Purification and characterization of recombinant pyrrolidone carboxyl peptidase of Bacillus subtilis.

Bacillus subtilis pyrrolidone carboxyl peptidase (Pcp) overexpressed in Escherichia coli was purified to homogeneity in less than 12 h using ammonium sulphate precipitation and hydrophobic interaction chromatography. The enzyme, which removes amino-terminal L-pyroglutamic acid from peptides, appears to be a tetramer of 25,200 molecular mass subunits. The protein cross-reacted with polyclonal antibodies raised against Pcp from Streptococcus pyogenes. The overexpressed enzyme exhibits an absolute substrate specificity towards N-terminal pyroglutamyl residues with a Michaelis constant of 1.04 mM for L-pyroglutamyl-beta-naphthylamide. The enzyme could be used for the removal of pyroglutamyl residues that block amino termini of proteins and peptides before performing Edman sequential degradation.     

Synthesis, structure, and characterization of new mononuclear Mn(II) complexes. Electrochemical conversion into new oxo-bridged Mn(2)(III,IV) complexes. Role of chloride ions

Two Mn(II) complexes are isolated and X-ray characterized, namely, cis-[(L(2))Mn(II)(Cl)(2)] (1) and [(L(3))Mn(II)Cl(OH(2))](ClO(4)) (2(ClO(4))), where L(2) and L(3) are the well-known tetradentate N,N'-dimethyl-N,N'-bis(2-pyridylmethyl)ethane-1,2-diamine and N,N'-dimethyl-N,N'-bis(2-pyridylmethyl)propane-1,3-diamine ligands, respectively. The crystal structure reveals that whereas the ligand L(2) is in the cis-alpha conformation in complex 1, the ligand L(3) is in the more unusual cis-beta conformation in 2. EPR spectra are recorded on frozen solutions for both complexes and are characteristic of Mn(II) species. Electrochemical behaviors are investigated on acetonitrile solution for both complexes and show that cation 2 exists as closely related Mn(II) species in equilibrium. For both complexes exhaustive bulk electrolyses of acetonitrile solution are performed at oxidative potential in various experimental conditions. In the presence of 2,6-lutidine and after elimination of chloride ligands, the formation of the di-mu-oxo mixed-valent complexes [(L(2))Mn(III)(mu-O)(2)Mn(IV)(L(2))](3+) (3a) and [(L(3))Mn(III)(mu-O)(2)Mn(IV)(L(3))](3+) (4) is confirmed by UV-vis and EPR spectroscopies and cyclic voltammetry. In addition crystals of 4(ClO(4))(3) were isolated, and the X-ray structure reveals the cis-alphaconformation of L(3). In the absence of 2,6-lutidine and without elimination of the exogenous chloride ions, the electrochemical oxidation of 1 leads to the formation of the mononuclear Mn(III) complex, namely, [(L(2))Mn(III)(Cl)(2)](+) (5), as confirmed by UV-vis as well as parallel mode EPR spectroscopy and cyclic voltammetry. In the same conditions, the electrochemical oxidation of complex 2 is more intricate, and a thorough analysis of EPR spectra establishes the formation of the binuclear mono-mu-oxo mixed-valent [(L(3))ClMn(III)(mu-O)Mn(IV)Cl(L(3))](3+) (6) complexes. Electrochemical conversion of Mn(II) complexes into mixed-valent Mn(2)(III,IV) oxo-bridged complexes in the presence of 2,6-lutidine is discussed. The role of the chloride ligands as well as that of L(3) in the building of oxo bridges is discussed. Differences in behavior between L(2) and L(3) are commented on.     

