Normal human dermal fibroblasts: proteomic analysis of cell layer and culture medium.

Proteins present within the cell layer and those released in the cell medium from in vitro cultured normal human dermal fibroblasts were separated and characterized in terms of their isoelectric point and molecular weight, by two-dimensional (2-D) gel electrophoresis. All spots in the synthetic gel were firstly analyzed by the Melanie 3 software and compared with those of breast cancer cells, colorectal epithelial cells, HL60, lymphoma cells, and platelets, already available on-line. From the identification of 144 spots from both the cell layer and the medium, we were able to recognize 89 different proteins, since a certain number of spots represented different isoforms of the same molecule. Identifications were performed by matching with on-line 2-D databases, and by matrix assisted laser-desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS), in order to confirm the identification by matching, or to identify new proteins. The procedure we used allows (i) to design a highly reproducible reference map of the proteome of adult human normal fibroblasts in culture, (ii) to evaluate protein species produced in the cell layer as well as those released in the culture medium, and (iii) to compare data from gel matching with those obtained by MS. This work represents an essential step for a better knowledge of mesenchymal cells, given the widespread use of this cell type in both clinical and experimental investigations.     

Isomers of C20: an energy profile III

Semiempirical calculations, at the PM3 level provided within the Winmopac v2.0 software package, are used to geometrically optimize and determine the absolute energies (heats of formation) of a variety of C(20) isomers that are predicted to exist in and around the ring and cage isomers. Using the optimized Cartesian coordinates for the ring and the cage isomers, a saddle-point calculation was performed. The resulting energy profile, consisting of a series of peaks and valleys, is used as a starting point for the identification and location of fifteen additional isomers of C(20) that are predicted to be energetically stable, both via geometry optimizations and force constant analysis. These additional isomers were subsequently determined to lie adjacent to one another on the potential surface and establish a step-wise transformation between the ring and the cage. Transition-state optimization of the Cartesian coordinates at the saddle point between adjacent isomers was performed to quantify the energy of the transition state. The step-wise process from one isomer to another, which extends out over the three-dimensional surface, is predicted to require approximately 15% less energy than that of the direct, two-dimensional transformation predicted in the bowl-cage profile. However, the net atomic rearrangement for the step-wise process is about four times greater than that of the direct process. Although less in energy, the amount of atomic rearrangement in the step-wise process would make the occurrence of such a route prohibitive. Utilizing the direct distance separating the three primary isomers (ring, bowl, cage), the method of triangulation is performed to quantitatively position other C(20) structures on the potential surface, relative to the ring, bowl, and cage isomers.     

Mechanistic studies on the formation and reactivity of dioxygen adducts of diiron complexes supported by sterically hindered carboxylates

Dioxygen activation by enzymes such as methane monooxygenase, ribonucleotide reductase, and fatty acid desaturases occurs at a nonheme diiron active site supported by two histidines and four carboxylates, typically involving a (peroxo)diiron(III,III) intermediate in an early step of the catalytic cycle. Biomimetic tetracarboxylatodiiron(II,II) complexes with the familiar "paddlewheel" topology comprising sterically bulky o-dixylylbenzoate ligands with pyridine, 1-methylimidazole, or THF at apical sites readily react with O(2) to afford thermally labile peroxo intermediates that can be trapped and characterized spectroscopically at low temperatures (193 K). Cryogenic stopped-flow kinetic analysis of O(2) adduct formation carried out for the three complexes reveals that dioxygen binds to the diiron(II,II) center with concentration dependences and activation parameters indicative of a direct associative pathway. The pyridine and 1-methylimidazole intermediates decay by self-decomposition. However, the THF intermediate decays much faster by oxygen transfer to added PPh(3), the kinetics of which has been studied with double mixing experiments in a cryogenic stopped-flow apparatus. The results show that the decay of the THF intermediate is kinetically controlled by the dissociation of a THF ligand, a conclusion supported by the observation of saturation kinetic behavior with respect to PPh(3), inhibition by added THF, and invariant saturation rate constants for the oxidation of various phosphines. It is proposed that the proximity of the reducing substrate to the peroxide ligand on the diiron coordination sphere facilitates the oxygen-atom transfer. This unique investigation of the reaction of an O(2) adduct of a biomimetic tetracarboxylatodiiron(II,II) complex provides a synthetic precedent for understanding the electrophilic reactivity of like adducts in the active sties of nonheme diiron enzymes.     

