Inhibition of protein ubiquitination by paraquat and 1-methyl-4-phenylpyridinium impairs ubiquitin-dependent protein degradation pathways

Intracytoplasmic inclusions of protein aggregates in dopaminergic cells (Lewy bodies) are the pathological hallmark of Parkinson’s disease (PD). Ubiquitin (Ub), alpha [α]-synuclein, p62/sequestosome 1 and oxidized proteins are major components of Lewy bodies. However, the mechanisms involved in the impairment of misfolded/oxidized protein degradation pathways in PD are still unclear. PD is linked to mitochondrial dysfunction and environmental pesticide exposure. In this work, we evaluated the effect of the pesticide paraquat (PQ) and the mitochondrial toxin 1-methyl-4-phenylpyridinium (MPP+) on Ub-dependent protein degradation pathways. No increase in the accumulation of Ub-bound proteins or aggregates was observed in dopaminergic cells (SK-N-SH) treated with PQ or MPP+, or in mice chronically exposed to PQ. PQ decreased Ub protein content, but not its mRNA transcription. Protein synthesis inhibition with cycloheximide depleted Ub levels and potentiated PQ–induced cell death. Inhibition of proteasomal activity by PQ was found to be a late event in cell death progression, and had no effect on either the toxicity of MPP+ or PQ, or the accumulation of oxidized sulfenylated, sulfonylated (DJ-1/PARK7 and peroxiredoxins) and carbonylated proteins induced by PQ. PQ- and MPP+-induced Ub protein depletion prompted the dimerization/inactivation of the Ub-binding protein p62 that regulates the clearance of ubiquitinated proteins by autophagic. We confirmed that PQ and MPP+ impaired autophagy flux, and that the blockage of autophagy by the overexpression of a dominant-negative form of the autophagy protein 5 (dnAtg5) stimulated their toxicity, but there was no additional effect upon inhibition of the proteasome. PQ induced an increase in the accumulation of α-synuclein in dopaminergic cells and membrane associated foci in yeast cells. Our results demonstrate that inhibition of protein ubiquitination by PQ and MPP+ is involved in the dysfunction of Ub-dependent protein degradation pathways

Enantiomeric separations by use of polymeric surfactant electrokinetic chromatography

This review surveys the enantiomeric separation of drugs by electrokinetic chromatography using polymeric chiral surfactant pseudostationary phases. These phases have recently been shown to provide better mass transfer and increased rigidity and stability than regular micelles in micellar capillary electrophoresis. Characterization of the polymeric chiral surfactants is presented. Solution interactions of the pseudostationary phases via thermodynamics and fluorescence probe studies are evaluated. Also, case studies of enantiomeric separation of drugs using a single amino acid surfactant and the synergistic effect of the addition of γ-cyclodextrin to the buffer is discussed. The use of dipeptide surfactants for chiral drug separations is described as well. Copyright (C) 2000 Elsevier Science B.V

Characterization and thermodynamic studies of the interactions of two chiral polymeric surfactants with model substances: Phenylthiohydantoin amino acids

Analytical ultracentrifugation is used for determination of the molecular weights and the sedimentation coefficients of poly(sodium undecanoyl-L-valinate) (PSUV) and poly(sodium undecanoyl-L-threoninate) (PSUT) at different temperatures. Plots of absorbance as a function of radius indicates that both PSUV and PSUT are highly monodispersed. A method for evaluating the partial specific volumes using density measurements is presented. The partial specific volumes of PSUV are slightly higher than those of PSUT. In addition, the temperature dependence of the retention factor in electrokinetic chromatography was used to estimate the enthalpy, the entropy, and the Gibbs free energy of the surfactant/analyte complexes. Five phenylthiohydantoin-DL-amino acids were separated and each enantiomeric pair was completely resolved. Comparison of the thermodynamic values obtained with PSUV vs PSUT using a van\u27t Hoff relationship suggests that PSUT, with a less favorable free energy change (i.e., less negative A(AG)), generates a more positive entropy change, hence slightly less chiral resolution

Capillary Zone Electrophoresis of Bile Acids with Indirect Photometric Detection

Investigations in our laboratory have demonstrated that capillary zone electrophoresis (CZE) with indirect photometric detection is a viable approach to the separation and identification of free bile acids along with their taurine and glycine derivatives. Various parameters such as pH, organic solvent concentrations, column temperature, and type of chromophore electrolyte were investigated to optimize the electrophoretic separation and to maximize the peak capacity. The quality of separation of bile acids can be dramatically improved by incorporating y-cyclodextrin (γ-CD) in the running electrolyte. This improvement in resolution is accompanied by a decreased migration time, suggestive of an increase in association of γ-CD with bile acids. As a result, a CZE separation of all 15 bile acids was possible in ∼30 min using 5 mM adenosine 5′-monophosphate, 7 mM γ-CD in 75% (v/v) methanol at pH 7.0