Supramolecular organization of alpha,alpha'-disubstituted sexithiophenes

The self-assembly of alpha,alpha'-linked sexithiophenes with chiral and achiral penta(ethylene glycol) chains attached at the alpha-positions of the terminal rings, that is, 2,2':5',2':5',2':5',2':5',2'-sexithiophene-5,5'-dicarboxylic acid-2S)-2-methyl-3,6,9,12,15-pentaoxahexadecyl ester (1) and 2,2':5',2':5',2':5',2':5',2'-sexithiophene-5,5'-dicarboxylic acid-3,6,9,12,15-pentaoxahexadecyl ester (2), respectively is described. Analysis of the UV/vis, fluorescence, circular dichroism, and circular polarization of luminescence spectroscopic data shows that these compounds form chiral aggregates in polar solvents and in the solid state. In n-butanol aggregation occurs at temperatures below 30 degrees C, while above this threshold temperature the aggregates break up without an intermediate disordered state of aggregation, and the compounds are molecularly dissolved. The "melting temperature" of the aggregates depends on the concentration of sexithiophene, indicating that the optical changes observed are a result of intermolecular processes. Mass spectrometric measurements reveal that 1 and 2 can form mixed aggregates. Analysis of the optical spectra reveals that in these mixed aggregates, chiral 1 molecules act as "sergeants" to direct the packing of the "soldiers" 2, illustrating cooperativity within the columns. In water, the same type of chiral aggregates are formed as in n-butanol below 30 degrees C; however, these aggregates are still present, but the chirality is lost above 30 degrees C. In spin-coated films of 1 chiral aggregates are present. AFM studies show that 1 self-organizes into chiral fiberlike structures in the solid state. Furthermore both 1 and 2 display thermotropic liquid crystalline behavior between 180 and 200 degrees C.     

Microphase separation of cationic poly(N-isopropylacrylamide) copolymers in water: Effect of the migration of charges

The structures of aqueous copolymer solutions have been examined through small angle neutron scattering. The copolymers contained mostly N-isopropylacrylamide (NIPAM) monomers. Poly (NIPAM) solutions have a lower critical solution temperature (LCST), above which the macromolecules separate from water. A small fraction of ionizable N,N-[(dimethylamino) propyl] methacrylamide (MADAP) monomers was introduced into the macromolecules. This had dramatic consequences on the solution behavior at temperatures above the LCST of PNIPAM, where phase separation would have been expected for the homopolymer. When all MADAP monomers were ionized, it was found that the solutions resisted the phase separation. At short spatial scales, the chains were collapsed but at large scales they formed branched aggregates that did not separate out of water. When only half of the MADAP monomers are ionized, the electrical charges were able to redistribute themselves along the chains. In this case, the rise in temperature caused a microphase separation where the electrical charges were relocated on a fraction of the chains that remained in solution.The other chains (or section of chains) formed large nodules of a polymer rich phase.     

Effect of gas pressure and pulse length on the isotopically selective multiphoton dissociation of CF3Cl under CO2 laser pulses

The multiphoton dissociation of CF₃Cl induced by TEA-CO₂ laser pulses has been studied in a focused beam geometry. TheR(10) [00°1–02°0] ( ) laser line was used, so as to dissociate preferentially the minor isotopic component¹³CF₃Cl. The isotopic selectivityS and the dissociation probability per pulse ω were measured in the pressure range between 0.25 and 8 Torr. With short laser pulses (90 ns FWHM),S is found to increase slightly with gas pressure up to 2 Torr, and ω, to increase almost linearly over the whole pressure range studied. A schematic model is proposed which satisfactorily explains these results if the transition rates across the energy level spectrum of the CF₃Cl molecules are assumed to increase with gas pressure.     

Experiments in mixed-integer linear programming using pseudo-costs

This paper discusses heuristic branch and bound methods for solving mixed integer linear programming problems. The research presented on here is the follow on to that recorded in [3].After a resumé of the concept of pseudo-costs and estimations, new heuristic rules for generating a tree which make use of pseudo-costs and estimations are presented. Experiments have shown that models having a low percentage of integer variables behave in a radically different way from models with a high percentage of integer variables. The new heuristic rules seem to apply generally to the first type of model.Later, other heuristic rules are presented that are used with models having a high percentage of integer variables and with models having a special structure (models including special ordered sets.)The rules introduced here have been implemented in the IBM Mathematical Programming System Extended/370. They are used to solve large mixed integer linear programming models.Numerical results that permit comparisons to be made among the different rules are provided and discussed.     

