Solution structure of the endonuclease domain from the master replication initiator protein of the nanovirus faba bean necrotic yellows virus and comparison with the corresponding geminivirus and circovirus structures

Nanoviruses are a family of plant viruses that possess a genome of multiple circular single-stranded DNA (ssDNA) components and are strikingly similar in their replication mode to the plant geminiviruses and to the circoviruses that infect birds or mammals. These viruses multiply by rolling circle replication using virus-encoded multifunctional replication initiator proteins (Rep proteins) that catalyze the initiation of replication on a double-stranded DNA (dsDNA) intermediate and the resolution of the ssDNA into circles. Here we report the solution NMR three-dimensional structure of the endonuclease domain from the master Rep (M-Rep) protein of faba bean necrotic yellows virus (FBNYV), a representative of the nanoviruses. The domain comprises amino acids 2-95 (M-Rep2-95), and its global fold is similar to those previously described for the gemini- and circovirus Rep endonuclease domains, consisting of a central 5-stranded antiparallel beta-sheet covered on one side by an alpha-helix and irregular loops and on the other, more open side of the domain, by an alpha-helix containing the catalytic tyrosine residue (the catalytic helix). Longer domain constructs extending to amino acids 117 and 124 were also characterized. They contain an additional alpha-helix, are monomeric, and exhibit catalytic activity indistinguishable from that of M-Rep2-95. The binding site for the catalytic metal was identified by paramagnetic broadening and maps to residues on the exposed face of the central beta-sheet. A comparison with the previously determined Rep endonuclease domain structures of tomato yellow leaf curl Sardinia virus (TYLCSV), a geminivirus, and that of porcine circovirus type 2 (PCV2) Rep allows the identification of a positively charged surface that is most likely involved in dsDNA binding, and reveals common features shared by all endonuclease domains of nanovirus, geminivirus, and circovirus Rep proteins.     

The key role of the largest extant Neotropical frugivore (Tapirus terrestris) in promoting admixture of plant genotypes across the landscape

The historical and contemporary loss of large‐bodied frugivores has disrupted many plant‐disperser mutualisms, with potentially profound consequences for plants. Although several aspects of seed dispersal by megafrugivores have already been examined, the role of these species in promoting seed‐mediated gene flow has remained unexplored. We evaluated the role of the Amazonian tapir (Tapirus terrestris), the largest Neotropical frugivore, in shaping plant genetic structure through seed‐mediated gene flow. We used microsatellites to analyze the genetic patterns of Syagrus romanzoffiana seedlings recruited in tapir latrines and around conspecific adult palms, the two sites where seeds and seedlings are most frequently found in this species. While the genetic diversity of seedlings was rather similar in both sites, the kinship structure was substantially weaker in latrines. Most seedlings recruited around adult palms were half‐ or full‐sibs originating from those adults. In contrast, seedlings recruited in latrines came from several (>5, on average) contributing mothers other than the nearest adult (95%) and were mostly non‐sibs (72%). Kinship patterns indicated that tapir‐mediated dispersal promotes the admixture of genotypes across space. Also, our results suggested that genetic diversity and the number of contributing mothers in latrines increase with the number of fruiting adults visited by tapirs before defecating and with the accumulation of feces over time. We provide evidence of the relevance of tapirs in mobilizing maternal progenies (and genotypes) across the landscape and recruiting clusters of unrelated seedlings. This study suggests a key role for plant–megafrugivore interactions in seed‐mediated gene flow and emphasizes the importance of preserving such mutualisms.     

EWI-2 association with α-actinin regulates T cell immune synapses and HIV viral infection

EWI motif-containing protein 2 (EWI-2) is a member of the Ig superfamily that links tetraspanin-enriched microdomains to the actin cytoskeleton. We found that EWI-2 colocalizes with CD3 and CD81 at the central supramolecular activation cluster of the T cell immune synapse. Silencing of the endogenous expression or overexpression of a cytoplasmic truncated mutant of EWI-2 in T cells increases IL-2 secretion upon Ag stimulation. Mass spectrometry experiments of pull-downs with the C-term intracellular domain of EWI-2 revealed the specific association of EWI-2 with the actin-binding protein α-actinin; this association was regulated by PIP2. α-Actinin regulates the immune synapse formation and is required for efficient T cell activation. We extended these observations to virological synapses induced by HIV and found that silencing of either EWI-2 or α-actinin-4 increased cell infectivity. Our data suggest that the EWI-2-α-actinin complex is involved in the regulation of the actin cytoskeleton at T cell immune and virological synapses, providing a link between membrane microdomains and the formation of polarized membrane structures involved in T cell recognition.     

