Presence of thiamine pyrophosphate in mammalian peroxisomes

Background

Thiamine pyrophosphate (TPP) is a cofactor for 2-hydroxyacyl-CoA lyase 1 (HACL1), a peroxisomal enzyme essential for the α-oxidation of phytanic acid and 2-hydroxy straight chain fatty acids. So far, HACL1 is the only known peroxisomal TPP-dependent enzyme in mammals. Little is known about the transport of metabolites and cofactors across the peroxisomal membrane and no peroxisomal thiamine or TPP carrier has been identified in mammals yet. This study was undertaken to get a better insight into these issues and to shed light on the role of TPP in peroxisomal metabolism.

Results

Because of the crucial role of the cofactor TPP, we reanalyzed its subcellular localization in rat liver. In addition to the known mitochondrial and cytosolic pools, we demonstrated, for the first time, that peroxisomes contain TPP (177 ± 2 pmol/mg protein). Subsequently, we verified whether TPP could be synthesized from its precursor thiamine, in situ, by a peroxisomal thiamine pyrophosphokinase (TPK). However, TPK activity was exclusively recovered in the cytosol.

Conclusion

Our results clearly indicate that mammalian peroxisomes do contain TPP but that no pyrophosphorylation of thiamine occurs in these organelles, implying that thiamine must enter the peroxisome already pyrophosphorylated. Consequently, TPP entry may depend on a specific transport system or, in a bound form, on HACL1 translocation.     

H2O nucleation around Au+

First principles electronic structure calculations have been carried out to investigate the ground state geometry, electronic structure, and the binding energy of [Au(H2O)n]+ clusters containing up to 10 H2O molecules. It is shown that the first coordination shell of Au+ contains two H2O molecules forming a H2O-Au+-H2O structure with C2 symmetry. Subsequent H2O molecules bind to the previous H2O molecules forming stable and fairly rigid rings, each composed of 4 H2O molecules, and leading to a dumbbell structure at [Au(H2O)8]+. The 9th and the 10th H2O molecules occupy locations above the Au+ cation mainly bonded to one H2O from each ring, leading to structures where the side rings are partially distorted and forming structures that resemble droplet formation around the Au+ cation. The investigations highlight quantum effects in nucleation at small sizes and provide a microscopic understanding of the observed incremental binding energy deduced from collision induced dissociation that indicates that [Au(H2O)n]+ clusters with 7-10 H2O molecules have comparable binding energy. The charge on the Au+ is shown to migrate to the outside H2O molecules, suggesting an interesting screening phenomenon.     

Liquid chromatography/tandem mass spectrometry determination of organophosphorus flame retardants and plasticizers in drinking and surface waters

A liquid chromatography/electrospray ionization tandem mass spectrometric method for analyzing organophophorus flame retardants and plasticizers in drinking and environmental waters was developed. Five alkyl phosphates, three chlorinated alkyl phosphates, two aryl phosphate and triphenylphosphine oxide were selected for this study. These compounds were extracted from water samples by a hydrophilic polymeric solid-phase extraction cartridge. Accuracy and precision were evaluated analyzing 0.5 L of water samples spiked at concentrations of 10 and 100 ng/L for drinking water and at 300 and 1000 ng/L for river water. Except for trimethyl phosphate, analyte recoveries were better than 80%, and were not dependent on the type of aqueous matrix in which they were dissolved. At the spike levels considered, within-day precision was between 3 and 12% for tap water and between 4 and 14% for river water, and estimated method quantification limits ranged from 0.2 to 3.9 ng/L. A short survey conducted by analyzing some river water samples (River Tiber) ascertained the presence of ten organophosphorus compounds at concentration levels ranging from a few nanograms per liter to 323 ng/L for tris(2-butoxyethyl) phosphate.     

Determination of aflatoxins in olive oil by liquid chromatography-tandem mass spectrometry

A liquid chromatography-tandem mass spectrometric with electrospray ionization (LC/ESI-MS/MS) method for determining the four naturally occurring aflatoxins (AFs) B1, B2, G1, and G2 in olive oil is proposed. AFs were extracted from oil sample by means of matrix solid phase dispersion (MSPDE), utilizing C18 as dispersing material. No further purification step, such as lipid removal, was performed. Aflatoxin M1, the hepatic metabolite of AFB1, was employed as internal standard. Olive oil extract was analyzed by LC/ESI-MS/MS in positive ionization mode, with multireaction monitoring acquisition. Due to a signal suppression ranging between 4 and 23%, quantitation was performed by matrix-matched calibration curves. The regression line coefficients of determination were above 0.9991. Sample recoveries ranged from 92 to 107%, with relative standard deviations below 13% for spiking levels between 0.5 and 5 ng g⁻¹; method quantification limits ranged between 0.04 and 0.12 ng g⁻¹. The developed LC/ESI-MS/MS method, although not as sensitive as LC coupled to fluorescence detection, is rapid, selective, accurate and precise, thus it can be used as confirmatory assay. The MSPDE appears suitable for application to other oleaginous matrices and for multiresidue investigation.     

