Oxidation of pharmaceutically active compounds by a ligninolytic fungal peroxidase

Pharmaceuticals are an important group of emerging pollutants with increasing interest due to their rising consumption and the evidence for ecotoxicological effects associated to trace amounts in aquatic environments. In this paper, we assessed the potential degradation of a series of pharmaceuticals: antibiotics (sulfamethoxazole), antidepressives (citalopram hydrobromide and fluoxetine hydrochloride), antiepileptics (carbamazepine), anti-inflammatory drugs (diclofenac and naproxen) and estrogen hormones (estrone, 17β-estradiol, 17α-ethinylestradiol) by means of a versatile peroxidase (VP) from the ligninolytic fungus Bjerkandera adusta. The effects of the reaction conditions: VP activity, organic acid concentration and H₂O₂ addition rate, on the kinetics of the VP based oxidation system were evaluated. Diclofenac and estrogens were completely degraded after only 5–25 min even with a very low VP activity (10 U l⁻¹). High degradation percentages (80%) were achieved for sulfamethoxazole and naproxen. Low or undetectable removal yields were observed for citalopram (up to 18%), fluoxetine (lower than 10%) and carbamazepine (not degraded).     

Synthesis, pharmacophore modeling and in vitro activity of 10,11-dihydrodibenzo[b,f]oxepine-4-carboxamide derivatives as novel and potent antagonists of the prostaglandin EP4 receptor

The construction of a EP(4) antagonists pharmacophore model and the discovery of a highly potent oxepinic series of EP(4) antagonists is discussed. Compound 1a exhibits an excellent selectivity profile toward EP(2) receptor subtype and low cytochrome P450 inhibition potential.     

Evaluation of a cell penetrating prenylated peptide lacking an intrinsic fluorophore via in situ click reaction

Protein prenylation involves the addition of either a farnesyl (C₁₅) or geranylgeranyl (C₂₀) isoprenoid moiety onto the C-terminus of many proteins. This natural modification serves to direct a protein to the plasma membrane of the cell. A recently discovered application of prenylated peptides is that they have inherent cell-penetrating ability, and are hence termed cell penetrating prenylated peptides. These peptides are able to efficiently cross the cell membrane in an ATP independent, non-endocytotic manner and it was found that the sequence of the peptide does not affect uptake, so long as the geranylgeranyl group is still present [Wollack, J. W.; Zeliadt, N. A.; Mullen, D. G.; Amundson, G.; Geier, S.; Falkum, S.; Wattenberg, E. V.; Barany, G.; Distefano, M. D. Multifunctional Prenylated Peptides for Live Cell Analysis. J. Am. Chem. Soc.2009, 131, 7293-7303]. The present study investigates the effect of removing the fluorophore from the peptides and investigating the uptake by confocal microscopy and flow cytometry. Our results show that the fluorophore is not necessary for uptake of these peptides. This information is significant because it indicates that the prenyl group is the major determinant in allowing these peptides to enter cells; the hydrophobic fluorophore has little effect. Moreover, these studies demonstrate the utility of the Cu-catalyzed click reaction for monitoring the entry of nonfluorescent peptides into cells.     

Photoaffinity labeling of Ras converting enzyme using peptide substrates that incorporate benzoylphenylalanine (Bpa) residues: improved labeling and structural implications

Rce1p catalyzes the proteolytic trimming of C-terminal tripeptides from isoprenylated proteins containing CAAX-box sequences. Because Rce1p processing is a necessary component in the Ras pathway of oncogenic signal transduction, Rce1p holds promise as a potential target for therapeutic intervention. However, its mechanism of proteolysis and active site have yet to be defined. Here, we describe synthetic peptide analogues that mimic the natural lipidated Rce1p substrate and incorporate photolabile groups for photoaffinity-labeling applications. These photoactive peptides are designed to crosslink to residues in or near the Rce1p active site. By incorporating the photoactive group via p-benzoyl-l-phenylalanine (Bpa) residues directly into the peptide substrate sequence, the labeling efficiency was substantially increased relative to a previously-synthesized compound. Incorporation of biotin on the N-terminus of the peptides permitted photolabeled Rce1p to be isolated via streptavidin affinity capture. Our findings further suggest that residues outside the CAAX-box sequence are in contact with Rce1p, which has implications for future inhibitor design.     

Polyoxazoline: chemistry, properties, and applications in drug delivery

Polyoxazoline polymers with methyl (PMOZ), ethyl (PEOZ), and propyl (PPOZ) side chains were prepared by the living cationic polymerization method and purified by ion-exchange chromatography. The following properties of polyoxazoline (POZ) were measured: apparent hydrodynamic radius by aqueous size-exclusion chromatography, relative lipophilicity by reverse-phase chromatography, and viscosity by cone-plate viscometry. The PEOZ polymers of different molecular weights were first functionalized and then conjugated to model biomolecules such as bovine serum albumin, catalase, ribonuclease, uricase, and insulin. The conjugates of catalase, uricase, and ribonuclease were tested for in vitro activity using substrate-specific reaction methods. The conjugates of insulin were tested for glucose lowering activity by injection to nai?ve Sprague-Dawley rats. The conjugates of BSA were injected into New Zealand white rabbits and serum samples were collected periodically and tested for antibodies to BSA. The safety of POZ was also determined by acute and chronic dosing to rats. The results showed that linear polymers of POZ with molecular weights of 1 to 40 kDa can easily be made with polydispersity values below 1.10. Chromatography results showed that PMOZ and PEOZ have a hydrodynamic volume slightly lower than PEG; PEOZ is more lipophilic than PMOZ and PEG; and PEOZ is significantly less viscous than PEG especially at the higher molecular weights. When PEOZ was attached to the enzymes catalase, ribonuclease, and uricase, the in vitro activity of the resultant bioconjugates depended on the extent of protein modification. POZ conjugates of insulin lowered blood glucose levels for a period of 8 h when compared to 2 h for insulin alone. PEOZ, like PEG, was also able to successfully attenuate the immunogenic properties of BSA. The POZ polymers (10 and 20 kDa) are safe when administered intravenously to rats, and the maximum tolerated dose (MTD) was greater than 2 g/kg. Blood counts, serum chemistry, organ weights, and the histopathology of key organs were normal. These results conclude that POZ has the desired drug delivery properties for a new biopolymer.     

