Common Antiviral Cytotoxic T-Lymphocyte Epitope for Diverse Arenaviruses

Members of the Arenaviridae family have been isolated from mammalian hosts in disparate geographic locations, leading to their grouping as Old World types (i.e., lymphocytic choriomeningitis virus [LCMV], Lassa fever virus [LFV], Mopeia virus, and Mobala virus) and New World types (i.e., Junin, Machupo, Tacaribe, and Sabia viruses) (C. J. Peters, M. J. Buchmeier, P. E. Rollin, and T. G. Ksiazek, p. 1521-1551, in B. N. Fields, D. M. Knipe, and P. M. Howley [ed.], Fields virology, 3rd ed., 1996; P. J. Southern, p. 1505-1519, in B. N. Fields, D. M. Knipe, and P. M. Howley [ed.], Fields virology, 3rd ed., 1996). Several types in both groups-LFV, Junin, Machupo, and Sabia viruses-cause severe and often lethal human diseases. By sequence comparison, we noted that eight Old World and New World arenaviruses share several amino acids with the nucleoprotein (NP) that consists of amino acids (aa) 118 to 126 (NP 118-126) (RPQASGVYM) of LCMV that comprise the immunodominant cytotoxic T-lymphocyte (CTL) epitope for H-2(d) mice (32). This L(d)-restricted epitope constituted >97% of the total bulk CTLs produced in the specific antiviral or clonal responses of H-2(d) BALB mice. NP 118-126 of the Old World arenaviruses LFV, Mopeia virus, and LCMV and the New World arenavirus Sabia virus bound at high affinity to L(d). The primary H-2(d) CTL anti-LCMV response as well as that of a CTL clone responsive to LCMV NP 118-126 recognized target cells coated with NP 118-126 peptides derived from LCMV, LFV, and Mopeia virus but not Sabia virus, indicating that a common functional NP epitope exists among Old World arenaviruses. Use of site-specific amino acid exchanges in the NP CTL epitope among these arenaviruses identified amino acids involved in major histocompatibility complex binding and CTL recognition.     

AP-1 and AP-3 mediate sorting of melanosomal and lysosomal membrane proteins into distinct post-Golgi trafficking pathways

The adaptor complexes AP-1 and AP-3 are localized to endosomes and/or the trans Golgi network (TGN). Because of limitations in analysing intracellular adaptor function directly, their site of function is a matter of ongoing uncertainty. To overcome this problem and to analyse adaptor sorting at the TGN, we reconstituted vesicle formation from Golgi/TGN-enriched membranes in a novel in vitro budding assay. Melanocytes were metabolically labelled followed by a 19°C temperature block to accumulate newly synthesized proteins in Golgi membranes, which were then enriched by subcellular fractionation and used as donor membranes for vesicle formation in vitro . The incorporation of the melanosomal proteins tyrosinase and tyrosinase-related protein 1 (TRP-1) as well as Lamp-1 and 46 kDa mannose-6-phosphate receptor (MPR46) into Golgi/TGN-derived vesicles was temperature, nucleotide, cytosol, ADP ribosylation factor 1 and adaptor dependent. We show that sorting of TRP-1 and MPR46 was AP-1 dependent, while budding of tyrosinase and Lamp-1 required AP-3. Depletion of clathrin inhibited sorting of all four cargo proteins, suggesting that AP-1 and AP-3 are involved in the formation of distinct types of clathrin-coated vesicles, each of which is characterized by the incorporation of specific cargo membrane proteins.     

