The role of cystine knots in collagen folding and stability,part I. Conformational properties of (Pro-Hyp-Gly)5 and (Pro-(4S)-FPro-Gly)5 model trimers with an artificial cystine knot

In analogy to the cystine knots present in natural collagens, a simplified disulfide cross-link was used to analyse the conformational effects of a C-terminal artificial cystine knot on the folding of collagenous peptides consisting of solely (Pro-Hyp-Gly) repeating units. Assembly of the alpha chains into a heterotrimer by previously applied regioselective disulfide-bridging strategies failed because of the high tendency of (Pro-Hyp-Gly)(5) peptides to self-associate and form homotrimers. Only when side-chain-protected peptides were used, for example in the Hyp(tBu) form, and a new protection scheme was adopted, selective interchain-disulfide cross-linking into the heterotrimer in organic solvents was successful. This unexpected strong effect of the conformational properties on the efficiency of well-established reactions was further supported by replacing the Hyp residues with (4S)-fluoroproline, which is known to destabilise triple-helical structures. With the related [Pro-(4S)-FPro-Gly](5) peptides, assembly of the heterotrimer in aqueous solution proceeded in a satisfactory manner. Both the intermediates and the final fluorinated heterotrimer are fully unfolded in aqueous solution even at 4 degrees C. Conversely, the disulfide-crossbridged (Pro-Hyp-Gly)(5) heterotrimer forms a very stable triple helix. The observation that thermal unfolding leads to scrambling of the disulfide bridges was unexpected. Although NMR experiments support an extension of the triple helix into the cystine knot, thermolysis is not associated with the unfolding process. In fact, the unstructured fluorinated trimer undergoes an equally facile thermodegradation associated with the intrinsic tendency of unsymmetrical disulfides to disproportionate into symmetrical disulfides under favourable conditions. The experimental results obtained with the model peptides fully support the role of triple-helix nucleation and stabilisation by the artificial cystine knot as previously suggested for the natural cystine knots in collagens.     

Electrochemical treatment of olive oil mill wastewater

The possibility of oxidizing at a PbO2 anode the phenols and polyphenols, present in the olive oil mill wastewater, has been studied as a pretreatment for the submission of such wastewater to the traditional biological treatments. The results obtained operating at current densities ranging 500 to 2000 A/m2 show that it is possible to reduce the concentration of the phenolic components, which interfere with the biological treatments, down to low values without decreasing too much the total organic content of the wastewater.     

Expanding the scope of C-H amination through catalyst design

Analysis of the mechanism for Rh-mediated C-H amination has led to the development of a remarkably effective dinuclear Rh catalyst derived from 1,3-benzenedipropionic acid. This unique complex, Rh2(esp)2, is capable of promoting both intra- and intermolecular C-H oxidation reactions, and in all cases is superior to Rh2(O2CtBu)4. For the first time, C-H insertion is described with urea and sulfamide substrates to give 1,2- and 1,3-diamine derivatives, respectively. In addition, intermolecular amination of benzylic and secondary C-H bonds is shown to proceed efficiently even under conditions in which the starting alkane is employed as the limiting reagent.     

Compatibilization of Poly(Lactic Acid) (PLA)/Plasticized Cellulose Acetate Extruded Blends through the Addition of Reactively Extruded Comb Copolymers

In the perspective of producing a rigid renewable and environmentally friendly rigid packaging material, two comb-like copolymers of cellulose acetate (AC) and oligo(lactic acid) OLA, feeding different percentages of oligo(lactic acid) segments, were prepared by chemical synthesis in solvent or reactive extrusion in the melt, using a diepoxide as the coupling agent and were used as compatibilizers for poly(lactic acid)/plasticized cellulose acetate PLA/pAC blends. The blends were extruded at 230 °C or 197 °C and a similar compatibilizing behavior was observed for the different compatibilizers. The compatibilizer C1 containing 80 wt% of AC and 14 wt% of OLA resulted effective in compatibilization and it was easily obtained by reactive extrusion. Considering these results, different PLAX/pAC(100-X) compounds containing C1 as the compatibilizer were prepared by extrusion at 197 °C and tested in terms of their tensile and impact properties. Reference materials were the uncompatibilized corresponding blend (PLAX/pAC(100-X)) and the blend of PLA, at the same wt%, with C1. Significant increase in Young’s modulus and tensile strength were observed in the compatibilized blends, in dependence of their morphologic features, suggesting the achievement of an improved interfacial adhesion thanks to the occurred compatibilization.     

