HIV-1 Vpu inhibits accumulation of the envelope glycoprotein within clathrin-coated, Gag-containing endosomes

Several viruses encode ion channels that both modulate the trafficking of envelope glycoprotein(s) and stimulate the release of virions from cells. HIV-1 Vpu enhances virion release and inhibits the endosomal accumulation of the viral structural protein Gag. We investigated whether Vpu affects the subcellular distribution of Env as well as Gag. Env and Vpu colocalized with each other, in part within the trans -Golgi network. In the absence of Vpu, Env accumulated more extensively within clathrin-coated endosomal structures. These structures had several features consistent with an endosomal viral assembly domain: they contained Gag, including proteolytically processed viral matrix protein; the tetraspanins CD63 and CD81; the adaptor protein complex AP-3; and AIP1/ALIX, a cellular cofactor for viral budding. These endosomes labelled incompletely with Env derived from the cell surface, suggesting that some Env reaches this compartment without transiting the plasma membrane. Consistent with this, endosomal accumulation of Env was not blocked by dominant-negative Eps15, an inhibitor of AP-2-mediated endocytosis. Although these data are potentially explained by greater endocytosis of mature virions in the absence of Vpu, they also raise the possibility that Vpu inhibits the transport of Env and Gag to late endosomes, leading to viral assembly at the plasma membrane.     

The interferon-induced protein BST-2 restricts HIV-1 release and is downregulated from the cell surface by the viral Vpu protein

The HIV-1 accessory protein Vpu counteracts a host factor that restricts virion release from infected cells. Here we show that the interferon-induced cellular protein BST-2/HM1.24/CD317 is such a factor. BST-2 is downregulated from the cell surface by Vpu, and BST-2 is specifically expressed in cells that support the vpu phenotype. Exogenous expression of BST-2 inhibits HIV-1 virion release, while suppression of BST-2 relieves the requirement for Vpu. Downregulation of BST-2 requires both the transmembrane/ion channel domain and conserved serines in the cytoplasmic domain of Vpu. Endogenous BST-2 colocalizes with the HIV-1 structural protein Gag in endosomes and at the plasma membrane, suggesting that BST-2 traps virions within and on infected cells. The unusual structure of BST-2, which includes a transmembrane domain and a lumenal GPI anchor, may allow it to retain nascent enveloped virions on cellular membranes, providing a mechanism of viral restriction counteracted by a specific viral accessory protein.     

Quantitative variation in monoterpenes in four species of conifers

Eighteen monoterpenes found in bole and foliage volatiles of four sympatric species of conifers, Douglas-fir, Pseudotsuga menziesii (Mirb.) Franco, lodgepole pine, Pinus contorta var. latifolia Engelm., interior spruce, Picea engelmannii×glauca, and interior fir, Abies lasiocarpa×bifolia, in three locations in British Columbia, Canada, were analysed for quantitative variation (N=10 trees per sample) using standard and chiral gas chromatography. There was significant variation in monoterpene composition between bole and foliage in all four species. Principal components analysis revealed that the monoterpene profile of coastal Douglas-fir was significantly different from trees in the interior, supporting the hypothesis that coastal and interior populations represent distinct chemotypes. Although the four species did not differ qualitatively, there were significant differences in their quantitative monoterpene profiles in both bole and foliage. These differences were large enough to suggest that host selection by four species of coniferophagous bark beetles (Coleoptera: Scolytidae), specific to each of the above tree species, may depend in part on perception of, and behavioural response to quantitatively distinct blends of monoterpenes.     

hnRNP H Is a Component of a Splicing Enhancer Complex That Activates a c-src Alternative Exon in Neuronal Cells