Extrathermodynamic study of surface diffusion in reversed-phase liquid chromatography with silica gels bonded with alkyl ligands of different chain lengths

Surface diffusion on adsorbents made of silica gels bonded to C1, C4, C8, and C18 alkyl ligands was studied in reversed-phase liquid chromatography (RPLC) from the viewpoints of two extrathermodynamic relationships: enthalpy-entropy compensation (EEC) and linear free-energy relationship (LFER). First, the values of the surface diffusion coefficient (D(s)), normalized by the density of the alkyl ligands, were analyzed with the modified Arrhenius equation, following the four approaches proposed in earlier research. This showed that an actual EEC resulting from substantial physicochemical effects occurs for surface diffusion and suggested a mechanistic similarity of molecular migration by surface diffusion, irrespective of the alkyl chain length. Second, a new model based on EEC was derived to explain the LFER between the logarithms of D(s) measured under different RPLC conditions. This showed that the changes of free energy, enthalpy, and entropy of surface diffusion are linearly correlated with the carbon number in the alkyl ligands of the bonded phases and that the contribution of the C18 ligand to the changes of the thermodynamic parameters corresponds to that of the C10 ligand. The new LFER model correlates the slope and intercept of the LFER to the compensation temperatures derived from the EEC analyses and to several parameters characterizing the molecular contributions to the changes in enthalpy and entropy. Finally, the new model was used to estimate D(s) under various RPLC conditions. The values of D(s) that were estimated from only two original experimental D(s) data were in agreement with corresponding experimental D(s) values, with relative errors of approximately 20%, irrespective of some RPLC conditions.     

Adhesion and proliferation of cells on new polymers modified biomaterials

Up to today, several techniques have been used to maintain cells in culture for studying many aspects of cell biology and physiology. More often, cell culture is dependent on proper anchorage of cells to the growth surface. Poly-l-lysine is commonly used as adhesive molecule. In this study, we present, as an alternative to poly-l-lysine, new polymer film substrates, realized by electropolymerization of different monomers on fluorine-doped tin oxide (FTO) surfaces since electropolymerization is a good method to coat selectively metallic or semiconducting electrodes with polymer films. So, the adhesion, proliferation and morphology of rat neuronal cell lines were investigated on polymer treated surfaces. Several amine-based biocompatible polymers were tested: polyethyleneimine (PEI), polypropyleneimine (PPI), polypyrrole (PPy) and poly(p-phenylenediamine) (PPPD). These polymer films were coated on FTO surfaces by electrochemical oxidation. After 8 h in a culture medium, a high percentage of cells was found to be attached to PEI and PPI compared to the other polymers and to the reference surfaces (glass and FTO uncovered). After 24 and 72 h in the culture medium, cells were found to proliferate faster on PEI and PPI than on other polymers and reference surfaces. Consequently, cells have a greater fold expansion on PEI and PPI than on PPPD, PPy or glass and FTO uncoated. From these results, we deduce that PEI and PPI can be useful as coating surface to cultivate neuronal cells.     

The use of two-dimensional gel electrophoresis in the analysis of organ-specific maize proteins

Two-dimensional gel electrophoresis with non-equilibrium pH gradient electrophoresis in the first dimension and sodium dodecyl sulfate-polyacrylamide gels in the second dimension has been used for the analysis of organ-specific proteins in maize. The method has been used to study the whole protein pattern of developing organs and adult leaves as well as protein patterns of in vitro translation. Examples of two-dimensional immunoblotting and in vitro translation of endosperm-specific proteins are also shown. Two-dimensional gel electrophoresis appears as an essential analytical step in the identification of organ-specific proteins and for the detection of the protein products related to organ-specific genes identified by other means.     

Highly stereoselective Friedel-Crafts type cyclization. Facile access to enantiopure 1,4-dihydro-4-phenyl isoquinolinones

The present report describes a stereoselective synthesis of 1,4-dihydro-4-phenyl isoquinolinones 5 based on a stereoselective Friedel-Crafts type cyclization. Cyclization precursors 1 were prepared in two steps, from the readily available (S)-mandelic acid, in 60-80% overall yield. The stereoselective electrophilic cyclization was accomplished in 20-86% yield and with 20-97% ee. In the course of this work, the presence of the amide carbonyl was found to be particularly important to guarantee a stereospecific process during the electrophilic aromatic substitution.