The anti-HIV pentameric pseudopeptide HB-19 binds the C-terminal end of nucleolin and prevents anchorage of virus particles in the plasma membrane of target cells

The multivalent pseudopeptide HB-19 that binds the cell-surface-expressed nucleolin is a potent inhibitor of human immunodeficiency virus (HIV) infection by blocking virus particle attachment and thus anchorage in the plasma membrane. We show that cross-linking of surface-bound HB-19A (like HB-19 but with a modified template) results in aggregation of HB-19A with surface nucleolin. Consistent with its specific action, HB-19A binding to different types of cells reaches saturation at concentrations that have been reported to result in inhibition of HIV infection. By using Chinese hamster ovary mutant cell lines, we confirm that the binding of HB-19A to surface nucleolin is independent of heparan and chondroitin sulfate proteoglycans. In vitro generated full-length nucleolin was found to bind HB-19A, whereas the N-terminal part containing the acidic amino acid stretches of nucleolin did not. The use of various deletion constructs of the C-terminal part of nucleolin then permitted the identification of the extreme C-terminal end of nucleolin, containing repeats of the amino acid motif, RGG, as the domain that binds HB-19A. Finally, a synthetic peptide corresponding to the last C-terminal 63 amino acids was able to inhibit HIV infection at the stage of HIV attachment to cells, thus suggesting that this domain could be functional in the HIV anchorage process.     

Interaction between two ubiquitin-protein isopeptide ligases of different classes,CBLC and AIP4/ITCH

In metazoans, CBL proteins are RING finger type ubiquitin-protein isopeptide (E3) ligases involved in the down-regulation of epidermal growth factor tyrosine kinase receptors (EGFR). Among the three CBL proteins described in humans, CBLC (CBL3) remains poorly studied. By screening in parallel a human and a Caenorhabditis elegans library using the two-hybrid procedure in yeast, we found a novel interaction between Hsa-CBLC and Hsa-AIP4 or its C. elegans counterpart Cel-WWP1. Hsa-AIP4 and Cel-WWP1 are also ubiquitin E3 ligases. They contain a HECT (homologous to E6-AP C terminus) catalytic domain and four WW domains known to bind proline-rich regions. We confirmed the interaction between Hsa-CBLC and Hsa-AIP4 by a combination of glutathione S-transferase pull-down, co-immunoprecipitation, and colocalization experiments. We show that these two E3 ligases are involved in EGFR signaling because both become phosphorylated on tyrosine following epidermal growth factor stimulation. In addition, we observed that CBLC increases the ubiquitination of EGFR, and that coexpressing the WW domains of AIP4 exerts a dominant negative effect on EGFR ubiquitination. Finally, coexpressing CBLC and AIP4 induces a down-regulation of EGFR signaling. In conclusion, our data demonstrate that two E3 ligases of different classes can interact and cooperate to down-regulate EGFR signaling.     

Low molecular weight peptides restore the procoagulant activity of factor VIII in the presence of the potent inhibitor antibody ESH8

Following repeated administration of factor VIII (FVIII), a significant number of hemophilia A patients develop antibodies (Abs), inhibiting the procoagulant activity of infused FVIII. We have designed an approach based on the blocking of the deleterious activity of these Abs by peptide decoys mimicking the anti-FVIII Ab epitopes. Here, the well characterized inhibitory monoclonal Ab ESH8 served as a model. Several phage peptide libraries were screened for specific binding to ESH8. Seven constrained dodecapeptide sequences were obtained. Six sequences carried the consensus motif, hydrophobic-(Y/F)GKTXL. This motif showed a certain similarity with the (2231)QVDFQKTMKV(2240) sequence of the C(2) domain. In the seventh sequence, YCNPSIGDKNCR, the residues GDKN are similar to the sequence (2267)DGHQ(2270). Upon inspection of the C(2) domain crystallographic structure, the two stretches QVDFQKTMKV and DGHQ appeared close together in space and might constitute a discontinuous epitope. Corresponding synthetic peptides were able to inhibit the binding of ESH8 to FVIII in a specific and dose-dependent manner. Moreover, the ability of the selected peptides to neutralize the inhibitory activity of ESH8 was demonstrated in functional tests as well as in vivo in a murine model of hemophilia A. This study demonstrates the potential of this approach to neutralize the activity of potent inhibitory Abs.     

