Synthetic erythropoietic proteins: tuning biological performance by site-specific polymer attachment

Chemical synthesis in combination with precision polymer modification allows the systematic exploration of the effect of protein properties, such as charge and hydrodynamic radius, on potency using defined, homogeneous conjugates. A series of polymer-modified synthetic erythropoiesis proteins were constructed that had a polypeptide chain similar to the amino acid sequence of human erythropoietin but differed significantly in the number and type of attached polymers. The analogs differed in charge from +5 to -26 at neutral pH and varied in molecular weight from 30 to 54 kDa. All were active in an in vitro cell proliferation assay. However, in vivo potency was found to be strongly dependent on overall charge and size. The trends observed in this study may serve as starting points for the construction of more potent synthetic EPO analogs in the future.     

Supramolecular helical mesomorphic polymers. Chiral induction through H-bonding

The work described here concerns a challenge of general interest in supramolecular chemistry: the achievement of chiral helical organizations with controlled structures. This work provides a strategy to obtain supramolecular polymers in which a chiral helical conformation has been induced by a noncovalent association, that is, through hydrogen bonding. Polycatenar 2,4,6-triarylamino-1,3,5-triazines, which organize into columnar mesophases and are susceptible to H-bonding interactions, were chosen as a starting point to build up the chiral supramolecular structure. The stacking of these mesogens has been forced to wind in a helical way by means of H-bond association with (R)-3-methyladipic acid, within the mesophase. The optically active columnar organization has been studied in depth by optical microscopy, differential scanning calorimetry (DSC), X-ray diffraction, and circular dichroism. Formation of stable complexes between the triazine units and (R)-3-methyladipic acid has also been investigated by means of NMR diffusion-ordered spectroscopy (DOSY) experiments in chloroform.     

Phase transition and mixed oxide-proton conductivity in germanium oxy-apatites

La_9.75□_0.25(Ge₆O₂₄)O_2.62 oxy-apatite shows a phase transition from triclinic to hexagonal symmetry at approximately 1020 K that has been characterised by high-temperature synchrotron X-ray and neutron powder diffraction, and ionic conductivity measurements. The crystal structure at 1073 K has been determined from joint Rietveld refinements of synchrotron X-ray and neutron powder diffraction data. The study shows that hexagonal-La_9.75□_0.25(Ge₆O₂₄)O_2.62 contains interstitial oxygen at the position previously reported for other oxy-germanates. Changes in the oxide conductivity associated with this structural transition are discussed. The thermal analyses showed a weight loss on heating close to 600 K very likely due to water release. The synchrotron thermodiffractometric study shows an anomaly in the cell parameters evolution at that temperature, which indicates that this residual water is located into the apatite channels. The electrical characterisation under different atmospheres (dry and wet synthetic air) indicates that there is a significant proton contribution to the overall conductivity below 600 K, mainly under wet atmosphere.     

Analysis of Colubroidea snake venoms by liquid chromatography with mass spectrometry: evolutionary and toxinological implications

The evolution of the venomous function of snakes and the diversification of the toxins has been of tremendous research interest and considerable debate. It has become recently evident that the evolution of the toxins in the advanced snakes (Colubroidea) predated the evolution of the advanced, front-fanged delivery mechanisms. Historically, the venoms of snakes lacking front-fanged venom-delivery systems (conventionally grouped into the paraphyletic family Colubridae) have been largely neglected. In this study we used liquid chromatography with mass spectrometry (LC/MS) to analyze a large number of venoms from a wide array of species representing the major advanced snake clades Atractaspididae, Colubrinae, Elapidae, Homalopsinae, Natricinae, Psammophiinae, Pseudoxyrhophiinae, Xenodontinae, and Viperidae. We also present the first sequences of toxins from Azemiops feae as well as additional toxin sequences from the Colubrinae. The large body of data on molecular masses and retention times thus assembled demonstrates a hitherto unsuspected diversity of toxins in all lineages, having implications ranging from clinical management of envenomings to venom evolution to the use of isolated toxins as leads for drug design and development. Although definitive assignment of a toxin to a protein family can only be done through demonstrated structural studies such as N-terminal sequencing, the molecular mass data complemented by LC retention information, presented here, do permit formulation of reasonable hypotheses concerning snake venom evolution and potential clinical effects to a degree not possible till now, and some hypotheses of this kind are proposed here. The data will also be useful in biodiscovery.     

A combinatorial approach to obtain affinity media with binding properties towards the aflatoxins

In our work we performed a combinatorial solid-phase synthesis in aqueous medium to prepare peptide libraries from which to select an amino acid sequence with binding properties towards aflatoxins.We used polystyrene beads, functionalised with carboxylic groups as solid support and eight amino acids as monomers. During the first step 64 different sequences of two amino acids were prepared by exploiting the principles of combinatorial chemistry; then the binding properties of all sequences towards aflatoxin B(1 )were checked. We determined binding constants towards aflatoxin B(1) and towards aflatoxins B(2), G(1) and G(2). Results were promising, so we prepared a new library by using the selected dipeptide as the starting solid phase. After selecting the best tetrapeptide sequences, we determined binding constants towards the quoted aflatoxins. We obtained binding constants ( K>10(4) M(-1)) similar to those shown by human serum albumin for similar compounds. Preliminary studies on an extraction column were promising for the development of an SPE system and for its application in food matrices.     