Synthesis and SAR of potent inhibitors of the Hepatitis C virus NS3/4A protease: Exploration of P2 quinazoline substituents

Novel NS3/4A protease inhibitors comprising quinazoline derivatives as P2 substituent were synthesized. High potency inhibitors displaying advantageous PK properties have been obtained through the optimization of quinazoline P2 substituents in three series exhibiting macrocyclic P2 cyclopentane dicarboxylic acid and P2 proline urea motifs. For the quinazoline moiety it was found that 8-methyl substitution in the P2 cyclopentane dicarboxylic acid series improved on the metabolic stability in human liver microsomes. By comparison, the proline urea series displayed advantageous Caco-2 permeability over the cyclopentane series. Pharmacokinetic properties in vivo were assessed in rat on selected compounds, where excellent exposure and liver-to-plasma ratios were demonstrated for a member of the 14-membered quinazoline substituted P2 proline urea series.     

Non-stationarity and local approaches to modelling the distributions of wildlife

Despite a growing interest in species distribution modelling, relatively little attention has been paid to spatial autocorrelation and non-stationarity. Both spatial autocorrelation (the tendency for adjacent locations to be more similar than distant ones) and non-stationarity (the variation in modelled relationships over space) are likely to be common properties of ecological systems. This paper focuses on non-stationarity and uses two local techniques, geographically weighted regression (GWR) and varying coefficient modelling (VCM), to assess its impact on model predictions. We extend two published studies, one on the presence–absence of calandra larks in Spain and the other on bird species richness in Britain, to compare GWR and VCM with the more usual global generalized linear modelling (GLM) and generalized additive modelling (GAM). For the calandra lark data, GWR and VCM produced better-fitting models than GLM or GAM. VCM in particular gave significantly reduced spatial autocorrelation in the model residuals. GWR showed that individual predictors became stationary at different spatial scales, indicating that distributions are influenced by ecological processes operating over multiple scales. VCM was able to predict occurrence accurately on independent data from the same geographical area as the training data but not beyond, whereas the GAM produced good results on all areas. Individual predictions from the local methods often differed substantially from the global models. For the species richness data, VCM and GWR produced far better predictions than ordinary regression. Our analyses suggest that modellers interpolating data to produce maps for practical actions (e.g. conservation) should consider local methods, whereas they should not be used for extrapolation to new areas. We argue that local methods are complementary to global methods, revealing details of habitat associations and data properties which global methods average out and miss.     

Compositional and immunobiological analyses of extracellular vesicles released by Candida albicans

The release of extracellular vesicles (EV) by fungal organisms is considered an alternative transport mechanism to trans‐cell wall passage of macromolecules. Previous studies have revealed the presence of EV in culture supernatants from fungal pathogens, such as Cryptococcus neoformans, Histoplasma capsulatum, Paracoccidioides brasiliensis, Sporothrix schenckii, Malassezia sympodialis and Candida albicans. Here we investigated the size, composition, kinetics of internalization by bone marrow‐derived murine macrophages (MO) and dendritic cells (DC), and the immunomodulatory activity of C. albicans EV. We also evaluated the impact of EV on fungal virulence using the Galleria mellonella larvae model. By transmission electron microscopy and dynamic light scattering, we identified two populations ranging from 50 to 100 nm and 350 to 850 nm. Two predominant seroreactive proteins (27 kDa and 37 kDa) and a group of polydispersed mannoproteins were observed in EV by immunoblotting analysis. Proteomic analysis of C. albicans EV revealed proteins related to pathogenesis, cell organization, carbohydrate and lipid metabolism, response to stress, and several other functions. The major lipids detected by thin‐layer chromatography were ergosterol, lanosterol and glucosylceramide. Short exposure of MO to EV resulted in internalization of these vesicles and production of nitric oxide, interleukin (IL)‐12, transforming growth factor‐beta (TGF‐β) and IL‐10. Similarly, EV‐treated DC produced IL‐12p40, IL‐10 and tumour necrosis factor‐alpha. In addition, EV treatment induced the up‐regulation of CD86 and major histocompatibility complex class‐II (MHC‐II). Inoculation of G. mellonella larvae with EV followed by challenge with C. albicans reduced the number of recovered viable yeasts in comparison with infected larvae control. Taken together, our results demonstrate that C. albicans EV were immunologically active and could potentially interfere with the host responses in the setting of invasive candidiasis.     

