Recovery of cytopathogenic and noncytopathogenic bovine viral diarrhea viruses from cDNA constructs.

After cDNA cloning of the genome of bovine viral diarrhea virus (BVDV) isolate CP7, a full-length cDNA clone was constructed. RNA transcribed in vitro from this construct was shown to direct the generation of infectious BVDV upon transfection into bovine cells. To confirm the de novo generation of infectious BVDV from cloned cDNA a genetically tagged virus was constructed. In comparison with parental BVDV, the recombinant virus was slightly retarded in growth. The NS2 coding region of the CP7 genome contains a duplication of 27 nucleotides which is not present in the genome of its noncytopathogenic counterpart, NCP7. Exchange of a small fragment harboring this insertion against the corresponding part of the NCP7 sequence led to recovery of noncytopathogenic BVDV. Alteration of the construct by introduction of a fragment derived from a cytopathogenic BVDV defective interfering particle resulted in a chimeric defective interfering particle which exhibits a cytopathogenic phenotype. These findings confirm the hypothesis that the recombination-induced alterations in the genomes of cytopathogenic BVDV are responsible for the induction of cell lysis.     

Early origin of foraminifera suggested by SSU rRNA gene sequences

Foraminifera are one of the largest groups of unicellular eukaryotes with probably the best known fossil record. However, the origin of foraminifera and their phylogenetic relationships with other eukaryotes are not well established. In particular, two recent reports, based on ribosomal RNA gene sequences, have reached strikingly different conclusions about foraminifera's evolutionary position within eukaryotes. Here, we present the complete small subunit (SSU) rRNA gene sequences of three species of foraminifera. Phylogenetic analysis of these sequences indicates that they branch very deeply in the eukaryotic evolutionary tree: later than those of the amitochondrial Archezoa, but earlier than those of the Euglenozoa and other mitochondria-bearing phyla. Foraminifera are clearly among the earliest eukaryotes with mitochondria, but because of the peculiar nature of their SSU genes we cannot be certain that they diverged first, as our data suggest.      

Climate change as a possible driver of invasion and differential in HSP70 expression in two genetically distinct populations of the invasive killer shrimp, <Emphasis Type="Italic">Dikerogammarus villosus</Emphasis>

Global climate change is known to affect physiological processes in charge of cellular stress response. That often results in forcing many organisms to shift their biogeographic distribution ranges. It also holds true for euryoecious and highly invasive species like the killer shrimp, Dikerogammarus villosus. In this study we compare the level of response to thermal stress in two genetically diversified populations of the amphipod D. villosus on the cellular level, namely HSP70 expression. The results show clear difference in HSP70 expression, that can be a direct consequence of the different climatic conditions both populations faced along their invasion routes. We conclude that the eastern population of D. villosus is more sensitive to thermal stress than the western population, hence its invasion potential may be lower than that of the latter. Considering the thermal tolerance of both populations and global warming, we can make some predictions about further spread of D. villosus, including the possibility of an emergence of the super-invader that may arise after cross-breeding of both populations, imposing even larger threat to the freshwater ecosystems.     

Cell cycle-regulated phosphorylation of p21-activated kinase 1

Mammalian p21-activated kinase 1 (Pak1) is a highly conserved effector for the small GTPases Cdc42 and Rac1. In lower eukaryotes, Pak1 homologs are regulated during the cell cycle by phosphorylation. Here, we show that Pak1 is phosphorylated during mitosis in mammalian fibroblasts. This phosphorylation occurs at a single site, Thr 212, within a domain that is unique to Pak1. Cdc2 phosphorylates Pak1 at the identical site in vitro, and inhibition of Cdc2 abolishes Pak1 mitotic phosphorylation in vivo, indicating that Cdc2 is the kinase responsible for phosphorylating Pak1 in mitotic cells. Expression of a Pak1 mutant in which Thr 212 is replaced with a phosphomimic (aspartic acid) has marked effects on the rate and extent of postmitotic spreading of fibroblasts. The mitotic phosphorylation of Pak1 does not alter the basal or Rac-stimulated activity of this kinase, but it does affect the coimmunoprecipitation of at least three proteins with Pak1. These findings are the first to implicate a mammalian Pak in cell cycle regulation and suggest that Pakl, as a result of phosphorylation by Cdc2, alters its association with binding partners and/or substrates that are relevant to the morphologic changes associated with cell division.     