Synthesis of some 4-substituted-2-(o-halogenophenyl)-1,2,3-triazoles

Mesoaldehyde 1,3-dioxime was treated with either 2,4,6-trichlorophenyl- (a), o-fluorophenyl- (b), or o-bromophenyl- (c) hydrazine to give the corresponding mesoaldehyde 1,3-dioxime-2-halogenophenylhydrazones (1a,b,c). The latter were O-acetylated with acetic anhydride, and cyclized to triazole 4-oximes (3b, c) or triazole 4-O-acetyloximes ( 6a,b,c ) with cesium carbonate, then converted to nitriles ( 7a,b,c ) by refluxing with acetic anhydride followed by pyrolysis, or to aldehydes ( 4a,b,c ) by hydrolysis. The nitriles ( 7a,b,c ) were also converted to acids ( 9a,b,c ), esters ( 10a,b,c ), amides ( 8a,c ), an alcohol (11a), and an amine ( 12a ). In addition, tetrazoles of two types were prepared. The first ( 13d,e ) were obtained from the acid chlorides by the action of 5-aminotetrazole, whereas the second ( 14f ) was produced from the respective nitrile by the action of ammonium azide.     

High performance liquid chromatography post-column chemiluminescence determination of sulfonamide residues in milk at low concentration levels using bis[4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)phenyl] oxalate as chemiluminescent reagent

The determination of seven sulfonamides by means of HPLC with chemiluminescence detection is proposed for the first time. The analytes are derivatized with fluorescamine, separated and subsequently they participate in the post-column chemiluminescence (CL) peroxyoxalate system using imidazole as a catalyst. Among the different peroxyoxalates tested, bis[4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)phenyl] oxalate provides higher sensitivities and stabilities, avoiding precipitation problems. A rigorous optimization of the significant variables by means of experimental designs has been developed in order to reconcile the chromatographic conditions with the CL reaction. The method provides detection limits in the low microgl(-1) range and has been satisfactorily applied to the analysis of spiked raw milk samples.     

Crystal structure of dicyclohexano-18-crown-6 potassium 2-nitrophenoxide

The first crystal structure of a potassium cation complex with dicyclohexano-18-crown-6 is reported. The potassium 2-nitrophenoxide complex ofsyn-cis-syn dicyclohexano-18-crown-6 crystallizes in the triclinic space group P with cell constantsa=8.604(2),b=10.772(4),C=16.123(5)Å, =73.86(3)°,=77.61(3)°, =82.68(3)° andZ=2 forD _c =1.31 g cm^–3. Least-squares refinement based on 2742 observed reflections led to a final conventionalR value of 0.040. Dicyclohexano-18-crown-6 has the shape of a saddle with the potassium cation sitting at the saddlepoint. The structure of the 2-nitrophenoxide anion is dominanted by the quinoid resonance contributor. Because the complex is devoid of significant intercomplex interactions, it is a prototypical 1:1:1 complex. Supplementary Data relating to this article are deposited with the British Library as Supplementary Publication No. SUP 82043 (26 pages).Now Mrs. K. M. Balo.     

Aerobic oxidation of methyl p-tolyl sulfide catalyzed by a remarkably labile heteroscorpionate RuII-aqua complex,fac-[RuII(H2O)(dpp)(tppm)]2+

fac-[RuII(Cl)(dpp)(L3)]+ (L3 = tris(pyrid-2-yl)methoxymethane (tpmm) = [1A]+ and tris(pyrid-2-yl)pentoxymethane (tppm) = [1B]+ and dpp = di(pyrazol-1-yl)propane) rapidly undergo ligand substitution with water to form fac-[RuII(H2O)(dpp)(L3)]2+ (L3 = tpmm = [2A]2+ and tppm = [2B]2+). In the structure of [2A]2+, the distorted octahedral arrangement of ligands around Ru is evident by a long Ru(1)-O(40) of 2.172(3) A and a large angle O(40)-Ru(1)-N(51) of 96.95(14) degrees . The remarkably short distance between O(40) of H2O and H(45a) of dpp confirms the heteroscorpionate ligand effect of dpp on H2O. [2B]2+ aerobically catalyzes methyl p-tolyl sulfide to methyl p-tolyl sulfoxide in 1,2-dichlorobenzene at 25.0 +/- 0.1 degrees C under 11.4 psi of O2. Experimental facts in support of this aerobic sulfide oxidation are the absence of H2O2 and the oxidative reactivity of the putative Ru(IV)-oxo intermediate toward methyl p-tolyl sulfide, 2-propanol, and allyl alcohol. This study provides the first documented example of aerobic-sulfide oxidation catalyzed by the remarkably labile heteroscorpionate Ru(II)-aqua complex without the formation of a highly reactive peroxide as an intermediate.     

Evaluation of a single-monochromator diode array spectroradiometer for sunbed UV-radiation measurements

The suitability of a new technology single-monochromator diode array spectroradiometer for UV-radiation safety measurements, in particular for sunbed measurements, was evaluated. The linearity, cosine response, temperature response, wavelength scale, stray-light and slit function of the spectroradiometer were determined and their effects on the measurement accuracy evaluated. The main error sources were stray-light and nonideal cosine response, for which correction methods are presented. Without correction, the stray-light may reduce the accuracy of the measurement excessively, particularly in the UV-B range. The expanded uncertainty of the corrected UV measurements is estimated to be 14%, which is confirmed with the comparative measurements carried out with a well-characterized double-monochromator spectroradiometer. The measurement accuracy is sufficient for sunbed measurements, provided that all corrections described above have been done and the user of the instrument has a good understanding of the instrument's operating principles and potential error sources. If these requirements are met, the tested spectroradiometer improves and facilitates market surveillance field measurements of sunbeds.