Stabilities of HIV-1 DIS type RNA loop-loop interactions in vitro and in vivo

RNA loop–loop interactions are a prevalent motif in the formation of tertiary structure and are well suited to trigger molecular recognition between RNA molecules. We determined the stabilities of several loop–loop interactions with a constant 6 bp core sequence and varying unpaired flanking nucleotides and found that the flanking bases have a strong influence on the stability and ion dependence of the kissing complex. In general, the stabilities determined in 1 M Na⁺ are equivalent to those in the presence of near physiological Mg²⁺ concentrations. Therefore we further tested whether the stabilities determined in vitro and within yeast cells correlate, using a recently developed yeast RNA-hybrid system. For the majority of the loop types analyzed here, the melting temperatures determined in vitro are in good agreement with the relative β-galactosidase activity in yeast cells, showing that data derived from in vitro measurements reflect in vivo properties. The most stable interactions are the naturally occurring HIV-1 DIS MAL and LAI derived loops with the motif (5′ A^A/_GN₆A 3′), emphasizing the crucial role of stable kissing complexes in HIV genome dimerization.     

Analysis of a rare melanoma patient with a spontaneous CTL response to a MAGE-A3 peptide presented by HLA-A1

We describe an HLA-A1 melanoma patient who has mounted a spontaneous cytolytic T cell (CTL) response against an antigenic peptide encoded by gene MAGE-A3 and presented by HLA-A1. The frequency of anti-MAGE-3.A1 CTLp was 5×10⁻⁷ of the blood CD8 cells, with a dominant clonotype which was present in six out of seven independent anti-MAGE-3.A1 CTL clones. After vaccination with a recombinant poxvirus coding for the MAGE-3.A1 antigen, the blood frequency of anti-MAGE-3.A1 CTLp increased tenfold. Twenty-two independent CTL clones were derived. Surprisingly, only one of them corresponded to the dominant clonotype present before vaccination. Two new clonotypes were repeated 12 and 7 times, respectively. Our interpretation of these results is that the spontaneous anti-MAGE-3.A1 CTL response pre-existing to vaccination was polyclonal, and that the vaccine restimulated only some of these clones. To estimate the incidence of spontaneous anti-MAGE-3.A1 CTL responses in melanoma patients with a tumor expressing gene MAGE-A3, we measured the blood frequency of anti-MAGE-3.A1 T cells in 45 patients, and found only two clear responses.     

Isoflavonoid exudation from white lupin roots is influenced by phosphate supply, root type and cluster-root stage

The internal concentration of isoflavonoids in white lupin (Lupinus albus) cluster roots and the exudation of isoflavonoids by these roots were investigated with respect to the effects of phosphorus (P) supply, root type and cluster-root developmental stage.To identify and quantify the major isoflavonoids exuded by white lupin roots, we used high-pressure liquid chromatography (HPLC) coupled to electrospray ionization (ESI) in mass spectrometry (MS).The major exuded isoflavonoids were identified as genistein and hydroxygenistein and their corresponding mono- and diglucoside conjugates. Exudation of isoflavonoids during the incubation period used was higher in P-deficient than in P-sufficient plants and higher in cluster roots than in noncluster roots. The peak of exudation occurred in juvenile and immature cluster roots, while exudation decreased in mature cluster roots.Cluster-root exudation activity was characterized by a burst of isoflavonoids at the stage preceding the peak of organic acid exudation. The potential involvement of ATP-citrate lyase in controlling citrate and isoflavonoid exudation is discussed, as well as the possible impact of phenolics in repelling rhizosphere microbial citrate consumers.     

Plant glutathione peroxidases are functional peroxiredoxins distributed in several subcellular compartments and regulated during biotic and abiotic stresses

We provide here an exhaustive overview of the glutathione (GSH) peroxidase (Gpx) family of poplar (Populus trichocarpa). Although these proteins were initially defined as GSH dependent, in fact they use only reduced thioredoxin (Trx) for their regeneration and do not react with GSH or glutaredoxin, constituting a fifth class of peroxiredoxins. The two chloroplastic Gpxs display a marked selectivity toward their electron donors, being exclusively specific for Trxs of the y type for their reduction. In contrast, poplar Gpxs are much less specific with regard to their electron-accepting substrates, reducing hydrogen peroxide and more complex hydroperoxides equally well. Site-directed mutagenesis indicates that the catalytic mechanism and the Trx-mediated recycling process involve only two (cysteine [Cys]-107 and Cys-155) of the three conserved Cys, which form a disulfide bridge with an oxidation-redox midpoint potential of -295 mV. The reduction/formation of this disulfide is detected both by a shift on sodium dodecyl sulfate-polyacrylamide gel electrophoresis or by measuring the intrinsic tryptophan fluorescence of the protein. The six genes identified coding for Gpxs are expressed in various poplar organs, and two of them are localized in the chloroplast, with one colocalizing in mitochondria, suggesting a broad distribution of Gpxs in plant cells. The abundance of some Gpxs is modified in plants subjected to environmental constraints, generally increasing during fungal infection, water deficit, and metal stress, and decreasing during photooxidative stress, showing that Gpx proteins are involved in the response to both biotic and abiotic stress conditions.     

