Overlapping motifs (PTAP and PPEY) within the Ebola virus VP40 protein function independently as late budding domains: involvement of host proteins TSG101 and VPS-4

The VP40 protein of Ebola virus can bud from mammalian cells in the form of lipid-bound, virus-like particles (VLPs), and late budding domains (L-domains) are conserved motifs (PTAP, PPxY, or YxxL; where "x" is any amino acid) that facilitate the budding of VP40-containing VLPs. VP40 is unique in that potential overlapping L-domains with the sequences PTAP and PPEY are present at amino acids 7 to 13 of VP40 (PTAPPEY). L-domains are thought to function by interacting with specific cellular proteins, such as the ubiquitin ligase Nedd4, and a component of the vacuolar protein sorting (vps) pathway, tsg101. Mutational analysis of the PTAPPEY sequence of VP40 was performed to understand further the contribution of each individual motif in promoting VP40 budding. In addition, the contribution of tsg101 and a second member of the vps pathway, vps4, in facilitating budding was addressed. Our results indicate that (i) both the PTAP and PPEY motifs contribute to efficient budding of VP40-containing VLPs; (ii) PTAP and PPEY can function as L-domains when separated and moved from the N terminus (amino acid position 7) to the C terminus (amino acid position 316) of full-length VP40; (iii) A VP40-PTAP/tsg101 interaction recruits tsg101 into budding VLPs; (iv) a VP40-PTAP/tsg101 interaction recruits VP40 into lipid raft microdomains; and (v) a dominant-negative mutant of vps4 (E228Q), but not wild-type vps4, significantly inhibited the budding of Ebola virus (Zaire). These results provide important insights into the complex interplay between viral and host proteins during the late stages of Ebola virus budding.     

A global study of relationships between leaf traits, climate and soil measures of nutrient fertility

Aim This first global quantification of the relationship between leaf traits and soil nutrient fertility reflects the trade‐off between growth and nutrient conservation. The power of soils versus climate in predicting leaf trait values is assessed in bivariate and multivariate analyses and is compared with the distribution of growth forms (as a discrete classification of vegetation) across gradients of soil fertility and climate. Location All continents except for Antarctica. Methods Data on specific leaf area (SLA), leaf N concentration (LNC), leaf P concentration (LPC) and leaf N:P were collected for 474 species distributed across 99 sites (809 records), together with abiotic information from each study site. Individual and combined effects of soils and climate on leaf traits were quantified using maximum likelihood methods. Differences in occurrence of growth form across soil fertility and climate were determined by one‐way ANOVA. Results There was a consistent increase in SLA, LNC and LPC with increasing soil fertility. SLA was related to proxies of N supply, LNC to both soil total N and P and LPC was only related to proxies of P supply. Soil nutrient measures explained more variance in leaf traits among sites than climate in bivariate analysis. Multivariate analysis showed that climate interacted with soil nutrients for SLA and area‐based LNC. Mass‐based LNC and LPC were determined mostly by soil fertility, but soil P was highly correlated to precipitation. Relationships of leaf traits to soil nutrients were stronger than those of growth form versus soil nutrients. In contrast, climate determined distribution of growth form more strongly than it did leaf traits. Main conclusions We provide the first global quantification of the trade‐off between traits associated with growth and resource conservation ‘strategies’ in relation to soil fertility. Precipitation but not temperature affected this trade‐off. Continuous leaf traits might be better predictors of plant responses to nutrient supply than growth form, but growth forms reflect important aspects of plant species distribution with climate.     