The regulation of the c-src N1 exon is mediated by an intronic splicing enhancer downstream of the N1 5′ splice site. Previous experiments showed that a set of proteins assembles onto the most conserved core of this enhancer sequence specifically in neuronal WERI-1 cell extracts. The most prominent components of this enhancer complex are the proteins hnRNP F, KSRP, and an unidentified protein of 58 kDa (p58). This p58 protein was purified from the WERI-1 cell nuclear extract by ammonium sulfate precipitation, Mono Q chromatography, and immunoprecipitation with anti-Sm antibody Y12. Peptide sequence analysis of purified p58 protein identified it as hnRNP H. Immunoprecipitation of hnRNP H cross-linked to the N1 enhancer RNA, as well as gel mobility shift analysis of the enhancer complex in the presence of hnRNP H-specific antibodies, confirmed that hnRNP H is a protein component of the splicing enhancer complex. Immunoprecipitation of splicing intermediates from in vitro splicing reactions with anti-hnRNP H antibody indicated that hnRNP H remains bound to the src pre-mRNA after the assembly of spliceosome. Partial immunodepletion of hnRNP H from the nuclear extract partially inactivated the splicing of the N1 exon in vitro. This inhibition of splicing can be restored by the addition of recombinant hnRNP H, indicating that hnRNP H is an important factor for N1 splicing. Finally, in vitro binding assays demonstrate that hnRNP H can interact with the related protein hnRNP F, suggesting that hnRNPs H and F may exist as a heterodimer in a single enhancer complex. These two proteins presumably cooperate with each other and with other enhancer complex proteins to direct splicing to the N1 exon upstream.     

Effects of life history variation on size and growth in stream-dwelling Atlantic salmon

A large size variation amongst life histories for stream-dwelling Atlantic salmon Salmo salar was found and the relative effect of life histories on size varied over time. As early as December (age 0+ years), fish that later smolted at age 2+ years were significantly larger than fish that did not smolt at age 2+ years. In contrast, there were no mass differences at age 0+ years between fish that would mature or not at age 1+ years (October). The mass differences between smolts and non-smolts persisted until smolting, and differences between mature and immature fish first appeared in May (age 1+ years). Following September (age 1+ years), there was also a significant interaction between smolting and maturity. Previously mature and immature age 2+ year smolts were not significantly different in size, but immature age 2+ year non-smolts were much lighter than mature age 2+ year non-smolts. Based on mass differences, the apparent 'decision' to smolt occurred c . 5 months before (winter, age 0+ years) the decision to mature (late spring, age 1+ years). In addition to strong seasonal growth variation, sizes of freshwater Atlantic salmon were largely structured by the complex interaction between smolt-age and maturity.     

Flow analysis by phase modulation of NMR signals

Time modulated field-gradient-sequences able to selectively phase encode spatial location or flow velocity in NMR signals are described. The specific phase modulation discriminates between stationary and mobile protons. The method is presented in the one dimensional case and has the following advantages: it is sensitive to flow direction and unaffected by the relaxation times.     

Natural phosphatidylcholine is actively translocated across the plasma membrane to the surface of mammalian cells

The cell surface of eukaryotic cells is enriched in choline phospholipids, whereas the aminophospholipids are concentrated at the cytosolic side of the plasma membrane by the activity of one or more P-type ATPases. Lipid translocation has been investigated mostly by using short chain lipid analogs because assays for endogenous lipids are inherently complicated. In the present paper, we optimized two independent assays for the translocation of natural phosphatidylcholine (PC) to the cell surface based on the hydrolysis of outer leaflet phosphoglycerolipids by exogenous phospholipase A2 and the exchange of outer leaflet PC by a transfer protein. We report that PC reached the cell surface in the absence of vesicular traffic by a pathway that involved translocation across the plasma membrane. In erythrocytes, PC that was labeled at the inside of the plasma membrane was translocated to the cell surface with a half-time of 30 min. This translocation was probably mediated by an ATPase, because it required ATP and was vanadate-sensitive. The inhibition of PC translocation by glibenclamide, an inhibitor of various ATP binding cassette transporters, and its reduction in erythrocytes from both Abcb1a/1b and Abcb4 knockout mice, suggest the involvement of ATP binding cassette transporters in natural PC cell surface translocation. The relative importance of the outward translocation of PC as compared with the well characterized fast inward translocation of phosphatidylserine for the overall asymmetric phospholipid organization in plasma membranes remains to be established.     

