonads. We analysed the sequence of the oprD gene in each of the 49 Pseudomonas isolates, mostly putida and fluorescens species, obtained from various environmental sources, including soil, rhizosphere and hospitals. Phylogeny based on OprD sequences distinguished three well-separated clusters in the P. fluorescens species whereas P. putida isolates formed only one cluster. The OprD sequences were generally well conserved within each cluster whereas on the opposite, they were highly variable from one cluster to another and particularly with regards to the cluster of P. aeruginosa. Predicted secondary structures, based on the topological model elaborated for P. aeruginosa, suggest signatures in the large extracellular loops of OprD, which are linked to the OprD-based clusters. Correlations between these OprD-based clusters and ecological niches, growth on various carbon sources and antibiotic sensitivity were investigated.     

Production of biologically active forms of recombinant hepcidin, the iron-regulatory hormone

Hepcidin is a liver produced cysteine-rich peptide hormone that acts as the central regulator of body iron metabolism. Hepcidin is synthesized under the form of a precursor, prohepcidin, which is processed to produce the biologically active mature 25 amino acid peptide. This peptide is secreted and acts by controlling the concentration of the membrane iron exporter ferroportin on intestinal enterocytes and macrophages. Hepcidin binds to ferroportin, inducing its internalization and degradation, thus regulating the export of iron from cells to plasma. The aim of the present study was to develop a novel method to produce human and mouse recombinant hepcidins, and to compare their biological activity towards their natural receptor ferroportin. Hepcidins were expressed in Escherichia coli as thioredoxin fusion proteins. The corresponding peptides, purified after cleavage from thioredoxin, were properly folded and contained the expected four-disulfide bridges without the need of any renaturation or oxidation steps. Human and mouse hepcidins were found to be biologically active, promoting ferroportin degradation in macrophages. Importantly, biologically inactive aggregated forms of hepcidin were observed depending on purification and storage conditions, but such forms were unrelated to disulfide bridge formation.     

Evaluation of cytotoxic properties of organometallic ferrocifens on melanocytes, primary and metastatic melanoma cell lines

Malignant melanoma is one of the most severe forms of skin cancer, and chemotherapeutic agents currently in use are poorly effective in curing the disease. Here we describe the properties of two organometallic ferrocenyl derivatives, ferrocifen (Fc-OH-Tam) and ferrociphenol (Fc-diOH) that show a specific antiproliferative effect on melanoma cells. After a short incubation period, Fc-OH-Tam is highly cytotoxic on melanoma cells but less toxic on melanocytes. Fc-diOH is slightly toxic at a high concentration but no discrepancy is observed between malignant and normal cells. After a long incubation time the latter is highly toxic for malignant cells but not for normal cells while the former was very highly toxic for primary malignant cells and significantly less toxic for normal cells. We also found that oxidative stress is not implicated in the mechanism of cytotoxicity, since both derivatives neither induce reactive oxygen species (ROS) level in melanocytes nor in melanoma cells. Finally, investigation on hair follicle growth revealed that the two organometallic derivatives induced an irreversible ejection of the hair shaft, thus predicting a potential hair loss side effect if used as a chemotherapeutic treatment.