Interactions of a fungistatic antibiotic, griseofulvin, with phospholipid monolayers used as models of biological membranes

Griseofulvin (GF) is an oral antibiotic for widely occurring superficial mycosis in man and animals caused by dermaphyte fungi; it is also used in agriculture as a fungicide. The mechanism of the biological activity of GF is poorly understood. Here, the interactions of griseofulvin with lipid membranes were studied using 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), and 1,2-myristoyl-sn-glycero-3-phosphoethanolamine (DMPE) monolayers spread at the air/water interface. Surface pressure (Pi), electric surface potential (Delta V), grazing incidence X-ray diffraction (GIXD), and Brewster angle microscopy (BAM) were used for studying pure phospholipid monolayers spread on GF aqueous solutions, as well as mixed phospholipid/GF monolayers spread on pure water subphase. Moreover, phospholipase A2 (PLA2) activity toward DLPC monolayers and molecular modeling of the GF surface and lipophilic properties were used to get more insight into the mechanisms of GF-membrane interactions. The results obtained show that GF has a meaningful impact on the film properties; we propose that nonpolar interactions are by and large responsible for GF retention in the monolayers. The modification of membrane properties can be detected using both physicochemical and enzymatic methods. The results obtained may be relevant for elaborating GF preparations with increased bioavailability.     

Reductive carbodiazenylation of nonactivated olefins via aryl diazonium salts

[Structure: see text] The reduction of aryl diazonium salts in the presence of nonactivated olefins provides rapid entry to carbodiazenylation products. The regioselective functionalization of double bonds is achieved in a one-pot process starting from aniline derivatives. Carboamination of olefins is possible via a two-step carbodiazenylation-hydrogenation sequence in which one aniline equivalent is recovered.     

Implementing tandem mass spectrometry as a routine tool for characterizing the complete purine and pyrimidine metabolic profile in urine samples

Purines and pyrimidines are the basic constituents of DNA and RNA and constitute the basis of at least 50 other important compounds that serve equally vital but separate roles as integral components of intracellular mononucleotide pools. They maintain the supply of these basic components to the different nucleotide pools through an extremely efficient mechanism involving the degradation and recycling of the daily waste products of normal cell turnover. We have developed an LC-MS/MS diagnostic and routine monitoring method for known defects due to both purine and pyrimidine metabolism in a single analysis. Precision tests were made by spiking several urine samples with different creatinine concentrations. For nonspiked low-creatinine urine, intraday precision was in the range of 0.1-9.8% and interday precision was between 1.6 and 14.1%. For nonspiked high-creatinine urine, intraday precision was in the range 0.5-17.2% and interday precision was between 1.5 and 29%. Limit-of-detection (LOD) was in the range 0.1-10 micromol/l and limit-of-quantification (LOQ) in the range of 0.2-15 micromol/l. The current 'dilute and shoot' approach monitors many metabolites, and utilizes a reverse phase chromatographic analysis with a detection requiring 17 min of analysis time. Tandem mass spectrometry and isotope dilution technique enable the accurate quantitation of more than 30 metabolites in one analysis.     

Distribution of humic substances dissolved and particulated in water column in Ross Sea, Antarctica

Dissolved (DFA) and particulate (PFA) fulvic acids were studied in some areas of the Ross Sea (Antarctica) characterised by different water masses. Our interest was focused on their distribution in the water column. Moreover, their correlation with fluorescence data and structural characterisation in relation to the water masses was also studied. We found that PFA concentration was mainly influenced by the primary production in surface layers and by stratification of water masses along the water column. Conversely, the DFA are less influenced by primary production and by the stratification of the water masses. An early stage of the humification process can be hypothesised for PFA related to DFA as shown by elemental analyses and spectroscopic data. PFA collected in the bottom depths of the water column showed a relationship with age of water mass (the highest concentration was found for the oldest circulating water mass (CDW)). This relationship was not observed for DFA. Moreover, any significant difference in structure was found in the DFA and PFA extracted from samples collected at bottom depths.     

A novel polyacrylamide gel system for proteomic use offering controllable pore expansion by crosslinker cleavage

SDS‐PAGE is still one of the most widespread separation techniques in proteomic research and usually coupled to subsequent MS measurement for protein identification. The proteins are digested while embedded in the gel matrix. The resultant peptides are eluted out of the gel and finally analyzed. The in‐gel digestion process suffers from several drawbacks which influence the experimental outcome with respect to protein sequence coverage and detection sensitivity. Limited accessibility of the protease to the substrate protein and insufficient peptide extraction represent the two major problems. To specifically target these issues, we established a novel partly reversible gel system, in which the gel matrix can be conditionally cleaved to increase the pore diameters. By using a crosslinker mixture consisting of Bis and ethylene‐glycol‐diacrylate the acrylamide filament interconnections can be partly hydrolyzed in alkaline solution. The new hybrid gels have been tested to be compatible with a variety of acidic staining techniques. They exhibit similar electrophoretic performance compared with regular solely Bis‐based gels, but yield significantly better MS results. Thus, the Bis/ethylene‐glycol‐diacrylate SDS‐PAGE gel system is a promising alternative for MS‐based in‐gel workflows and might be transferred to other gel‐electrophoretic applications.     