A truncated SV40 large T antigen lacking the p53 binding domain overcomes p53-induced growth arrest and immortalizes primary mesencephalic cells

As an alternative to primary fetal tissue, immortalized central nervous system (CNS)-derived cell lines are useful for in vitro CNS model systems and for gene manipulation with potential clinical use in neural transplantation. However, obtaining immortalized cells with a desired phenotype is unpredictable, because the molecular mechanisms of growth and differentiation of CNS cells are poorly understood. The SV40 large T antigen is commonly used to immortalize mammalian cells, but it interferes with multiple cell-cycle components, including p53, p300, and retinoblastoma protein, and usually produces cells with undifferentiated phenotypes. In order to increase the phenotypic repertoire of immortalized CNS cells and to address the molecular mechanisms underlying immortalization and differentiation, we constructed an expression vector containing a truncated SV40 large T gene that encodes only the amino-terminal 155 amino acids (T155), which lacks the p53-binding domain. Constructs were first transfected into a p53-temperature-sensitive cell line, T64-7B. Colonies expressing T155 proliferated at the growth-restrictive temperature. T155 was then transfected into primary cultures from embryonic day-14 rat mesencephalon. Two clonal cell lines were derived, AF-5 and AC-10, which co-expressed T155 and mature neuronal and astrocytic markers. Thus, the amino-terminal portion of SV40 large T is sufficient to: (1) overcome p53-mediated growth arrest despite the absence of a p53-binding region, and (2) immortalize primary CNS cells expressing mature markers while actively dividing. T155 and T155-transfectants may be useful for further studies of cell-cycle mechanisms and phenotyic expression in CNS cells or for further gene manipulation to produce cells with specific properties.     

Ontogeny and disease responses of Langerhans-like cells in lymphoid tissues of salmonid fish

The ontogeny and disease responses of Langerhans-like cells within lymphoid tissues of Atlantic salmon, Salmo salar, and rainbow trout, Oncorhynchus mykiss, were investigated. These cells were studied in situ with the use of two markers: the ultrastructural presence of Birbeck-like granules and immunohistochemistry with an antibody against human langerin/CD207 that cross-reacts with salmonid tissues. The appearance of Birbeck-like granules was observed in rainbow trout at 2 weeks post-hatch (PH) in the thymus and anterior kidney prior to the development of the spleen. Spleen first appeared at 3 weeks PH in both Atlantic salmon and rainbow trout, and Birbeck-like granules were observed within cells of the newly developed spleens. The cross-reactivity of langerin as seen by immunohistochemistry was not clearly observed in kidney and spleen until 9 weeks PH, when a strong cytoplasmic reaction was observed. To study langerin-positive cells in spleen and kidney during disease, microsporidial gill disease (MGD) in rainbow trout was used as a known disease model inducing a strong cell-mediated adaptive immune response. Langerin-positive cells in healthy fish were seen predominantly in the spleen, and only low numbers were present in the anterior kidney. During MGD, langerin-positive cell numbers were elevated in the anterior kidney and were significantly higher during 5, 6, and 10 weeks post-exposure (PE) compared with healthy control tissue. During MGD, the distribution of langerin-positive cells in the spleen and anterior kidney shifted from having significantly higher numbers of cells in the spleen than in the kidney in controls and at 1 and 4 weeks PE to having a similar distribution of the cells in the two organs at 2, 3, 5, and 6 weeks PE. By 10 weeks PE, significantly higher numbers of langerin-positive cells occurred in the anterior kidney compared with the spleen.     

Telomere Biology and Cellular Aging in Nonhuman Primate Cells

To determine how cellular aging is conserved among primates, we analyzed the replicative potential and telomere shortening in skin fibroblasts of anthropoids and prosimians. The average telomere length of the New World primates Ateles geoffroyi (spider monkey) and Saimiri sciureus (squirrel monkey) and the Old World primates Macaca mulatta (rhesus monkey), Pongo pygmaeus (orangutan), and Pan paniscus (pigmy chimpanzee) ranged from 4 to 16 kb. We found that telomere shortening limits the replicative capacity of anthropoid fibroblasts and that the expression of human telomerase produced telomere elongation and the extension of their in vitro life span. In contrast the prosimian Lemur catta (ring-tailed lemur) had both long and short telomeres and telomere shortening did not provide an absolute barrier to immortalization. Following a transient growth arrest a subset of cells showing a reduced number of chromosomes overgrew the cultures without activation of telomerase. Here we show that the presence of continuous TTAGGG repeats at telomeres and rigorous control of replicative aging by telomere shortening appear to be conserved among anthropoid primates but is less effective in prosimian lemurs.     

