Synthesis and chloroquine-enhancing activity of Na-deacetyl-ferrocenoyl-strychnobrasiline

Several strychnobrasiline derivatives have been synthesized to overcome the lack of in vivo reversal activity of the parent compound. In the present study, N(a)-deacetyl-ferrocenoyl-strychnobrasiline was synthesized by condensing N(a)-deacetyl-strychnobrasiline with ferrocenic acid previously treated with oxalyl chloride. While the in vitro antiplasmodial activity of the test compound (IC(50)=4.83 microg/mL) was increased 15-fold compared to that of strychnobrasiline, and the in vitro enhancing activity was found to be similar to that of the parent compound, the compound was devoid of any in vivo potentiating effect, and an antagonistic effect was even observed at higher doses. Based on the overall results on the hemisynthesis of strychnobrasiline derivatives for better reversal activity, this strategy has appeared to be of little value for useful drugs.     

The neuronal choline transporter CHT1 is regulated by immunosuppressor-sensitive pathways

The immunosuppressor cyclosporin A inhibits the peptidyl-prolyl-cis/trans-isomerase activity of cyclophilins and the resulting complex inhibits the phosphatase activity of calcineurin. Both enzymes were detected in peripheral nerve endings isolated from the electric organ of Torpedo and shown to be affected by 10 micro m cyclosporin A. Among the cholinergic properties studied, choline uptake was specifically inhibited by cyclosporin A to a maximum of 40%. Cyclosporin A decreased the rate of choline transport but not the binding of the non-transportable choline analogue hemicholinium-3, indicating that the number of membrane transporters was not affected. Through the use of two other immunosuppressors, FK506, which also inhibits calcineurin, and rapamycin, which does not, two different mechanisms of choline uptake inhibition were uncovered. FK506 inhibited the rate of choline transport, whereas rapamycin diminished the affinity for choline. The Torpedo homologue of the high affinity choline transporter CHT1 was cloned and its activity was reconstituted in Xenopus oocytes. Choline uptake by oocytes expressing tCHT1 was inhibited by all three immunosuppressors and also by microinjection of the specific calcineurin autoinhibitory domain A457-481, indicating that the phosphatase calcineurin regulates CHT1 activity and could be the common target of cyclosporin and FK506. Rapamycin, which changed the affinity of the transporter, may have acted through an immunophilin on the isomerization of critical prolines that are found in the tCHT1 sequence.     

Use of whole genome amplification to rescue DNA from plasma samples

While DNA of good quality and sufficient amount can be obtained easily from whole blood, buccal swabs, surgical specimens, or cell lines, these DNA-rich sources are not always available. This is particularly the case in studies for which biological specimens were collected when genotyping assays were not widely available. In those studies, serum or plasma is often the only source of DNA. Newly developed whole genome amplification (WGA) methods, based on phi29 polymerase, may play a significant role in recovering DNA in such instances. We tested a total of 528 plasma samples kept in storage at -40 degrees C for approximately 10 years for 8 single nucleotide polymorphisms (SNPs) using the 5' exonuclease (TaqMan) assay. These specimens yielded undetectable levels of DNA following extraction with an affinity column but produced an average 52.7 microg (standard deviation of 31.2 microg) of DNA when column-extracted DNA was used as a template for WGA. This increased the genotyping success rate from 54% to 93%. There were only 3 disagreements out of 364 paired genotyping results for pre- and post-WGA DNAs, indicating an error rate of 0.82%. These results are encouraging for expanding the use of poor DNA resources in genotyping studies.     

Role of the Crumbs complex in the regulation of junction formation in Drosophila and mammalian epithelial cells

The formation of a belt-like junctional complex separating the apical from the lateral domain is an essential step in the differentiation of epithelial cells. Thus protein complexes regulating this event are of first importance for the development of cell polarity and physiological functions of epithelial tissues. In Drosophila, the discovery of a gene, crb, controlling the coalescence of the spots of zonula adherens (ZA) into a adhesive ring around the cells was a major step. We know now that Crumbs, the product of crb is an apical transmembrane protein conserved in mammals and that it interacts by its cytoplasmic domain with two cortical modular proteins, Stardust (Sdt) and Discs lost (Dlt) that are also essential for the correct assembly of the ZA. These two proteins are also conserved in mammals and it is most likely that the Crumbs complex plays a similar role in very different species. Recently, we have shown that Crumbs interacts with the cortical cytoskeleton made of DMoesin and beta heavy-Spectrin and this connection could explain in part the role of Crumbs in building the ZA. Future work will help to understand several aspects of the Crumbs complex that are still unknown, like the role of the large extracellular domain or the precise function of Sdt and Dlt in the building of the ZA. Finding an answer to these questions will help to find new therapies for Retinitis pigmentosa and other retina degeneration in which CRB1, the human homologue of crb, has been involved.     