Cooccurring Gentiana verna and Gentiana acaulis and Their Neighboring Plants in Two Swiss Upper Montane Meadows Harbor Distinct Arbuscular Mycorrhizal Fungal Communities

The community composition of arbuscular mycorrhizal fungi (AMF) was analyzed in roots of Gentiana verna, Gentiana acaulis, and accompanying plant species from two species-rich Swiss alpine meadows located in the same area. The aim of the study was to elucidate the impact of host preference or host specificity on the AMF community in the roots. The roots were analyzed by nested PCR, restriction fragment length polymorphism screening, and sequencing of ribosomal DNA small-subunit and internal transcribed spacer regions. The AMF sequences were analyzed phylogenetically and used to define monophyletic sequence types. The AMF community composition was strongly influenced by the host plant species, but compositions did not significantly differ between the two sites. Detailed analyses of the two cooccurring gentian species G. verna and G. acaulis, as well as of neighboring Trifolium spp., revealed that their AMF communities differed significantly. All three host plant taxa harbored AMF communities comprising multiple phylotypes from different fungal lineages. A frequent fungal phylotype from Glomus group B was almost exclusively found in Trifolium spp., suggesting some degree of host preference for this fungus in this habitat. In conclusion, the results indicate that within a relatively small area with similar soil and climatic conditions, the host plant species can have a major influence on the AMF communities within the roots. No evidence was found for a narrowing of the mycosymbiont spectrum in the two green gentians, in contrast to previous findings with their achlorophyllous relatives.     

Potential effects of oilseed rape expressing oryzacystatin-1 (OC-1) and of purified insecticidal proteins on larvae of the solitary bee Osmia bicornis

Despite their importance as pollinators in crops and wild plants, solitary bees have not previously been included in non-target testing of insect-resistant transgenic crop plants. Larvae of many solitary bees feed almost exclusively on pollen and thus could be highly exposed to transgene products expressed in the pollen. The potential effects of pollen from oilseed rape expressing the cysteine protease inhibitor oryzacystatin-1 (OC-1) were investigated on larvae of the solitary bee Osmia bicornis (= O. rufa). Furthermore, recombinant OC-1 (rOC-1), the Bt toxin Cry1Ab and the snowdrop lectin Galanthus nivalis agglutinin (GNA) were evaluated for effects on the life history parameters of this important pollinator. Pollen provisions from transgenic OC-1 oilseed rape did not affect overall development. Similarly, high doses of rOC-1 and Cry1Ab as well as a low dose of GNA failed to cause any significant effects. However, a high dose of GNA (0.1%) in the larval diet resulted in significantly increased development time and reduced efficiency in conversion of pollen food into larval body weight. Our results suggest that OC-1 and Cry1Ab expressing transgenic crops would pose a negligible risk for O. bicornis larvae, whereas GNA expressing plants could cause detrimental effects, but only if bees were exposed to high levels of the protein. The described bioassay with bee brood is not only suitable for early tier non-target tests of transgenic plants, but also has broader applicability to other crop protection products.     

Interaction network of the ribosome assembly machinery from a eukaryotic thermophile

Ribosome biogenesis in eukaryotic cells is a highly dynamic and complex process innately linked to cell proliferation. The assembly of ribosomes is driven by a myriad of biogenesis factors that shape pre‐ribosomal particles by processing and folding the ribosomal RNA and incorporating ribosomal proteins. Biochemical approaches allowed the isolation and characterization of pre‐ribosomal particles from Saccharomyces cerevisiae, which lead to a spatiotemporal map of biogenesis intermediates along the path from the nucleolus to the cytoplasm. Here, we cloned almost the entire set (～180) of ribosome biogenesis factors from the thermophilic fungus Chaetomium thermophilum in order to perform an in‐depth analysis of their protein–protein interaction network as well as exploring the suitability of these thermostable proteins for structural studies. First, we performed a systematic screen, testing about 80 factors for crystallization and structure determination. Next, we performed a yeast 2‐hybrid analysis and tested about 32,000 binary combinations, which identified more than 1000 protein–protein contacts between the thermophilic ribosome assembly factors. To exemplary verify several of these interactions, we performed biochemical reconstitution with the focus on the interaction network between 90S pre‐ribosome factors forming the ctUTP‐A and ctUTP‐B modules, and the Brix‐domain containing assembly factors of the pre‐60S subunit. Our work provides a rich resource for biochemical reconstitution and structural analyses of the conserved ribosome assembly machinery from a eukaryotic thermophile.     