The impact of aromatase mechanism on other P450s

Experimental findings from a number of laboratories have converged to show that the conversion of androgens into oestrogen, catalysed by aromatase, involves three distinct reactions which occur at a single active site. That each one of these reactions belongs to a different generic type was revealed by chemical consideration, together with our ¹⁸O-experiments. In particular, these findings high-lighted the fact that the third reaction in the sequence occurs by a novel process for which a number of plausible mechanisms have been considered. The scrutiny of these mechanisms has involved either studies on aromatase itself, or on related enzymes which catalyse the aromatase type of cleavage reaction as generalized in equation 1:

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The acyl-carbon cleavage reaction of equation 1 is catalysed by sterol 14-demethylases, accounts for several side-chain fission products formed by CYP17 (17-hydroxylase-17,20-lyase), and constitutes a weak property of certain drug metabolizing P450s, when given aliphatic aldehydes as substrates. From cumulative studies on these enzymes, consensus is beginning to emerge that the acyl-carbon fission may be promoted by the Fe^III---OOH intermediate, formed during the catalytic cycles of P450s. The precedent for the direct involvement of the Fe^III---OOH species in the reaction of equation 1 is influencing our thinking regarding the mechanism of the conventional hydroxylation reaction. The status of knowledge surrounding the current debate on these issues will be reviewed.     

l-Alanine uptake in brush border membrane vesicles from the gill of a marine bivalve

Summary Brush border membrane vesicles were prepared from mussel gills using differential and sucrose density gradient centrifugation. These vesicles contained both the maximal Na⁺-dependent alanine transport activity found in the gradient and the maximal activities of -glutamyl transpeptidase and alkaline phosphatase. Electron micrographs showed closed vesicles of approximately 0.1–0.5 m diameter. Transport experiments using these vesicles demonstrated a transient 18-fold overshoot in intravesicular alanine concentration in the presence of an inwardly directed Na⁺ gradient, but not under Na⁺ equilibrium conditions. A reduced overshoot (10-fold) was seen with an inwardly directed K⁺ gradient. Further studies revealed a broad cation selectivity, with preference for Na⁺, which was characteristic of alanine transport but not glucose transport in these membranes. The apparent amino acid specificity of the uptake pathway(s) was similar to that of intact gills and supported the idea of at least four separate pathways for amino acid transport in mussel gill brush border membranes. The apparent Michaelis constant for alanine uptake was approximately 7m, consistent with values forK _t determined with intact tissue.     

Visual and Olfactory Female-Borne Cues Evoke Male Courtship in the Aphid Parasitoid Aphidius colemani Viereck (Hymenoptera: Braconidae)

We investigated the courtship and mating behavior of the pan-tropical polyphagous endoparasitoid Aphidius colemani Viereck. The courtship and mating displays, the magnitude of male-male sexual approaches and the role of female-borne cues evoking male courtship behavior were quantified. The sequence of events leading to copulation in this parasitoid did not differ from that found for other braconids. Females refused to copulate more than once. Same-sex courtships were observed among males and their possible role in an adaptive context is discussed. Olfactory female-borne cues played a key role in eliciting the courtship responses of males. Males were attracted by freshly dead females, but not by dead females soaked in hexane, nor by visual cues from females alone. Intense male wing fanning behavior was elicited by crushed abdomens of virgin females, suggesting that the female abdomen is the source of a short-distance pheromone crucial in evoking male courtship. Further studies are required to clarify the exact nature of the chemicals involved.