Ferrocene derivatives supported on poly(N-vinylpyrrolidin-2-one) (PVP): Synthesis of new water-soluble electrochemically active probes for biomolecules

Carboxy-terminated polyvinylpyrrolidin-2-one (PVP) has been used as a new water-soluble and biocompatible polymeric support for a series of ferrocene labeled amino acid and peptide nucleic acid (PNA) monomer derivatives 4-7. The organometallic polymer-conjugates thus obtained are new and potentially useful as water-soluble electrochemically active probes for biomolecules. In view of such application, their electrochemical activity has been evaluated and has proved very high notwithstanding the complexity and bulkiness of the molecule, affording detection limits down to 10⁻⁸ M in the aqueous medium.     

Origin of enantioselectivity in palladium-catalyzed asymmetric allylic alkylation reactions using chiral N,N-ligands with different rigidity and flexibility

The chiral bidentate-N,N ligands, (S(a))-1, (S(a))-2, (S,S)-3 and (S,S)-4, were synthesized. They were shown to contain rigid 2-pyridinyl or 8-quinolinyl building blocks and the C(2)-symmetric chiral frameworks trans-2,5-dimethylpyrrolidinyl or (S)-(+)-2,2'-(2-azapropane-1,3-diyl)-1,1'-binaphthalene. In the (S(a))-2, and (S,S)-4 ligands pair, the 8-quinolinyl skeleton is directly bonded to the C(2)-symmetric chiral frameworks (S)-(+)-2,2'-(2-azapropane-1,3-diyl)-1,1'-binaphthalene or trans-2,5-dimethylpyrrolidinyl. This feature induces rigidity in this pair of ligands upon the N,N-framework. However, this does not occur for the (S(a))-1 and (S,S)-3 ligands, in which the presence of the -CH(2)- spacer between the frameworks bearing the nitrogen atom donors gives greater flexibility to the ligand. A further difference between the pairs of ligands is significant from the electronic properties of the chiral framework N-donor atom. The coordinating properties and the specific steric structural features of the (S(a))-1, (S(a))-2, (S,S)-3, and (S,S)-4 ligands are explained by their reactions with the [Pd(PhCN)(2)Cl(2)] and [Pd(eta(3)-PhCHCHCHPh)(mu-Cl)](2) substrates, in which the reported ligands form chelate complexes, with the exception of (S(a))-2, which failed to react with [Pd(eta(3)-PhCHCHCHPh)(mu-Cl)](2). The ligands were used in the palladium-allyl catalyzed substitution reaction of 1,3-diphenylallyl acetate with dimethylmalonate, with the best result being obtained using the (S(a))-1 ligand, giving the substitution product 2-(1,3-diphenylallyl)dimethylmalonate with an enantiomeric excess of 82% in the S form and a yield of 96%. The work demonstrates that in the presence of a steric ligand control, the electronic properties of the ligand donor atoms play a role though not significant in determining the enantioselectivity of palladium(II) catalyzed allylic substitution reactions. The results of the catalytic reaction do not provide a convincing explanation considering the coordinated chiral ligand features, as rigidity or flexibility and electronic properties of the N-donor atoms. A rationalization of the results is proposed on the basis of NMR studies and DFT calculation on the cationic complexes [Pd(eta(3)-PhCHCHCHPh)(N-N*)]CF(3)SO(3), (N-N* = (S(a))-1, 9; (S,S)-3, 10; (S,S)-4, 11).     

Experimental Investigations on Highly Conducting Solid Bio-Polymer Electrolytes Based on Latex of Calotropis gigantea Plant

The study has prepared highly conducting polymer electrolyte films using solution cast technique with poly(vinylidene fluoride-co-hexafluoropropylene) PVDF-HFP, mixture of ethylene carbonate (EC), and propylene carbonate (PC) as plasticizer and latex of Calotropis gigantea (CGL) as an ionic source. In this study, four films are prepared using PVDF-HFP:CGL in ratio 1:1 with the increasing concentration of EC+PC as 1, 2, 3, and 4 M named as 1:1:1, 1:1:2, 1:1:3, 1:1:4. The prepared polymer electrolyte is examined by polarized optical microscopy (POM), elemental dispersive X-ray technique (EDX), and complex impedance spectroscopy. EDX and POM are studied for the surface morphology of all prepared samples and to investigate the porous nature of films. The enhancement in ionic conductivity occurs due to CGL and increasing amount of EC-PC. Conductivity of highest composition (1:1:4) polymer electrolyte film is found to be ≈10⁻³ S cm⁻¹. The optimized polymer electrolyte film is considered as a promising candidate for application in supercapacitors.     

Biodegradable hybrid polymer films based on poly(vinyl alcohol) and collagen hydrolyzate

Hybrid blends of poly(vinyl alcohol) (PVA) and collagen hydrolyzate (CH), an added value waste from leather indutry, have been converted by blown molding extrusion, to environmentally degradable films. Blown extruded films comprising 5-15% of CH, were tested as sel fertilizing mulching films and analyzed for their propensity to enviromental degradation. PVA/CH films rapidly disintegrate when buried in soil, and resulted promising for application such as transplanting films, with additional fertilizing action of CH.