Resveratrol-induced limitation of dysfunction of mitochondria isolated from rat brain in an anoxia-reoxygenation model

Resveratrol protection on the main functions of purified rat brain mitochondria submitted to anoxia-reoxygenation was investigated. Resveratrol (<0.1 microM) reversed partly (23.3%) the respiratory control ratio (RCR) decrease by protecting both states 3 and 4. This effect was both observed when resveratrol was added before anoxia or reoxygenation. Resveratrol fully inhibited the release of cytochrome c in a concentration-dependent manner and significantly decreased the superoxide anion (O2(0-)) production at a concentration of 1 nM. The mitochondrial membranes damaged after the anoxia-reoxygenation were partly protected (about 70%) by resveratrol at 0.1 microM. The oxygen consumption of mitochondria in presence of NADH and cytochrome c was significantly inhibited by resveratrol with a low EC50 of 18.34 pM. Resveratrol inhibited the CCCP-induced uncoupling from about 20%. The effects of resveratrol on oxidative phosphorylation parameters were also investigated in rats after pretreatment (0.4, 2 and 10 mg/kg/day) for one week. After the isolation of brain mitochondria, the RCR was significantly less decreased in the resveratrol group compared to the control group. These results showed that resveratrol could preserve the mitochondrial functions with at least three mechanisms: antioxidant properties, action on complex III and a membrane stabilizing effect.     

Aluminum in the opal silica reticule of phytoliths: a new tool in palaeoecological studies

X-ray microanalysis was employed to screen biogenic plant silica extracted from the aboveground tissues of 20 species (Gramineae, Cyperaceae, Ericaceae, and Coniferae) occurring in subalpine and alpine grasslands, heaths, and woodlands on siliceous bedrock in the Valaisan Swiss Alps. Among the taxa investigated, only woody species produced a high proportion of phytoliths containing aluminum in the form of aluminosilicates. This difference between the chemical composition of wood and that of herbaceous phytoliths has important implications for the sourcing of phytoliths. As applications for palaeoenvironmental studies can be expected to be far-reaching, the potential of this microanalytical technique is discussed.     

New mutations of CIAS1 that are responsible for Muckle-Wells syndrome and familial cold urticaria: a novel mutation underlies both syndromes

Mutations of CIAS1 have recently been shown to underlie familial cold urticaria (FCU) and Muckle-Wells syndrome (MWS), in three families and one family, respectively. These rare autosomal dominant diseases are both characterized by recurrent inflammatory crises that start in childhood and that are generally associated with fever, arthralgia, and urticaria. The presence of sensorineural deafness that occurs later in life is characteristic of MWS. Amyloidosis of the amyloidosis-associated type is the main complication of MWS and is sometimes associated with FCU. In FCU, cold exposure is the triggering factor of the inflammatory crisis. We identified CIAS1 mutations, all located in exon 3, in nine unrelated families with MWS and in three unrelated families with FCU, originating from France, England, and Algeria. Five mutations--namely, R260W, D303N, T348M, A439T, and G569R--were novel. The R260W mutation was identified in two families with MWS and in two families with FCU, of different ethnic origins, thereby demonstrating that a single CIAS1 mutation may cause both syndromes. This result indicates that modifier genes are involved in determining either a MWS or a FCU phenotype. The finding of the G569R mutation in an asymptomatic individual further emphasizes the importance of such modifier a gene (or genes) in determining the disease phenotype. Identification of this gene (or these genes) is likely to have significant therapeutic implications for these severe diseases.     