Selection of Brassica rapa genotypes for tolerance to boron toxicity

Although boron (B) is a micronutrient essential for the growth of vascular plants, it reduces growth and seed yield when present in excessive amounts. A hydroponic assay of nineteen Brassica rapa genotypes resulted in the identification of two tolerant genotypes, WWY Sarson and Local at a range of boron concentrations (15–165 μM). The most tolerant and sensitive genotypes were assessed for shoot boron concentrations in a soil assay with 4, 29 and 54 mg B kg⁻¹ soil. The soil assay confirmed the results of the hydroponic screening. Shoot boron uptake was at least three times lower and shoot boron concentrations about 10 times lower in the tolerant than sensitive genotypes, indicating that boron tolerance involved boron exclusion from the shoot.     

Synthesis, conformational characteristics and anti-influenza virus A activity of some 2-adamantylsubstituted azacycles

The broad-spectrum antiviral activity of 2-(2-adamantyl)piperidines 11, 13a,b, and 15, 3-(2-adamantyl)pyrrolidines 27, 21a-g and 2-(2-adamantylmethyl)piperidines 30, 32a-c, and 35a-d was examined. Several compounds in the new series were potent against influenza A H(3)N(2) virus. When 1-aminoethyl pharmacophore group of 2-rimantadine 4 (2-isomer of rimantadine) is included into a saturated nitrogen heterocycle, see compound 11, potency was retained. The diamine derivatives 21e-g and particularly 35a-c possessing three pharmocophoric groups, that is, the adamantyl and the two amine groups, exhibited high potency. The new compounds did not afford specific activity at non-toxic concentrations against any of the other viruses tested. According to NMR spectroscopy and molecular mechanics calculations it is striking that the parent structures 11 and 27 adopt a fixed trans conformation around C2-C2' bond. In the parent amines, which proved to be active compounds, the distance between nitrogen and adamantyl pharmacophoric groups was different; N-C2' distance is 3.7, 3.8 A for 27, 30 and 2.5 A for 11 suggesting that M2 receptor site can accommodate different in size and orientation lipophilic cages.     

Modo-UG, a marsupial nonclassical MHC class I locus

Modo-UG is a class I gene located in the MHC of the marsupial Monodelphis domestica, the gray, short-tailed opossum. Modo-UG is expressed as three alternatively spliced mRNA forms, all of which encode a transmembrane form with a short cytoplasmic tail that lacks phosphorylation sites typically found in classical class I molecules. The three alternative mRNAs would encode a full-length form, an isoform lacking the α2 domain, and one lacking both α2 and α3 domains. Genotyping both captive-bred and wild M. domestica from different geographic regions revealed no variation in the residues that make up Modo-UG’s peptide-binding groove. Modo-UG’s low polymorphism is contrasting to that of a nearby class I locus, Modo-UA1, which has a highly polymorphic peptide-binding region. Absence of functional polymorphism in Modo-UG is therefore not a general feature of opossum class I genes but the result of negative selection. Modo-UG is the first MHC linked marsupial class I to be described that appears to clearly have nonclassical features.Electronic Supplementary Material Supplementary material is available for this article at     

Calcium store contents control the expression of TRPC1, TRPC3 and TRPV6 proteins in LNCaP prostate cancer cell line

The mammalian homologues of the Drosophila transient receptor potential (TRP) represent a superfamily of ion channels involved in Ca(2+) homeostasis. Several members of this family are activated either by a depletion of the internal stores of Ca(2+) or by stimulation of G protein-coupled receptors. In androgen responsive prostate cancer cell line LNCaP, TRPC1, TRPC4 and/or TRPV6 have been reported to function as store-operated channels (SOCs) while TRPC3 might be involved in the response to agonist stimulation, possibly through the induction of diacylglycerol production by phospholipase C. However, the control of expression of these TRP proteins is largely unknown. In the present study, we have investigated if the expression of the TRP proteins possibly involved in the capacitative influx of calcium is influenced by the contents of Ca(2+) in the endoplasmic reticulum. Using real-time PCR and Western blot techniques, we show that the expression of TRPC1, TRPC3 and TRPV6 proteins increases after a prolonged (24-48 h) depletion of the stores with thapsigargin. The upregulation of TRPC1 and TRPC3 depends on the store contents level and involves the activation of the Ca(2+)/calmodulin/calcineurin/NFAT pathway. Functionally, cells overexpressing TRPC1, TRPC3 and TRPV6 channels after a prolonged depletion of the stores showed an increased [Ca(2+)](i) response to alpha-adrenergic stimulation. However, the store-operated entry of calcium was unchanged. The isolated overexpression of TRPV6 (without overexpression of TRPC1 and TRPC3) did not produce this increased response to agonists, therefore suggesting that TRPC1 and/or TRPC3 proteins are responsible for the response to alpha-adrenergic stimulation but that TRPC1, TPRC3 and TRPV6 proteins, expressed alone or concomitantly, are not sufficient for SOC formation.