Design, synthesis, and biological characterization of potential antiatherogenic nitric oxide releasing tocopherol analogs

Synthesis and biological characterization of a series of alpha-tocopherol analogs with NO-releasing capacity are reported. The selected NO-donor moieties were nitrooxy and furoxan. All products were tested for their in vitro NO-releasing capacities, vasodilating properties, and antiplatelet activity. They were also capable of preventing LDL oxidation.     

Expression and phylogenetic analysis of two new lycopene β‐cyclases from Citrus paradisi

Two new lycopene β‐cyclases (LCYBs) were cloned and characterized from grapefruit (Citrus paradisi Macf.). During fruit ripening, CpLCYB1 expression did not show significant differences between ‘Flame’ (red flesh) and ‘Marsh’ (white flesh), and was much lower than CpLCYB2 and nearly constant; however, CpLCYB2 expression dramatically changed in a similar tendency in the pulp of both grapefruit cultivars, but the relative abundance of mRNA in ‘Flame’ was significantly lower than in ‘Marsh’. Phylogenetically and structurally, CpLCYB1 was a chloroplast‐specific member and CpLCYB2 a chromoplast‐specific member, the two subfamilies of all the LCYB genes. An intron was found in the 5′‐untranslated region of CpLCYB1 and in two other Citrus LCYB1 genes (CcLCYB1 and CsLCYB1‐2), resulting in an extra 20 amino acids, compared with all the other LCYB1s. It suggested that a different genomic event, in addition to gene duplication, has contributed to the evolution of these LCYB genes, and likewise, the change of their functions.     

Long-term climate record inferred from early-middle Pleistocene amphibian and squamate reptile assemblages at the Gran Dolina Cave, Atapuerca, Spain

The Gran Dolina cave site is famous for having delivered some of the oldest hominin remains of Western Europe (Homo antecessor, ca. 960 ka). Moreover, the evidence of lithic industries throughout the long vertical section suggests occupation on the part of hominins from the latest early Pleistocene (levels TD3/4, TD5, and TD6) to the late middle Pleistocene (level TD10). The Gran Dolina Sondeo Sur (TDS) has furnished a great number of small-vertebrate remains; among them some 40,000 bones are attributed to amphibians and squamates. Although they do not differ specifically from the extant herpetofauna of the Iberian Peninsula, the overlap of their current distribution areas (= mutual climatic range method) in Spain can provide mean annual temperatures (MAT), the mean temperatures of the coldest (MTC) and warmest (MTW) months, and mean annual precipitation (MAP) estimations for each sub-level, and their change can be studied throughout the sequence. Results from the squamate and amphibian study indicate that during hominin occupation the MAT (10-13 °C) was always slightly warmer than at present in the vicinity of the Gran Dolina Cave, and the MAP (800-1000 mm) was greater than today in the Burgos area. Climatic differences between “glacial” and “interglacial” phases are poorly marked. Summer temperatures (MTW) show stronger oscillations than winter temperatures (MTC), but seasonality remains almost unchanged throughout the sequence. These results are compared with those for large mammals, small mammals, and pollen analysis, giving a scenario for the palaeoclimatic conditions that occurred during the early to middle Pleistocene in Atapuerca, and hence a scenario for the hominins that once lived in the Sierra de Atapuerca.     

SPE-39 Family Proteins Interact with the HOPS Complex and Function in Lysosomal Delivery

