Expression studies of the core+1 protein of the hepatitis C virus 1a in mammalian cells. The influence of the core protein and proteasomes on the intracellular levels of core+1

Recent studies have suggested the existence of a novel protein of hepatitis C virus (HCV) encoded by an ORF overlapping the core gene in the +1 frame (core+1 ORF). Two alternative translation mechanisms have been proposed for expression of the core+1 ORF of HCV-1a in cultured cells; a frameshift mechanism within codons 8-11, yielding a protein known as core+1/F, and/or translation initiation from internal codons in the core+1 ORF, yielding a shorter protein known as core+1/S. To date, the main evidence for the expression of this protein in vivo has been the specific humoral and cellular immune responses against the protein in HCV-infected patients, inasmuch as its detection in biopsies or the HCV infectious system remains elusive. In this study, we characterized the expression properties of the HCV-1a core+1 protein in mammalian cells in order to identify conditions that facilitate its detection. We showed that core+1/S is a very unstable protein, and that expression of the core protein in addition to proteosome activity can downregulate its intracellular levels. Also, we showed that in the Huh-7/T7 cytoplasmic expression system the core+1 ORF from the HCV-1 isolate supports the synthesis of both the core+1/S and core+1/F proteins. Finally, immunofluorescence and subcellular fractionation analyses indicated that core+1/S and core+1/F are cytoplasmic proteins with partial endoplasmic reticulum distribution in interphase cells, whereas in dividing cells they also localize to the microtubules of the mitotic spindle.     

Physiological Adaptations in Response to Environmental Stress During an N2-Fixing Anabaena Bloom

Anabaena spiroides has the ability to maintain intense biomass production for extensive periods in the epilimnion of a small eutrophic lake characterized by conditions shown to cause photooxidative death in a number of other phytoplankton. By the enhancement of carotenoid synthesis chlorophyll a was protected from photooxidation and prevented from catalyzing other photooxidative reactions within the cells. By temporally separating CO₂ and N₂ fixation, maximum utilization of photosynthetically active radiation was achieved. Because CO₂ fixation was more sensitive than N₂ fixation to a high oxygen concentration, the former was maximized during morning hours, before the afternoon buildup of dissolved oxygen. The diurnal partitioning of carbon and N₂ fixation has two additional advantages; possible competition for reductant-generating compounds is minimized, and adequate endogenous pools of carbon skeletons are assured to accept newly fixed ammonia. Hence, Anabaena, far from undergoing photooxidative death, appears to utilize a physiological strategy which allows optimization of radiant energy use for reductive processes and dominance of surface waters and shading of deeper phytoplankton during summer blooms.     

The genetic status and conservation management of two cultivated bulb species extinct in the wild: Tecophilaea cyanocrocus (Chile) and Tulipa sprengeri (Turkey)

Tecophilaea cyanocrocus and Tulipasprengeri are both extinct in the wild as aresult of overharvesting by commercialcollectors. Stocks of both species survive incultivation in amateur collections, commercialnurseries and botanic garden collections.Whilst both species share a similarconservation history, the distribution ofgenetic diversity within cultivated materialwas unknown. To support the long-termconservation of both species the geneticdiversity of the surviving stocks was assessedusing amplified fragment length polymorphisms(AFLPs). This study revealed different geneticstructures for the two bulb species. The Kewcollections of T. cyanocrocus, originallyobtained from a commercial nursery, VanTubergen, are genetically highly uniform andthe addition of samples from other collectionshas dramatically increased the level ofvariation. In contrast, the collections ofT. sprengeri held at Kew since the early20th century include representativegenotypes covering the whole range of geneticvariation found in this species and areprobably the source of all other cultivatedmaterial. The results are discussed in relationto the management of these species to ensuretheir continued survival in cultivation.     

Isolation of microsatellites in a parthenogenetic lizard Menetia greyii (Scincidae) and their utility in sexual species of the Menetia greyii complex

Tetra and tri‐nucleotide microsatellite loci were isolated from a parthenogenetic form of the Australian lizard Menetia greyii, using an enrichment method with polymerase chain reaction (PCR)‐based colony screening. Primers for 23 loci were designed and 11 of these loci amplified well in a panel of 10 parthenogenetic individuals from a single population. Seven loci were further characterized and six successfully amplified and were polymorphic in all five sexual species of the Menetia greyii complex they were tested in. These loci will be used to investigate the parental ancestry and clonal diversity of various parthenogenetic forms within the Menetia greyii species complex.     