Simultaneous determination of nine endogenous steroids in human urine by polymeric-mixed micelle capillary electrophoresis

A new CE system based on the use of polymeric-mixed micelles (cholic acid, SDS and the poloxamine Tetronic(?) 1107) was developed for the simultaneous determination of nine steroids in human urine. This method allows the baseline separation and quantitation of cortisol, androstenedione, estriol, dehydroepiandrosterone sulfate, testosterone, dehydroepiandrosterone, estrone, progesterone and estradiol in less than 25 min showing to be sensitive enough to detect low concentrations of these steroids in urine samples (5-45 ng/mL). The optimized electrophoretic conditions were performed using a 50 cm × 75 μm capillary, 18 kV, 25°C, with 44 mM cholic acid, 10 mM SDS, 0.05% w/v tetronic(?) 1107, 2.5% v/v methanol, 2.5% v/v tetrahydrofuran in 5 mM borate - 5 mM phosphate buffer (pH=8.0) as a background electrolyte and a dual 210/254 UV-detection. The method can simultaneously determine 0.1-120 μg/mL, which corresponds to 5-6000 ng/mL of steroids in 2 mL urine. The recoveries ranged between 82.4 and 101.5%. Due to its simplicity, speed, accuracy and reliability, the proposed method could be a potential alternative to the traditional methodologies used with clinical purposes.     

Measurement of PAHs in environmental matrices: results from an interlaboratory comparison on the different steps of the measurement procedure

Proficiency testing (PT) is becoming a feature of laboratory accreditation and the PT results are used to assess the technical competence of the participating laboratories. ISPRA (former APAT) plays an important role in supporting the Italian laboratories belonging to the network of the Regional Environmental Agencies to improve the quality of their analytical measurements. As a consequence, ISPRA organized an interlaboratory comparison to assess the performance of the laboratories on PAH measurement procedure. The interlaboratory comparison was drawn separating the different steps of the measurement (from the extraction to the instrumental measurement). Two matrix reference materials: (1) a polluted soil and (2) an extract reference material of the same polluted soil and a “blind” PAH mixture standard stock solution were distributed to 59 Italian laboratories. The results of interlaboratory comparison showed a significant dispersion of the PAH measurements that masks the effects of the different extraction and cleanup procedures used, but it is consistent with the results of other European interlaboratory comparisons.     

Pd complexes of iminophosphine ligands: A homogeneous molecular catalyst for Suzuki–Miyaura cross-coupling reactions under mild conditions

The complexes formed by combining Pd(OAc)₂ and iminophosphine ligands (P^N) are active catalysts in Suzuki–Miyaura cross-coupling reactions under mild conditions. Aryl bromides and iodides, as well as benzyl chlorides give the corresponding coupled products in high yields at low temperatures (25–50 °C) using these catalysts. Iminophosphines containing the most sterically demanding groups attached to the N-imino moiety were the most effective ligands. New divalent Pd complexes of known iminophosphines were synthesised and their activity was compared with the in situ generated catalyst system. The complex resulting from the oxidative addition of 4-bromo anisole [Pd(4-CH₃OC₆H₄)Br(P^N)] was more active than the in situ generated system. However, palladacycles containing the iminophosphine ligand (e.g., {[C₆H₄CH(Me)₂St-Bu]Pd(P^N)}⁺PF₆⁻) were less active than the in situ generated catalyst due to the greater stability of the complexes that involve two bidentate ligands. Poisoning tests demonstrated that homogeneous mononuclear palladium species containing the iminophsophine ligand were responsible for the catalytic activity.     

Synthesis,Crystal Structure and Magnetic Properties of a Trinuclear Copper(II) Complex Based on P-Cresol-Substituted Bis(α-Nitronyl Nitroxide) Biradical

Trinuclear copper(II) complex [Cu^II₃(NIT₂PhO)₂Cl₄] was synthesized with p-cresol-substituted bis(α-nitronyl nitroxide) biradical: 4-methyl-2,6-bis(1-oxyl-3-oxido-4,4,5,5-tetramethyl-2-imidazolin-2-yl)phenol (NIT₂PhOH). The crystal structure of this heterospin complex was determined using single-crystal X-ray diffraction analysis and exhibits four unusual seven-membered metallocycles formed from the coordination of oxygen atoms of the N-O groups and of bridging phenoxo (µ-PhO⁻) moieties with copper(II) ions. The crystal structure analysis reveals an incipient agostic interaction between a square planar copper center and a hydrogen-carbon bond from one methyl group carried on the coordinated nitronyl-nitroxide radical. The intramolecular Cu∙∙∙H-C interaction involves a six-membered metallocycle and may stabilize the copper center in square planar coordination mode. From the magnetic susceptibility measurements, the complex, which totals seven S = 1/2 spin carriers, has almost a ground state spin S = 1/2 at room temperature ascribed to strong antiferromagnetic interaction between the nitronyl nitroxide moieties and the copper(II) centers and in between the copper(II) centers through the bridging phenoxo oxygen atom.     

A New Alternative Tool to Analyse Glycosylation in Monoclonal Antibodies Based on Drop-Coating Deposition Raman imaging: A Proof of Concept

Glycosylation is considered a critical quality attribute of therapeutic proteins as it affects their stability, bioactivity, and safety. Hence, the development of analytical methods able to characterize the composition and structure of glycoproteins is crucial. Existing methods are time consuming, expensive, and require significant sample preparation, which can alter the robustness of the analyses. In this context, we developed a fast, direct, and simple drop-coating deposition Raman imaging (DCDR) method combined with multivariate curve resolution alternating least square (MCR-ALS) to analyze glycosylation in monoclonal antibodies (mAbs). A database of hyperspectral Raman imaging data of glycoproteins was built, and the glycoproteins were characterized by LC-FLR-MS as a reference method to determine the composition in glycans and monosaccharides. The DCDR method was used and allowed the separation of excipient and protein by forming a “coffee ring”. MCR-ALS analysis was performed to visualize the distribution of the compounds in the drop and to extract the pure spectral components. Further, the strategy of SVD-truncation was used to select the number of components to resolve by MCR-ALS. Raman spectra were processed by support vector regression (SVR). SVR models showed good predictive performance in terms of RMSECV, R²_CV.