Latitudinal shifts of forest and savanna in N. W. Africa during the Brunhes chron: further marine palynological results from site M 16415 (9°;N 19°W)

Palynological data of the marine core M 16415-2 show latitudinal shifts of the northern fringe of the tropical rain forest in north-west Africa during the last 700 ka. Savanna and dry open forest expanded southwards and tropical rain forest expanded northwards during dry and humid periods, respectively. Until 220 ka B.P., the tropical rain forest probably kept its zonal character in West Africa during glacials and interglacials. It is only during the last two glacial periods that the rain forest possibly fragmented into refugia. Throughout the Brunhes chron, pollen and spore transport was mainly by trade winds.     

On gene flow between Tetranychus urticae Koch, 1836 and Tetranychus cinnabarinus (Boisduval) Boudreaux, 1956 (Acari: Tetranychidae): Sononomy between the two species

It was found that the reproductive separation between the two-spotted spider mite (Tetranychus urticae Koch) and the carmine spider mite (Tetranychus cinnabarinus (Boisduval) is far from complete. Hybrid strains were established which possessed the morphological characteristics of T. cinnabarinus, but which were homozygous for a marker gene originating from T. urtica. In addition, short-day treatment leads to diapause not only in the species from cold climates (T. urticae), but also in a population of T. cinnabarinus. It is questioned whether T. cinnabarinus deserves ranking at species level.

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Résumé Ce travail montre que l'isolement reproductif entre Tetranychus uriticae Koch et Tetranychus cinnabarinus (Boisduval), est loin d'être complet. L'auteur a obtenu des souches hybrides présentant les caractéristiques morphologiques de T. cinnabarinus, mais homozygotes pour un gêne marqueur provenant de T. urticae. De plus, une diminution de la photopériode entraîne l'entrée en diapause des espèces provenant de régions à climat froid (T. urticae), mais aussi d'une population de T. cinnabarinus.Il est finalemant proposé de considérer Tetranychus cinnabarinus (Boisduval) Boudreaux, 1956, comme un synonyme de Tetranychus urticae Koch, 1836.

     

A new method of purification of proteasome substrates reveals polyubiquitination of 20 S proteasome subunits

The 26 S proteasome is implicated in the control of many major biological functions but a reliable method for the identification of its major substrates, i.e. polyubiquitin (Ub) conjugates, is still lacking. Based on the steps present in cells, i.e. recognition and deubiquitination, we developed an affinity matrix-based purification of polyUb conjugates suitable for any biological sample. Ub-conjugates were first purified from proteasome inhibitor-treated C2C12 cells using the Ub binding domains of the S5a proteasome subunit bound to an affinity matrix and then deubiquitinated by the catalytic domain of the USP2 enzyme. This two step purification of proteasome substrates involving both protein-protein interactions and enzyme-mediated release allowed highly specific isolation of polyUb 26 S proteasome substrates, which were then resolved on two-dimensional gels post-deubiquitination. To establish our method, we focused on a gel area where spots were best resolved. Surprisingly, spot analysis by mass spectrometry identified alpha2, alpha6, alpha7, beta2, beta3, beta4, and beta5 20 S proteasome subunits as potential substrates. Western blots using an anti-beta3 proteasome subunit antibody confirmed that high molecular weight forms of beta3 were present, particularly in proteasome inhibitor-treated cells. Sucrose gradients of cell lysates suggested that the proteasome was first disassembled before subunits were polyubiquitinated. Altogether, we provide a technique that enables large scale identification of 26 S proteasome substrates that should contribute to a better understanding of this proteolytic machinery in any living cell and/or organ/tissue. Furthermore, the data suggest that proteasome homeostasis involves an autoregulatory mechanism.     

Structural insights into the innate immune recognition specificities of L- and H-ficolins

Innate immunity relies critically upon the ability of a few pattern recognition molecules to sense molecular markers on pathogens, but little is known about these interactions at the atomic level. Human L- and H-ficolins are soluble oligomeric defence proteins with lectin-like activity, assembled from collagen fibers prolonged by fibrinogen-like recognition domains. The X-ray structures of their trimeric recognition domains, alone and in complex with various ligands, have been solved to resolutions up to 1.95 and 1.7 A, respectively. Both domains have three-lobed structures with clefts separating the distal parts of the protomers. Ca(2+) ions are found at sites homologous to those described for tachylectin 5A (TL5A), an invertebrate lectin. Outer binding sites (S1) homologous to the GlcNAc-binding pocket of TL5A are present in the ficolins but show different structures and specificities. In L-ficolin, three additional binding sites (S2-S4) surround the cleft. Together, they define an unpredicted continuous recognition surface able to sense various acetylated and neutral carbohydrate markers in the context of extended polysaccharides such as 1,3-beta-D-glucan, as found on microbial or apoptotic surfaces.