Lipid partitioning at the nuclear envelope controls membrane biogenesis

Partitioning of lipid precursors between membranes and storage is crucial for cell growth, and its disruption underlies pathologies such as cancer, obesity, and type 2 diabetes. However, the mechanisms and signals that regulate this process are largely unknown. In yeast, lipid precursors are mainly used for phospholipid synthesis in nutrient-rich conditions in order to sustain rapid proliferation but are redirected to triacylglycerol (TAG) stored in lipid droplets during starvation. Here we investigate how cells reprogram lipid metabolism in the endoplasmic reticulum. We show that the conserved phosphatidate (PA) phosphatase Pah1, which generates diacylglycerol from PA, targets a nuclear membrane subdomain that is in contact with growing lipid droplets and mediates TAG synthesis. We find that cytosol acidification activates the master regulator of Pah1, the Nem1-Spo7 complex, thus linking Pah1 activity to cellular metabolic status. In the absence of TAG storage capacity, Pah1 still binds the nuclear membrane, but lipid precursors are redirected toward phospholipids, resulting in nuclear deformation and a proliferation of endoplasmic reticulum membrane. We propose that, in response to growth signals, activation of Pah1 at the nuclear envelope acts as a switch to control the balance between membrane biogenesis and lipid storage.     

TRAF family proteins link PKR with NF-kappa B activation

The double-stranded RNA (dsRNA)-dependent protein kinase PKR activates NF-kappa B via the I kappa B kinase (IKK) complex, but little is known about additional molecules that may be involved in this pathway. Analysis of the PKR sequence enabled us to identify two putative TRAF-interacting motifs. The viability of such an interaction was further suggested by computer modeling. Here, we present evidence of the colocalization and physical interaction between PKR and TRAF family proteins in vivo, as shown by immunoprecipitation and confocal microscopy experiments. This interaction is induced upon PKR dimerization. Most importantly, we show that the binding between PKR and TRAFs is functionally relevant, as observed by the absence of NF-kappa B activity upon PKR expression in cells genetically deficient in TRAF2 and TRAF5 or after expression of TRAF dominant negative molecules. On the basis of sequence information and mutational and computer docking analyses, we favored a TRAF-PKR interaction model in which the C-terminal domain of TRAF binds to a predicted TRAF interaction motif present in the PKR kinase domain. Altogether, our data suggest that TRAF family proteins are key components located downstream of PKR that have an important role in mediating activation of NF-kappa B by the dsRNA-dependent protein kinase.     

The role of divalent cations in structure and function of murine adenosine deaminase.

For murine adenosine deaminase, we have determined that a single zinc or cobalt cofactor bound in a high affinity site is required for catalytic function while metal ions bound at an additional site(s) inhibit the enzyme. A catalytically inactive apoenzyme of murine adenosine deaminase was produced by dialysis in the presence of specific zinc chelators in an acidic buffer. This represents the first production of the apoenzyme and demonstrates a rigorous method for removing the occult cofactor. Restoration to the holoenzyme is achieved with stoichiometric amounts of either Zn2+ or Co2+ yielding at least 95% of initial activity. Far UV CD and fluorescence spectra are the same for both the apo- and holoenzyme, providing evidence that removal of the cofactor does not alter secondary or tertiary structure. The substrate binding site remains functional as determined by similar quenching measured by tryptophan fluorescence of apo- or holoenzyme upon mixing with the transition state analog, deoxycoformycin. Excess levels of adenosine or N6- methyladenosine incubated with the apoenzyme prior to the addition of metal prevent restoration, suggesting that the cofactor adds through the substrate binding cleft. The cations Ca2+, Cd2+, Cr2+, Cu+, Cu2+, Mn2+, Fe2+, Fe3+, Pb2+, or Mg2+ did not restore adenosine deaminase activity to the apoenzyme. Mn2+, Cu2+, and Zn2+ were found to be competitive inhibitors of the holoenzyme with respect to substrate and Cd2+ and Co2+ were noncompetitive inhibitors. Weak inhibition (Ki > or = 1000 microM) was noted for Ca2+, Fe2+, and Fe3+.     

The menstrual cycle of the spider monkey (Ateles geoffroyi)

The ovarian cycles of four adult female spider monkeys (Ateles geoffroyi) were followed daily throughout 30 days by means of vaginal swabs and blood samplings. Cytological analyses of the vaginal swabs and radioimmunoassay determination of the daily levels of estradiol-17β (E₂) and progesterone (P₄) were done in order to classify the kind of ovarian cycle of this species. Our results show that Ateles geoffroyi females display menstrual cycles of about 24 days on average. By comparison with the well-known menstrual cycles of women, apes, and Old World monkeys, the four distinctive cytological phases (bleeding, follicular, periovulatory, and luteal) could be recognized; mid-cycle E₂ peaks followed by mid-luteal increases of the same hormone were present in all four females. P₄ levels were higher after the E₂ peak, although both hormones were present throughout the cycles. Also, age-dependent features, hormone profiles, and changes in menstrual phases lengths were detected. Am. J. Primatol. 44:183–195, 1998. © 1998 Wiley-Liss, Inc.