β-thymosins and interstitial lung disease: study of a scleroderma cohort with a one-year follow-up

Background

β-thymosins play roles in cytoskeleton rearrangement, angiogenesis, fibrosis and reparative process, thus suggesting a possible involvement in the pathogenesis of systemic sclerosis. The aim of the study was to investigate the presence of thymosins β₄, β₄ sulfoxide, and β₁₀ in bronchoalveolar lavage fluid of scleroderma patients with interstitial lung disease and the relation of these factors with pulmonary functional and radiological parameters.

Methods

β-thymosins concentrations were determined by Reverse Phase-High Performance Liquid Chromatography-Electrospray-Mass Spectrometry in the bronchoalveolar lavage fluid of 46 scleroderma patients with lung involvement and of 15 controls.

Results

Thymosin β_4, β₄ sulfoxide, and β₁₀ were detectable in bronchoalveolar lavage fluid of patients and controls. Thymosin β₄ levels were significantly higher in scleroderma patients than in controls. In addition, analyzing the progression of scleroderma lung disease at one-year follow-up, we have found that higher thymosin β₄ levels seem to have a protective role against lung tissue damage. Thymosin β₄ sulfoxide levels were higher in the smokers and in the scleroderma patients with alveolitis.

Conclusions

We describe for the first time β-thymosins in bronchoalveolar lavage fluid and their possible involvement in the pathogenesis of scleroderma lung disease. Thymosin β₄ seems to have a protective role against lung tissue damage, while its oxidation product mirrors an alveolar inflammatory status.     

Inter-plant vibrational communication in a leafhopper insect

Vibrational communication is one of the least understood channels of communication. Most studies have focused on the role of substrate-borne signals in insect mating behavior, where a male and a female establish a stereotyped duet that enables partner recognition and localization. While the effective communication range of substrate-borne signals may be up to several meters, it is generally accepted that insect vibrational communication is limited to a continuous substrate. Until now, interplant communication in absence of physical contact between plants has never been demonstrated in a vibrational communicating insect. With a laser vibrometer we investigated transmission of natural and played back vibrational signals of a grapevine leafhopper, Scaphoideus titanus, when being transmitted between leaves of different cuttings without physical contact. Partners established a vibrational duet up to 6 cm gap width between leaves. Ablation of the antennae showed that antennal mechanoreceptors are not essential in detection of mating signals. Our results demonstrate for the first time that substrate discontinuity does not impose a limitation on communication range of vibrational signals. We also suggest that the behavioral response may depend on the signal intensity.     

Redox proteomics of fat globules unveils broad protein lactosylation and compositional changes in milk samples subjected to various technological procedures

The Maillard reaction between lactose and proteins occurs during thermal treatment of milk and lactosylated β-lactoglobulin, α-lactalbumin and caseins have widely been used to monitor the quality of dairy products. We recently demonstrated that a number of other whey milk proteins essential for nutrient delivery, defense against bacteria/virus and cellular proliferation become lactosylated during milk processing. The extent of their modification is associated with the harshness of product manufacturing. Since fat globule proteins are also highly important for the health-beneficial properties of milk, an evaluation of their lactosylation is crucial for a complete understanding of aliment nutritional characteristics. This is more important when milk is the unique dietary source, as in the infant diet. To this purpose, a sequential proteomic procedure involving an optimized milk fat globule (MFG) preparation/electrophoretic resolution, shot-gun analysis of gel portions for protein identification, selective trapping of lactosylated peptides by phenylboronate chromatography and their analysis by nanoLC-ESI-electron transfer dissociation (ETD) tandem MS was used for systematic characterization of fat globule proteins in milk samples subjected to various manufacturing procedures. Significant MFG protein compositional changes were observed between samples, highlighting the progressive adsorption of caseins and whey proteins on the fat globule surface as result of the technological process used. A significant lactosylation of MFG proteins was observed in ultra-high temperature sterilized and powdered for infant nutrition milk preparations, which well paralleled with the harshness of thermal treatment. Globally, this study allowed the identification of novel 157 non-redundant modification sites and 35 MFG proteins never reported so far as being lactosylated, in addition to the 153 ones ascertained here as present on other 21 MFG-adsorbed proteins whose nature was already characterized. Novel MFG proteins include components involved in nutrient delivery, defense response against pathogens and cellular proliferation/differentiation. Nutritional, biological and toxicological consequences of these findings are here discussed, highlighting their possible impact on children's diet.