Capillary electrophoretic separation of binaphthyl enantiomers with two polymeric chiral surfactants: \u3csup\u3e1\u3c/sup\u3eH-nuclear magnetic resonance and fluorescence spectroscopy study

The use of the water-soluble polymeric chiral surfactants (PCS), sodium N-undecanoyl-L-valinate (poly-L-SUV) and sodium undecanoyl-L-isoleucinate (poly-L-SUI) as buffer additives in electrokinetic chromatography (EKC) afforded the separation of racemic mixtures of 2,2\u27-dihydroxy-1,1\u27-binaphthyl (BOH) and 1,1\u27-binaphthyl-2,2\u27-diyl hydrogen phosphate (BNP). The apparent binding constants of the PCS to the enantiomers of BNP and BOH were obtained through 1H-nuclear magnetic resonance (1H-NMR) titrations and fluorescence spectroscopy, respectively. The 1H-NMR titration studies show that the BNP enantiomers are localized in the hydrophobic micellar pockets of PCS and form complexes of a 1:1 stoichiometry. The binding constants of PCS of BOH were determined from a Benesi-Hildebrand treatment of the fluorescence data. The EKC data corroborate those of the binding constants, supporting the formation of inclusion complexes. A model rationalizing the chiral discrimination of the enantiomers of BNP is proposed based on the intermolecular interactions observed in 1H-NMR data

Chiral separation with dipeptide-terminated polymeric surfactants: The effect of an extra heteroatom on the polar head group

Chiral recognition of two binaphthyl derivatives and three benzodiazepines were studied by use of polymeric surfactants in electrokinetic chromatography. Four specific dipeptide terminated (multichiral) micelle polymers were synthesized for this study. These include poly (sodium-N-undecanoyl-L-alanyl-leucinate)-(poly L-SUAL), poly (sodium-N- undecanoyl-L-valyl-leucinate) (poly L-SUVL), poly (sodium-N-undecanoyl-L- seryl-leucinate) (poly L-SUSL), and poly(sodium-N-undecanoyl-L-threonyl- leucinate) (poly L-SUTL). In addition to the chiral separation study, the physicochemical properties (critical micelle concentration and specific rotation) of each polymer were investigated. The molecular weights of the various dipeptide-terminated micelle polymers were determined using analytical ultracentrifugation. These dipeptide-terminated micelle polymers were designed to study the effect of the extra heteroatom at the polar head group of the micelle polymer (i.e., poly L-SUSL compared to poly L-SUAL and poly L-SUTL compared to poly L-SUVL) on the enantiomeric separation of the binaphthyl derivatives and benzodiazepines. The synergistic effect of three chiral centers (poly L-SUTL) provided improved resolution over that of two chiral centered dipeptide-terminated micelle polymer in the case of (±)- temazepam, (±)-oxazepam, (±)binaphthol, and (±)-binaphthol phosphate. The chiral recognition mechanisms in these cases were additionally controlled by the presence of the extra heteroatom located on the polar head group of the micelle polymers

GR 24 enantiomers: Synthesis, NMR spectroscopy, X-ray crystallography, and separation by chiral electrokinetic capillary chromatography

Chiral discrimination of enantiomers of 2-methyl-4-(2-oxo-2,3,3a,8b-tetrahydro-4H-indeno[1,2b]furan-3-yl-idenemethoxy )but-2-en-4-olide (commonly referred to as GR 24) by three polymeric chiral surfactants (PCS) is studied by use of chiral polymeric surfactant capillary electrophoresis (CPSCE). The CPSCE results indicate that the optical configurations of valine residues on the PCS backbone affect chiral resolution and elution order of GR24 stereoisomers. The L- and D-forms of poly(sodium N-undecanoyl valinate) provide baseline separation of all four enantiomers while the DL-form separates diastereomers of GR 24 (1). A model is presented rationalizing the migration behavior and chiral resolution of 1 in CPSCE. The actual configuration of the stereogenic centers of GR 24 and 3-[(2,5-dihydro-3-methyl-2-oxo-5-furanyl)oxo]-methylene-3,3a,6,6a-tetrahydro- 2H-cyclopenta[b]furan-2-one (GR 7) is established by a concerted application of high-resolution nuclear magnetic resonance spectroscopy and X-ray crystallography