Water deficit during root development: effects on the growth of roots and osmotic water permeability of isolated root protoplasts

The effect of low water potentials on root growth of flax (Linum usitatissimum L. cv. Ariane), rape (Brassica napus L. de Candolle, cv. Bristol), hard wheat (Triticum turgidum L. cv. Cham1) and soft wheat (Triticum aestivum L. cv. Ritmo) was studied by measuring the osmotic water permeability (Pos) of root protoplasts and the protein abundance of PIP1 and PIP2 aquaporins. These different species require more or less water, the most sensitive to water deficit being flax and rape. Ritmo, is a cultivar of wheat adapted to temperate zones, while the other cultivar Cham1 is adapted to low-rainfall areas. The seedlings were germinated and grown in water, salt or sugar solutions at different water potentials. The values of Pos for flax, rape and Chaml wheat were normally distributed and could be characterized by mean +/- SD. Root protoplasts from water-grown seedlings had Pos values of 485+/-159 microm s(-1) (flax), 582+/-100 microm s(-1) (rape), and 6.3+/-3.5 microm s(-1) (Cham1). At the same age, the protoplasts from Ritmo exhibited a much wider range of values than the protoplasts of Cham1. When seedlings were grown under conditions of osmotic or salt stress, the mass of the roots was reduced for all species. With 0.25 mol kg(-1) sorbitol or 0.125 M NaCl, the Pos for flax, rape and Cham1 remained constant or slightly increased, while for Ritmo the reduction in the mass of the roots was paralleled by a reduction in Pos. Only Cham1 and Ritmo were able to germinate at a lower potential (0.5 mol kg(-1) sorbitol). For Ritmo the reduction in the mass of the roots was paralleled by a reduction in Pos when grown in this stress condition and both wheats exhibited low Pos values. The expression of the PIP1 and PIP2 aquaporins families was also studied by immunoblotting. We did not observe any difference in protein expression for any of the species, whatever the growing conditions. We suggest that the high Pos values for flax and rape could play a role in the sensitivity of these plants at low water potential. The low native Pos for Cham1 in spite of the expression of both families of aquaporins may reflect its adaptation to low-rainfall conditions by a functional regulation of the water channels. For a similar reason, the low-water-potential-induced Pos of Ritmo may also correspond to a down-regulation of the aquaporins, reflecting adaptation of this wheat to water-deficit conditions.     

Ligand modulation of [35S]GTPgammaS binding at human alpha(2A), alpha(2B) and alpha(2C) adrenoceptors

Affinities and efficacies of chemically diverse ligands--some of them used as clinical agents--were examined, employing [3H]RX821,002 and [35S]GTPgammaS binding assays, respectively, at human (h) cloned, halpha(2A), halpha(2B) and halpha(2C) adrenoceptors (AR) expressed in Chinese hamster ovary (CHO) cells. As compared to noradrenaline (NA, efficacy defined as 100%), the majority of the 13 agonists tested generally behaved as partial agonists. Amongst 18 antagonists, pK(B) and pK(i) values, which were highly correlated for each alpha(2)-AR subtype, failed to reveal any strikingly selective agents. Inverse agonist properties were not detected for any antagonist, consistent with a lack of constitutive activity suggested by the monophasic inhibition of [35S]GTPgammaS binding by GTPgammaS. These data should facilitate interpretation of experimental and clinical actions of adrenergic agonists. Moreover, they emphasize the continuing need for alpha(2)-AR subtype-selective antagonists in order to define further the roles and therapeutic relevance of halpha(2A)-, halpha(2B)-, and halpha(2C)-AR.     

Global Positioning System (GPS) location accuracy improvement due to selective availability removal

Global Positioning System (GPS) is an important new technology for spatio-temporal behaviour studies of animals. Differential correction improves location accuracy. Previously, it mostly removed partially the influence of Selective Availability (SA). SA was deactivated in May 2000. The aim of this study was to quantify the influence of SA cancellation on location accuracy of various GPS receivers. We tested the accuracy of locations obtained from non-differential and differential GPS animal collars before and after SA removal. We found a significant improvement in accuracy for both types of GPS collars. However, differential GPS still provides more accurate locations.