Dynamics of an oligotrophic bacterial aquifer community during contact with a groundwater plume contaminated with benzene, toluene, ethylbenzene, and xylenes: an in situ mesocosm study

An in situ mesocosm system was designed to monitor the in situ dynamics of the microbial community in polluted aquifers. The mesocosm system consists of a permeable membrane pocket filled with aquifer material and placed within a polypropylene holder, which is inserted below groundwater level in a monitoring well. After a specific time period, the microcosm is recovered from the well and its bacterial community is analyzed. Using this system, we examined the effect of benzene, toluene, ethylbenzene, and xylene (BTEX) contamination on the response of an aquifer bacterial community by denaturing gradient gel electrophoresis analysis of PCR-amplified 16S rRNA genes and PCR detection of BTEX degradation genes. Mesocosms were filled with nonsterile or sterile aquifer material derived from an uncontaminated area and positioned in a well located in either the uncontaminated area or a nearby contaminated area. In the contaminated area, the bacterial community in the microcosms rapidly evolved into a stable community identical to that in the adjacent aquifer but different from that in the uncontaminated area. At the contaminated location, bacteria with tmoA- and xylM/xylE1-like BTEX catabolic genotypes colonized the aquifer, while at the uncontaminated location only tmoA-like genotypes were detected. The communities in the mesocosms and in the aquifer adjacent to the wells in the contaminated area consisted mainly of Proteobacteria. At the uncontaminated location, Actinobacteria and Proteobacteria were found. Our results indicate that communities with long-term stability in their structures follow the contamination plume and rapidly colonize downstream areas upon contamination.     

Synthetic nanopores as a test case for ion channel theories: the anomalous mole fraction effect without single filing

The predictions of a theory for the anomalous mole fraction effect (AMFE) are tested experimentally with synthetic nanopores in plastic. The negatively charged synthetic nanopores under consideration are highly cation selective and 50 Å in diameter at their smallest point. These pores exhibit an AMFE in mixtures of Ca²⁺ and monovalent cations. An AMFE occurs when the conductance through a pore is lower in a mixture of salts than in the pure salts at the same concentration. For ion channels, the textbook interpretation of the AMFE is that multiple ions move through the pore in coordinated, single-file motion. However, because the synthetic nanopores are so wide, their AMFE shows that single filing is not necessary for the AMFE. It is shown that the AMFE in the synthetic nanopores is explained by a theory of preferential ion selectivity. The unique properties of the synthetic nanopores allow us to experimentally confirm several predictions of this theory. These same properties make synthetic nanopores an interesting new platform to test theories of ion channel permeation and selectivity in general.     

Modeling Neisseria meningitidis B metabolism at different specific growth rates

Neisseria meningitidis is a human pathogen that can infect diverse sites within the human host. The major diseases caused by N. meningitidis are responsible for death and disability, especially in young infants. At the Netherlands Vaccine Institute (NVI) a vaccine against serogroup B organisms is currently being developed. This study describes the influence of the growth rate of N. meningitidis on its macro-molecular composition and its metabolic activity and was determined in chemostat cultures. In the applied range of growth rates, no significant changes in RNA content and protein content with growth rate were observed in N. meningitidis. The DNA content in N. meningitidis was somewhat higher at the highest applied growth rate. The phospholipid and lipopolysaccharide content in N. meningitidis changed with growth rate but no specific trends were observed. The cellular fatty acid composition and the amino acid composition did not change significantly with growth rate. Additionally, it was found that the PorA content in outer membrane vesicles was significantly lower at the highest growth rate. The metabolic fluxes at various growth rates were calculated using flux balance analysis. Errors in fluxes were calculated using Monte Carlo Simulation and the reliability of the calculated flux distribution could be indicated, which has not been reported for this type of analysis. The yield of biomass on substrate (Y(x/s)) and the maintenance coefficient (m(s)) were determined as 0.44 (+/-0.04) g g(-1) and 0.04 (+/-0.02) g g(-1) h(-1), respectively. The growth associated energy requirement (Y(x/ATP)) and the non-growth associated ATP requirement for maintenance (m(ATP)) were estimated as 0.13 (+/-0.04) mol mol(-1) and 0.43 (+/-0.14) mol mol(-1) h(-1), respectively. It was found that the split ratio between the Entner-Doudoroff and the pentose phosphate pathway, the sole glucose utilizing pathways in N. meningitidis, had a minor effect on ATP formation rate but a major effect on the fluxes going through for instance the citric-acid cycle. For this reason, we presented flux ranges for underdetermined parts of metabolic network rather than presenting single flux values, which is more commonly done in literature.     

