Characterization of mature rat oligodendrocytes: a proteomic approach

Oligodendrocytes are glial cells responsible for the synthesis and maintenance of myelin in the central nervous system (CNS). Oligodendrocytes are vulnerable to damage occurring in a variety of neurological diseases. Understanding oligodendrocyte biology is crucial for the dissemination of de- and remyelination mechanisms. The goal of the present study is the construction of a protein database of mature rat oligodendrocytes. Post-mitotic oligodendrocytes were isolated from mature Wistar rats and subjected to immunocytochemistry. Proteins were extracted and analyzed by means of two-dimensional gel electrophoresis and two-dimensional liquid chromatography, both coupled to mass spectrometry. The combination of the gel-based and gel-free approach resulted in confident identification of a total of 200 proteins. A minority of proteins were identified in both proteomic strategies. The identified proteins represent a variety of functional groups, including novel oligodendrocyte proteins. The results of this study emphasize the power of the applied proteomic strategy to study known or to reveal new proteins and to investigate their regulation in oligodendrocytes in different disease models.     

Expression of 25 human ABC transporters in the yeast Pichia pastoris and characterization of the purified ABCC3 ATPase activity

Human ATP-binding cassette (ABC) transporters comprise a family of 48 membrane-spanning transport proteins, many of which are associated with genetic diseases or multidrug resistance of cancers. In this study, we present a comprehensive approach for the cloning, expression, and purification of human ABC transporters in the yeast Pichia pastoris. We analyzed the expression of 25 proteins and demonstrate that 11 transporters, including ABCC3, ABCB6, ABCD1, ABCG1, ABCG4, ABCG5, ABCG8, ABCE1, ABCF1, ABCF2, and ABCF3, were expressed at high levels comparable to that of ABCB1 (P-glycoprotein). As an example of the purification strategy via tandem affinity chromatography, we purified ABCC3 (MRP3) whose role in the transport of anticancer drugs, bile acids, and glucuronides has been controversial. The yield of ABCC3 was 3.5 mg/100 g of cells in six independent purifications. Purified ABCC3, activated with PC lipids, exhibited significant ATPase activity with a Vmax of 82 +/- 32 nmol min-1 mg-1. The ATPase activity was stimulated by bile acids and glucuronide conjugates, reaching 170 +/- 28 nmol min-1 mg-1, but was not stimulated by a variety of anticancer drugs. The glucuronide conjugates ethinylestradiol-3-glucuronide and 17beta-estradiol-17-glucuronide stimulated the ATPase with relatively high affinities (apparent Km values of 2 and 3 microM, respectively) in contrast to bile acids (apparent Km values of >130 microM), suggesting that glucuronides are the preferred substrates for this transporter. Overall, the availability of a purification system for the production of large quantities of active transporters presents a major step not only toward understanding the role of ABCC3 but also toward future structure-function analysis of other human ABC transporters.     

Synthesis of 3-O-methylviridicatin analogues with improved anti-TNF-alpha properties

We synthesized 3-O-methylviridicatin and several analogues of this fungal metabolite. We showed that replacement of the methoxy moiety by a thiomethyl enhanced dramatically its ability to inhibit TNF-alpha secretion. These results strongly suggest that 4-phenyl-3-methylthioquinolinone may provide the basis for the development of new anti-inflammatory agents.     

Perceptual interactions between characteristic notes smelled above aqueous solutions of odorant mixtures

Twenty-two experienced panelists rated odor intensity of aqueous solutions of citral, octen-1-ol-3, and hexanal. The panel assessed unmixed components and mixtures (9 binary and 4 ternary). In sensory sessions dedicated to mixtures (n = 6), evaluation was focused on one target odor, presented at a fixed concentration. All components had lower odor intensity on mixed presentations. In many cases, information obtained from simpler systems was not extended to complex mixtures. In a mixture, the competition between odorant molecules on qualitative aspects (dominance/suppression) imbalanced components contribution, anticipated from the quantitative distribution. Hexanal appeared to be the potentially weaker odorant in paired combinations, whereas octen-1-ol-3 had a lower relative impact on ternary systems. Suppression of the odor of octen-1-ol-3 and a concomitant increase in the odor of hexanal was common to all ternary mixtures. Reciprocal inhibition of octen-1-ol-3 and citral odors through perceptual interactions was suspected. Mutual suppression is suspected to have eased the perception of hexanal intensity.     

Chromosome segregation: spindle mechanics come to life

Chromosome segregation is a mechanical process, and the spindle generates, and is subject to, mechanical force. A recent study probes how the mechanical architecture of the spindle allows it to maintain mechanical integrity despite these forces.     

Covalent cell surface functionalization of human fetal osteoblasts for tissue engineering

The chemical functionalization of cell-surface proteins of human primary fetal bone cells with hydrophilic bioorthogonal intermediates was investigated. Toward this goal, chemical pathways were developed for click reaction-mediated coupling of alkyne derivatives with cellular azido-expressing proteins. The incorporation via a tetraethylene glycol linker of a dipeptide and a reporter biotin allowed the proof of concept for the introduction of cell-specific peptide ligands and allowed us to follow the reaction in living cells. Tuning the conditions of the click reaction resulted in chemical functionalization of living human fetal osteoblasts with excellent cell survival.