Über die Zinkresistenz der Schwermetallform von Silene cucubalus var. humilis im Laufe der Vegetationsperiode

• 1 . Die Zinkresistenz der Schwermetallpflanzen wurde am Beispiel von Silene cucubalus var. humilis dargestellt.
• 2 . Die Toleranzgrenze ändert sich im Laufe der Vegetationsperiode. Entwicklungsphysiologisch ältere Gewebe sind widerstandsfähiger als junge.
• 3 . Die mit der protoplasmatischen Methode erfaßbare Zinkverträglichkeit der Schwermetallpflanzen muß im Zusammenhang mit dem Zinkgehalt der Gewebe gesehen werden, da die Resistenzgrenze der Pflanzen aus beiden Größen resultiert.

     

Association of nickel versus transport of cadmium and calcium in tonoplast vesicles of oat roots

The plant vacuole has long been suspected of being a site for accumulation of Ni in plant roots, but testing this hypothesis directly by vacuole isolation is technically difficult and has not been reported. Here, we have attempted to determine if Ni can be transported into isolated oat (Avena sativa L.) root tonoplast vesicles as an alternative approach toward understanding the importance of the vacuole in Ni accumulation in roots. We found that, in contrast to Ca and Cd, Ni did not affect the proton gradient of vesicles (MgATP energized or artificially created), and further, that Cd/H antiport activity was not affected by the presence of Ni. Nickel was associated with vesicles, but relative rates of accumulation/association of metals with vesicles were Ca > Cd Ni. Protonophores and the potential Ni ligands citrate and histidine, and nucleoside triphosphates or PPi did not stimulate Ni association with vesicles. Comparison of Ni versus Ca and Cd associated with vesicles using various membrane perturbants indicated that while Ca and Cd are rapidly and principally antiported to the vesicle sap, Ni is only slowly associated with the membrane in a not-easily dissociated condition. Our results indicate the absence of an Ni/H antiport or Ni-nucleotide-dependent pump in oat root tonoplasts, and support the contention that the vacuole is not a major compartment for Ni accumulation in oat roots. Received: 2 June 1997 / Accepted: 17 July 1997     

Interaction of LDL, Lp[a], and reduced Lp[a] with monoclonal antibodies against apoB

Five monoclonal antibodies (2A, 9A, 6B, L3, L7) produced in mice against human apolipoprotein B were investigated by competitive and inhibitive electroimmunoassay (EIA) for their reactivity with low density lipoprotein (LDL), lipoprotein[a] (Lp[a]), and reduced Lp[a]. All of the antibodies reacted with apoB of the different lipoproteins indicated by very similar slopes of the binding curves. None of them gave a positive reaction with apolipoprotein[a]. The amount of apoB required for 50% inhibition of antibody binding varied for the different antibodies and lipoproteins. Antibody 9A showed almost the same affinity for LDL, Lp[a], and reduced Lp[a]. Antibodies 2A and 6B bound about twofold better to LDL and reduced Lp[a] than to untreated Lp[a]. Antibodies L3 and L7 needed nearly threefold higher amounts of Lp[a]-apoB for 50% inhibition of antibody binding than of apoB of LDL and reduced Lp[a]. The amount of apoB required for 50% inhibition of antibody binding was somewhat higher in inhibitive assay than in competitive assay. We suggest that apo[a] covers certain epitopes of apoB in native Lp[a] leading to a reduced reaction with the monoclonal antibodies. However, it could also be that the binding of the [a]antigen to apoB via disulfide bridges causes profound conformational changes of the apoB region exposed to the surface.