Novel Copolymers of Styrene. 16. Halogen Ring-Disubstituted Methyl 2-Cyano-3-Phenyl-2-Propenoates

Electrophilic trisubstituted ethylenes, ring-disubstituted methyl 2-cyano-3-phenyl-2-propenoates, RPhCH?C(CN)CO₂CH₃, where R is 2,5-dichloro, 3,5-dichloro, 2,3-difluoro, 3-chloro-2-fluoro, 3-chloro-4-fluoro, 4-chloro-3-fluoro, 2-chloro-5-nitro, and 2-chloro-6-nitro were prepared and copolymerized with styrene. The monomers were synthesized by the piperidine catalyzed Knoevenagel condensation of ring-disubstituted benzaldehydes and methyl cyanoacetate, and characterized by CHN analysis, IR, ¹H and ¹³C-NMR. All the ethylenes were copolymerized with styrene (M₁) in solution with radical initiation (ABCN) at 70°C. The compositions of the copolymers were calculated from nitrogen analysis and the structures were analyzed by IR, ¹H and ¹³C-NMR. The order of relative reactivity (1/r₁) for the monomers is 4-Cl-3-F (4.87) > 2,3-F₂ (4.49) > 3-Cl-4-F (3.50) > 3-Cl-2-F (2.96) > 2-Cl-5-NO₂ (2.02) > 2,5-Cl₂ (1.54) > 2-Cl-6-NO₂ (1.00) > 3,5-Cl₂ (0.41). Relatively high T_g of the copolymers in comparison with that of polystyrene indicates a decrease in chain mobility of the copolymer due to the high dipolar character of the trisubstituted ethylene monomer unit. Decomposition of the copolymers in nitrogen occurred in two steps, first in the 200–500ºC range with residue (1.5–34.5% wt), which then decomposed in the 500-800ºC range.     

Novel Copolymers of 4-Fluorostyrene. 11. Some Ring-substituted 1,1-dicyano-2-(1-naphthyl)ethylenes

Novel copolymers of trisubstituted ethylene monomers, ring-substituted 1,1-dicyano-2-(1-naphthyl)ethylenes, RC₁₀H₆CH?C(CN)₂ (where R is H, 2-OCH₃, 4-OCH₃) and 4-fluorostyrene were prepared by solution copolymerization in the presence of a radical initiator (ABCN) at 70°C. The composition of the copolymers was calculated from nitrogen analysis, and the structures were analyzed by IR, ¹H and ¹³C-NMR. The order of relative reactivity (1/r ₁) for the monomers is (5.86) > 2-CH₃O (4.28) > 4-CH₃O (2.87). Relatively high T_g of the copolymers in comparison with that of poly(4-fluorostyrene) indicates a decrease in chain mobility of the copolymer due to the high dipolar character of the trisubstituted ethylene monomer unit. Decomposition of the copolymers in nitrogen occurred in two steps, first in the 200–500°C range with residue (7.3–7.7% wt.), which then decomposed in the 500–800°C range.     

Sensitivity enhancement by exchange mediated magnetization transfer of the xenon biosensor signal

Hyperpolarized xenon associated with ligand derivatized cryptophane-A cages has been developed as a NMR based biosensor. To optimize the detection sensitivity we describe use of xenon exchange between the caged and bulk dissolved xenon as an effective signal amplifier. This approach, somewhat analogous to 'remote detection' described recently, uses the chemical exchange to repeatedly transfer spectroscopic information from caged to bulk xenon, effectively integrating the caged signal. After an optimized integration period, the signal is read out by observation of the bulk magnetization. The spectrum of the caged xenon is reconstructed through use of a variable evolution period before transfer and Fourier analysis of the bulk signal as a function of the evolution time.     

Dispersion measurements using time-of-flight remote detection MRI

Remote detection nuclear magnetic resonance and magnetic resonance imaging can be used to study fluid flow and dispersion in a porous medium from a purely Eulerian point of view (i.e., in a laboratory frame of reference). Information about fluid displacement is obtained on a macroscopic scale in a long-time regime, while local velocity distributions are averaged out. It is shown how these experiments can be described using the common flow propagator formalism and how experimental data can be analyzed to obtain effective porosity, flow velocity inside the porous medium, fluid dispersion and flow tracing of fluid.     

High pressure and thermal pasteurization effects on sweet cherry juice microbiological stability and physicochemical properties

This study evaluated high pressure processing (P1 – 400?MPa/5?min; P2 – 550?MPa/2?min) and thermal pasteurization (TP – 70°C/30?s) effects on sweet cherry juice's microbiological and physicochemical parameters, during four weeks of refrigerated storage. All treatments reduced the microbiological load to undetectable levels not affecting total soluble solids and titratable acidity. The pH increased with all treatments, however, it decreased during storage. Phenols were differently affected: TP increased them by 6%, P1 had no effect while P2 decreased them by 11%. During storage, phenols in control and TP samples decreased by 26% and 20%, P1 samples decreased them by 11% whereas P2 showed no variation. TP had no effect on anthocyanins, while pressure treatments increased them by 8%. Anthocyanins decreased during storage, particularly in the control and P1 (decreasing 41%). All treatments had no effect on antioxidant activity until the 14th day, thereafter high pressure processing samples showed the highest antioxidant activity.