Yeast and animal homotypic fusion and vacuole protein sorting (HOPS) complexes contain conserved subunits, but HOPS-mediated traffic in animals might require additional proteins. Here, we demonstrate that SPE-39 homologues, which are found only in animals, are present in RAB5-, RAB7-, and RAB11-positive endosomes where they play a conserved role in lysosomal delivery and probably function via their interaction with the core HOPS complex. Although Caenorhabditis elegans spe-39 mutants were initially identified as having abnormal vesicular biogenesis during spermatogenesis, we show that these mutants also have disrupted processing of endocytosed proteins in oocytes and coelomocytes. C. elegans SPE-39 interacts in vitro with both VPS33A and VPS33B, whereas RNA interference of VPS33B causes spe-39–like spermatogenesis defects. The human SPE-39 orthologue C14orf133 also interacts with VPS33 homologues and both coimmunoprecipitates and cosediments with other HOPS subunits. SPE-39 knockdown in cultured human cells altered the morphology of syntaxin 7-, syntaxin 8-, and syntaxin 13-positive endosomes. These effects occurred concomitantly with delayed mannose 6-phosphate receptor-mediated cathepsin D delivery and degradation of internalized epidermal growth factor receptors. Our findings establish that SPE-39 proteins are a previously unrecognized regulator of lysosomal delivery and that C. elegans spermatogenesis is an experimental system useful for identifying conserved regulators of metazoan lysosomal biogenesis.     

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude stringent methods for the isolation and identification of protein networks around a protein of interest. The use of chemical crosslinkers allows the selective stabilization of labile interactions, thus bypassing biochemical limitations for purification. Here we present a protocol amenable for cells in culture that uses a homobifunctional crosslinker with a spacer arm of 12 Å, dithiobis-(succinimidyl proprionate) (DSP). DSP is cleaved by reduction of a disulphide bond present in the molecule. Cross-linking combined with immunoaffinity chromatography of proteins of interest with magnetic beads allows the isolation of protein complexes that otherwise would not withstand purification. This protocol is compatible with regular western blot techniques and it can be scaled up for protein identification by mass spectrometry¹.Stephanie A. Zlatic and Pearl V. Ryder contributed equally to this work.Open in a separate windowClick here to view.^{(62M, flv)}     

CD8 T cell-intrinsic GITR is required for T cell clonal expansion and mouse survival following severe influenza infection

The regulation of T cell expansion by TNFR family members plays an important role in determining the magnitude of the immune response to pathogens. As several members of the TNFR family, including glucocorticoid-induced TNFR-related protein (GITR), are found on both regulatory and effector T cells, there is much interest in understanding how their effects on these opposing arms of the immune system affect disease outcome. Whereas much work has focused on the role of GITR on regulatory T cells, little is known about its intrinsic role on effector T cells in an infectious disease context. In this study, we demonstrate that GITR signaling on CD8 T cells leads to TNFR-associated factor (TRAF) 2/5-dependent, TRAF1-independent NF-κB induction, resulting in increased Bcl-x(L). In vivo, GITR on CD8 T cells has a profound effect on CD8 T cell expansion, via effects on T cell survival. Moreover, GITR is required on CD8 T cells for enhancement of influenza-specific CD8 T cell expansion upon administration of agonistic anti-GITR Ab, DTA-1. Remarkably, CD8 T cell-intrinsic GITR is essential for mouse survival during severe, but dispensable during mild respiratory influenza infection. These studies highlight the importance of GITR as a CD8 T cell costimulator during acute viral infection, and argue that despite the similarity among several TNFR family members in inducing T lymphocyte survival, they clearly have nonredundant functions in protection from severe infection.     

Identification of the sex pheromone of Copitarsia decolora (Lepidoptera: Noctuidae)

Chemical and electrophysiological analyses and field trials were used to identify the female sex pheromone of Copitarsia decolora (Guenée) (Lepidoptera: Noctuidae). Coupled gas chromatographic-electroantennographic detection (GC-EAD) analysis of the female gland extract showed the presence of two EAD-active peaks, which were identified by GC-mass spectrometric (MS) analysis as (Z)-9-tetradecenyl acetate (Z9-14:Ac) and (Z)-9-tetradecenol (Z9-14:OH). The field evaluation of the EAD-active compounds indicated that traps baited with either Z9-14:Ac or Z9-14:OH caught few males. In contrast, traps baited with the binary blend of both components caught significantly more males than traps baited with the single compounds. Captures in traps baited with a mixture of Z9-14:Ac and Z9 -14:OH at 4:1, 10:1, and 100:1 ratios were not significantly different from the catches in traps baited with virgin females. Few males were captured in traps baited with a blend of Z9-14:Ac and Z9-14:OH at 1:4, 1:10, and 1:100 ratios.