Splice variants of mda-7/IL-24 differentially affect survival and induce apoptosis in U2OS cells

Interleukin-24 (mda-7/IL-24) is a cytokine in the IL-10 family that has received a great deal of attention for its properties as a tumor suppressor and as a potential treatment for cancer. In this study, we have identified and characterized five alternatively spliced isoforms of this gene. Several, but not all of these isoforms induce apoptosis in the osteosarcoma cell line U2OS, while none affect the survival of the non-cancerous NOK cell line. One of these isoforms, lacking three exons and encoding the N-terminal end of the mda-7/IL-24 protein sequence, caused levels of apoptosis that were higher than those caused by the full-length mda-7/IL-24 variant. Additionally, we found that the ratio of isoform expression can be modified by the splice factor SRp55. This regulation suggests that alternative splicing of mda-7/IL-24 is under tight control in the cell, and can be modified under various cellular conditions, such as DNA damage. In addition to providing new insights into the function of an important tumor suppressor gene, these findings may also point toward new avenues for cancer treatment.     

Liquid serial dilution is inferior to solid media for isolation of cultures representative of the phylum-level diversity of soil bacteria

Representatives of only four well-characterized bacterial phyla were isolated from a pasture soil by using liquid serial dilution culture. In contrast, members of Acidobacteria, Verrucomicrobia, and Gemmatimonadetes and of other poorly represented bacterial lineages were isolated in earlier experiments with solidified versions of the same media. We conclude that, contrary to expectation, liquid serial dilution culture is inferior to culturing on solid media for isolating representatives of many bacterial phyla from soil.     

Human pregnancy: the role of chemokine networks at the fetal-maternal interface

Chemokines are multifunctional molecules initially described as having a role in leukocyte trafficking and later found to participate in developmental processes such as differentiation and directed migration. Similar events occur in pregnancy during development of the fetal-maternal interface, where there is extensive leukocyte trafficking and tissue morphogenesis, and this is accompanied by abundant chemokine expression. The relationship between chemokines, leukocytes and placental development is beginning to be delineated. During pregnancy a specialised population of maternal leukocytes infiltrates the implantation site. These leukocytes are thought to sustain the delicate balance between protecting the developing embryo/fetus and tolerating its hemiallogeneic tissues. A network of chemokine expression by both fetal and maternal components in the pregnant uterus functions in establishing this leukocyte population. Intriguingly, experiments investigating immune cell recruitment revealed the additional possibility that chemokines influence aspects of placental development. Specifically, cytotrophoblasts, the effector cells of the placenta, express chemokine receptors that can bind ligands found at key locations, implicating chemokines as regulators of cytotrophoblast differentiation and migration. Thus, as in other systems, at the fetal-maternal interface chemokines might regulate multiple functions.     

Inhibition of CLC-2 chloride channel expression interrupts expansion of fetal lung cysts

Normal lung morphogenesis is dependent on chloride-driven fluid transport. The molecular identity of essential fetal lung chloride channel(s) has not been elucidated. CLC-2 is a chloride channel, which is expressed on the apical surface of the developing respiratory epithelium. CLC-2-like pH-dependent chloride secretion exists in fetal airway cells. We used a 14-day fetal rat lung submersion culture model to examine the role of CLC-2 in lung development. In this model, the excised fetal lung continues to grow, secrete fluid, and become progressively cystic in morphology (26). We inhibited CLC-2 expression in these explants, using antisense oligonucleotides, and found that lung cyst morphology was disrupted. In addition, transepithelial voltage (V(t)) of lung explants transfected with antisense CLC-2 was inhibited with V(t) = -1.5 +/- 0.2 mV (means + SE) compared with -3.7 +/- 0.3 mV (means + SE) for mock-transfected controls and -3.3 +/- 0.3 mV (means + SE) for nonsense oligodeoxynucleotide-transfected controls. This suggests that CLC-2 is important for fetal lung fluid production and that it may play a role in normal lung morphogenesis.     

Serpin polymerization is prevented by a hydrogen bond network that is centered on his-334 and stabilized by glycerol

Polymerization of serpins commonly results from mutations in the shutter region underlying the bifurcation of strands 3 and 5 of the A-sheet, with entry beyond this point being barred by a H-bond network centered on His-334. Exposure of this histidine in antithrombin, which has a partially opened sheet, allows polymerization and peptide insertion to occur at pH 6 or less when His-334 will be predictably protonated with disruption of the H-bond network. Similarly, thermal stability of antithrombin is pH-dependent with a single unfolding transition at pH 6, but there is no such transition when His-334 is buried by a fully closed A-sheet in heparin-complexed antithrombin or in alpha(1)-antitrypsin. Replacement of His-334 in alpha(1)-antitrypsin by a serine or alanine at pH 7.4 results in the same polymerization and loop-peptide acceptance observed with antithrombin at low pH. The critical role of His-334 and the re-formation of its H-bond network by the conserved P8 threonine, on the full insertion of strand 4, are relevant for the design of therapeutic blocking agents. This is highlighted here by the crystallographic demonstration that glycerol, which at high concentrations blocks polymerization, can replace the P8 threonine and re-form the disrupted H-bond network with His-334.