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.     

Transition metal-catalysed sequences of nucleophilic addition

Summary In presence of nickel or palladium catalysts, nucleophiles can attack vinylcyclopropanes with concomitant ring cleavage. With palladium catalysts in presence of appropriate substituents the terminal carbon atom of the resulting open chain is able to add to two molecules of a conjugated diene giving rise to long-chain unsaturated compounds.     

Isoelectric focusing identification of four freshwater fish commercially labeled "perch"

Isoelectric focusing (IEF) was used to distinguish 4 freshwater fish species that are sold in the European Union under the generic label of "perch": Perca fluviatilis (European perch), Lates niloticus (Nile perch), Stizostedion lucioperca (European pikeperch), and Morone chrysops x saxatilis (Sunshine bass). These species have different commercial values but are easily interchangeable because they are sold already filleted, in view of the numerous bones of the whole fish. IEF of the water-soluble proteins extracted from fish muscle resulted in species-specific patterns. None of the bands was common to all 4 species. Intraspecies polymorphism was low and did not concern the bands identified as characteristic of the species.     

Immunodetection of lactosylated proteins as a useful tool to determine heat treatment in milk samples

This paper reports the optimisation of a competitive immunoassay (ELISA) to detect lactosylated proteins in milk samples. The assay employs monoclonal antibodies for lactosylated proteins produced in our laboratory and requires no pre-treatment of the samples other than a dilution step. Monoclonal antibodies were fully characterised in terms of selectivity and cross-reactivity with structurally related molecules and used in a competitive assay format with lactosylated standard proteins (lactosylated ovalbumin). The detection limit for lactosylated ovalbumin was 0.015 microgram ml-1 and the working range was from 0.010 to 40 micrograms ml-1. The data obtained indicate that the ELISA developed is applicable to diluted milk samples and is able to distinguish between milk samples that have undergone different heat treatments (UHT and pasteurised milk).     

Expression of the small tyrosine phosphatase (Stp1) in Saccharomyces cerevisiae: a study on protein tyrosine phosphorylation

Small tyrosine phoshatase 1 (Stp1) is a Schizosaccharomyces pombe low-molecular-mass phosphotyrosine-phosphatase 50% identical to Saccharomyces cerevisiae Ltp1. In order to investigate the role of Stp1 in yeast, a mutant was generated having the characteristic of a dominant negative molecule. Changes in protein tyrosine phosphorylation in S. cerevisiae proteome in response to Stp1 or its dominant negative mutant expression were analyzed by high-resolution two-dimensional (2-D) electrophoresis. The most remarkable result is the modification by phosphorylation on tyrosine of several proteins involved in carbohydrate metabolism. Twelve proteins were identified on the basis of their positions in the anti-phosphotyrosine immunoblot of the 2-D electrophoresis. Ten of these present tyrosyl residues that are within the consensus sequence for protein kinase CK2 (casein kinase-2). These data open the possibility for the identification of Stp1 substrates in yeast and provide hints about the nature of tyrosine phosphorylating agents in yeast and in other organisms where bona fide tyrosine kinases are lacking.     

Synthesis and characterization of gold nanoparticles stabilized by palladium(II) phosphine thiol

This work is focused on the synthesis of innovative hybrids made by linking gold nanoparticles to protected organometallic Pd(II) thiolate. The organometallic protected Pd(II) thiolate, i.e. trans-thioacetate-ethynylphenyl-bis(tributylphosphine)palladium(II) has been synthesized, in situ deprotected and linked to Au nanoparticles. In this way new hybrid, with a direct link between Pd(II) and Au nanoparticles through a single S bridge, has been isolated. The combination of the organometallic Pd(II) thiol with gold nanoparticles allows the enhancement and tailoring of electronic and optical properties of the new organic-inorganic nano-compound. Single-crystal gold nanoparticles, uniform in shape and size were obtained by applying a modified two-phase method (improved Brust-Schiffrin reaction). In addition, the chemical environment of the Au nanoparticles was investigated and a covalent bonding between Au nanoparticles and the organometallic thiols was observed.     

Raman and SERS study on cimetidine–metal complexes with biomedical interest

Cimetidine (cim) is one of the most potent histamine H₂‐receptor antagonists for inhibiting excessive acid secretion caused by histamine; it has been hypothesized that the therapeutic effects can be related to its interactions with metal ions. Raman spectra of the solid cim with Co(II), Cu(II), Ni(II) and Zn(II) metal complexes show that they can adopt two different structures: one is octahedral and the other, with Zn(II), is probably tetrahedral. The octahedral structure appears to be distorted both by the different metal ions as well as by the different anion present. The study was extended to very dilute solutions (ppm range) by using the surface‐enhanced Raman scattering (SERS) technique, mimicking the physiological concentrations of cim and its metal complexes. SERS spectra suggest that, upon the binding of cim to silver colloids, the formation of stable 1:2 cim–metal complexes is excluded, the formation of 1:1 adduct appearing more probable; in this product the metal reaches its total coordination shell by complexion with water molecules. To better explain the binding mechanism of cim to a metal (Ag) surface, we performed theoretical B3LYP calculations on cim alone as well as on cim bonded to an Ag₂ metal cluster in presence of water, observing a sufficiently good agreement between experimental and theoretical wavenumbers. Copyright © 2010 John Wiley & Sons, Ltd.     

13C and 17O chemical shifts and conformational analysis of mono- and di-methyl-substituted thiane 1-oxide and thiane 1,1-dioxide

The ¹³C and ¹⁷O (natural abundance) chemical shifts of several mono- and di-methyl ring-substituted thiane 1-oxides and thiane 1,1-dioxides are reported. The cis and trans isomers of methyl-substituted thiane 1-oxide are readily identified by ¹³C and ¹⁷O NMR. In particular, the ¹⁷O NMR signals of axial SO groups are found several ppm upfield of those of the equatorial counterparts. The proportion of axial and equatorial conformers of thiane 1-oxide in different solvents has been measured by low-temperature ¹³C NMR. In THF the proportion of the axial conformer is higher than in CD₂Cl₂ whereas in CDCl₃ or CHF₂Cl the conformational preference is reversed and the equatorial conformer is slightly favoured.