Macrophage-specific responses to human- and animal-adapted tubercle bacilli reveal pathogen and host factors driving multinucleated cell formation

The Mycobacterium tuberculosis complex (MTBC) is a group of related pathogens that cause tuberculosis (TB) in mammals. MTBC species are distinguished by their ability to sustain in distinct host populations. While Mycobacterium bovis (Mbv) sustains transmission cycles in cattle and wild animals and causes zoonotic TB, M. tuberculosis (Mtb) affects human populations and seldom causes disease in cattle. The host and pathogen determinants underlying host tropism between MTBC species are still unknown. Macrophages are the main host cell that encounters mycobacteria upon initial infection, and we hypothesised that early interactions between the macrophage and mycobacteria influence species-specific disease outcome. To identify factors that contribute to host tropism, we analysed blood-derived primary human and bovine macrophages (hMϕ or bMϕ, respectively) infected with Mbv and Mtb. We show that Mbv and Mtb reside in different cellular compartments and differentially replicate in hMϕ whereas both Mbv and Mtb efficiently replicate in bMϕ. Specifically, we show that out of the four infection combinations, only the infection of bMϕ with Mbv promoted the formation of multinucleated giant cells (MNGCs), a hallmark of tuberculous granulomas. Mechanistically, we demonstrate that both MPB70 from Mbv and extracellular vesicles released by Mbv-infected bMϕ promote macrophage multinucleation. Importantly, we extended our in vitro studies to show that granulomas from Mbv-infected but not Mtb-infected cattle contained higher numbers of MNGCs. Our findings implicate MNGC formation in the contrasting pathology between Mtb and Mbv for the bovine host and identify MPB70 from Mbv and extracellular vesicles from bMϕ as mediators of this process.     

A novel model of cryoinjury-induced myocardial infarction in the mouse: a comparison with coronary artery ligation

Mouse myocardial infarction (MI) models are frequently used research tools. The most commonly applied model is coronary artery ligation. However, coronary ligation often gives rise to apical aneurysmatic infarcts of variable size. Other infarct models include cryoinfarction, which produces reproducible infarcts of the anterior wall. Thus far, this model has not been extensively described in mice. Therefore, we developed a murine cryoinfarction model and compared it with coronary ligation. Studies were performed under isoflurane anesthesia with a follow-up of 4 and 8 wk. Cryoinfarction was induced using a 2- or 3-mm cryoprobe. Two-dimensional guided M-mode echocardiography was used to assess fractional shortening and left ventricular (LV) dimensions at baseline and end point. At end point, hemodynamics were assessed using a 1.4-Fr Millar catheter. Pressure-diameter relations were constructed by combining echocardiography and hemodynamic data. Histological and morphometric analyses of infarct and remote areas were performed. At 4 wk, 3-mm cryoinfarction resulted in decreased LV fractional shortening as well as decreased global LV contractility and relaxation, which was comparable with coronary ligation. No adverse remodeling was observed at this time point, in contrast with the ligation model. However, progressive LV remodeling occured between 4 and 8 wk after cryoinfarction with a further decline in hemodynamic parameters and LV pump function. Histologically, cryoinfarction resulted in highly reproducible, transmural, cone-shaped infarcts with reperfusion at the macrovascular level. These results indicate that the cryoinfarction model represents the anterior myocardial infarct with modest adverse remodeling and may thus be representative for infarcts encountered in clinical practice.