Confirmatory identification of sixteen non-steroidal anti-inflammatory drug residues in raw milk by liquid chromatography coupled with ion trap mass spectrometry

The European Council Decision 2002/657/EC established that group B substances detected in foods must be identified and confirmed on the basis of their molecular structure. To this aim, we have developed a panel of methods for unambiguous determination of sixteen non-steroidal anti-inflammatory drugs (NSAIDs) in cattle and buffalo raw milk. A multi-residue reversed-phase high-performance liquid chromatography method with photodiode array detection is described for quantitative screening analysis. For confirmatory purposes, two multi-residue reversed-phase ion trap liquid chromatography/electrospray ionisation tandem mass spectrometry (LC/ESI-MS/MS) methods were developed: the former to identify salicylic acid, naproxen, carprofen, flurbiprofen, ibuprofen, meclofenamic acid, niflumic acid, flunixin and its metabolite 5-hydroxyflunixin in the negative ion mode; the latter to identify ketoprofen, suxibutazone, diclofenac, mefenamic acid, tolfenamic acid, phenylbutazone and its metabolite oxyphenbutazone in the positive ion mode. These drugs are representative of different subclasses of NSAIDs not chemically related. The methods were in-house validated, evaluating specificity and calculating the mean recoveries, repeatability, within-laboratory reproducibility, and limits of quantification. For all the NSAIDs, apart from salicylic acid and 5-hydroxyflunixin, mean recoveries ranging between 69.0% and 96.7% were measured. The qualitative identification of all drugs was attained by their MS/MS spectra in the concentration range studied. Similarly, at 5 microg/kg all NSAIDs, apart from flurbiprofen, were unambiguously confirmed.     

Infinite Classes of Dihedral Snarks

Flower snarks and Goldberg snarks are two infinite families of cyclically 5–edge–connected cubic graphs with girth at least five and chromatic index four. For any odd integer k, k > 3, there is a Flower snark, say J _k , of order 4k and a Goldberg snark, say B _k , of order 8k. We determine the automorphism groups of J _k and B _k for every k and prove that they are isomorphic to the dihedral group D _4k of order 4k. Research performed within the activity of INdAM–GNSAGA with the financial support of the Italian Ministry MIUR, project “Strutture Geometriche, Combinatoria e loro Applicazioni”.     

The Use of Bovine Serum Albumin as a Ligand in Affinity Chromatographic Clean-up of Non-Steroidal Anti-Inflammatory Drugs from Bovine Plasma

The European Union regulates the use of non-steroidal anti-inflammatory drugs (NSAID) in animal production and official national analytical monitoring programmes have been set up in each member state to detect residues of the drugs in bovine plasma. In this work, we describe the development and application of affinity chromatography for molecular recognition of NSAID in bovine plasma. Bovine serum albumin (BSA), the plasma protein which binds such drugs, was covalently bound to a polymeric support via its glycosyl moieties. Loading capacities and specificity were tested both on standard solutions and on spiked bovine plasma for nine drugs—ketoprofen, naproxen, phenylbutazone, oxyphenylbutazone, acetylsalicylic acid, salicylic acid, tolfenamic acid, diclofenac, and nimesulide—chosen as being the most representative of the different chemical sub-classes of NSAID. The BSA columns could bind up to 150 × 10^–6 g total of a mixture of these nine NSAID, with mean recoveries ranging from 74.0% (tolfenamic acid) to 96.0% (nimesulide). HPLC separation on an RP C₁₈ column with a linear gradient enabled resolution of the drugs; identification was achieved by coupling with photodiode array detection (DAD) operated at 240 and 280 nm. Results showed that all the compounds except acetylsalicylic acid were bound effectively by the BSA affinity columns. When plasma samples were spiked at 2.5 g mL^–1 with a mixture of the NSAID the chromatographic profile and UV spectra recorded (in the range 220–350 nm) indicated the procedure was sufficiently selective for identification of the drugs at the concentrations expected according to their pharmacokinetics. No memory effects and matrix interferences were observed.Revised: 18 December 2003 and 16 April 2004     

Study on the photostability of guaiazulene by high-performance liquid chromatography/mass spectrometry and gas chromatography/mass spectrometry

The photostability of guaiazulene (1,4-dimethyl-7-isopropylazulene; GA), a natural azulenic compound used in cosmetic and health-care products, as well as in pharmaceutical preparations, was investigated in solution (methanol, ethanol, acetonitrile), by different techniques: gas chromatography/mass spectrometry (GC/MS) and high-performance liquid chromatography combined with atmospheric pressure chemical ionization mass spectrometry and UV detection (LC/APCI-MS and HPLC/UV). A solar simulator (xenon-arc lamp) was used as UV-A radiation source. The study involved: monitoring compound decomposition, identifying products of photodegradation (PPs), assessing the role of oxygen and evaluating the kinetics of the process. Minor PPs are volatile compounds and were characterized by GC/MS, while oligomeric polyoxygenated compounds, tentatively characterized on the basis of MS and MS/MS spectra, were found to be the main photoproducts. The photodegradation was found to be enhanced by the presence of oxygen; nevertheless, determination of the singlet oxygen quantum yield for GA gave a lower value than that for the reference standard Rose Bengal. The obtained results and the developed stability-indicating methods (GC/MS and LC/MS) are of interest for stability studies and/or quality control purposes of GA as raw material or cosmetic products.     

Determination of 30 elements in colorectal biopsies by sector field inductively coupled plasma mass spectrometry: method development and preliminary baseline levels

An analytical procedure applicable to restricted sample sizes was developed and applied to the analysis of 30 chemical elements in colorectal biopsies of healthy patients. Acidic microwave digestion processed 相似文献