The zoogeography of algae-associated peracarids along the Pacific coast of Chile

Abstract Aim To describe the zoogeography of the algae‐associated peracarid crustaceans from exposed rocky shores along the SE‐Pacific. Location Chile, 18° S to 42° S. Methods A standardized sampling programme was used at all sites. Samples of macroalgae were taken at twenty sites distributed along the entire study area. Quantitative samples (n = 6 replicates of 8 cm^?2 surface area each) of calcareous and non‐calcareous red algae were taken in the low intertidal, preserved immediately in 4%‐formalin and washed over a 0.2‐mm mesh before sorting. All peracarid individuals were sorted, identified to the species level and then categorized in separate functional groups according to their feeding habits. Graphical representations of species replacement within each functional group along the latitudinal gradient are provided. A classification analysis employing the unweighted paired group method using arithmetic average (UPGMA) was conducted in order to reveal the main zoogeographical zones. Results A total of forty epifaunal peracarid species was found. A gradual replacement of species within different functional groups (grazing and suspension‐feeding species) was observed in the central region (c. 26° S?37° S). In this central region, species with northern and those with southern distribution overlapped, while other species were only found here, resulting in high species richness. The number of species/site/algal species in the northern (north of c. 25.5° S) and southern region (south of c. 38.5° S) was considerably lower than in the central region. The distribution of most grazing peracarids showed a more continuous pattern than that of suspension‐feeding amphipods. The distribution of the remaining species (predators, scavengers, deposit‐feeders, unknowns) was scattered along the examined sites. The cluster analysis for the epifaunal peracarid assemblage confirmed the separation of a northern and southern zone connected by a central (transitional) zone between c. 26° S and c. 37° S. Similar zonation patterns have been found by most other studies on the zoogeography of the Chilean coast, although little agreement exists about the exact limits of this transitional zone. It is discussed that the distribution limits of algae‐associated peracarids (and other macroinvertebrates) – particularly in the transitional zone – may show interannual variations as a result of varying oceanographic conditions. The large affinity of the algae‐associated peracarid fauna from the central and southern Chilean coast to those of other regions indicates that dispersal may be facilitated by rafting with floating algae transported in the Antarctic Circumpolar Current. Main conclusions The zoogeographical analysis of algae‐associated peracarids confirms the existence of a northern and a southern zone connected by an extensive transitional zone. General biology, habitat use and the abundant presence of dispersal vectors such as floating macroalgae may affect the zoogeography of species living in transitional zones with strong interannual variations in current regimes. In these areas, species associated with substrata of high dispersal potential may show different distribution patterns than species inhabiting other substrata.     

Highly Predictive Model for a Protective Immune Response to the A(H1N1)pdm2009 Influenza Strain after Seasonal Vaccination

Understanding the immune response after vaccination against new influenza strains is highly important in case of an imminent influenza pandemic and for optimization of seasonal vaccination strategies in high risk population groups, especially the elderly. Models predicting the best sero-conversion response among the three strains in the seasonal vaccine were recently suggested. However, these models use a large number of variables and/or information post- vaccination. Here in an exploratory pilot study, we analyzed the baseline immune status in young (<31 years, N = 17) versus elderly (≥50 years, N = 20) donors sero-negative to the newly emerged A(H1N1)pdm09 influenza virus strain and correlated it with the serological response to that specific strain after seasonal influenza vaccination. Extensive multi-chromatic FACS analysis (36 lymphocyte sub-populations measured) was used to quantitatively assess the cellular immune status before vaccination. We identified CD4⁺ T cells, and amongst them particularly naive CD4⁺ T cells, as the best correlates for a successful A(H1N1)pdm09 immune response. Moreover, the number of influenza strains a donor was sero-negative to at baseline (NSSN) in addition to age, as expected, were important predictive factors. Age, NSSN and CD4⁺ T cell count at baseline together predicted sero-protection (HAI≥40) to A(H1N1)pdm09 with a high accuracy of 89% (p-value = 0.00002). An additional validation study (N = 43 vaccinees sero-negative to A(H1N1)pdm09) has confirmed the predictive value of age, NSSN and baseline CD4⁺ counts (accuracy = 85%, p-value = 0.0000004). Furthermore, the inclusion of donors at ages 31–50 had shown that the age predictive function is not linear with age but rather a sigmoid with a midpoint at about 50 years. Using these results we suggest a clinically relevant prediction model that gives the probability for non-protection to A(H1N1)pdm09 influenza strain after seasonal multi-valent vaccination as a continuous function of age, NSSN and baseline CD4 count.     

Comparative effectiveness of sugar beet microsatellite markers isolated from genomic libraries and GenBank ESTs to map the sugar beet genome

Sugar beet (Beta vulgaris) is an important root crop for sucrose production. A study was conducted to find a new abundant source of microsatellite (SSR) markers in order to develop marker assistance for breeding. Different sources of existing microsatellites were used and new ones were developed to compare their efficiency to reveal diversity in mapping population and mapping coverage. Forty-one microsatellite markers were isolated from a B. vulgaris ssp maritima genomic library and 201 SSRs were extracted from a B. vulgaris ssp vulgaris library. Data mining was applied on GenBank B. vulgaris expressed sequence tags (ESTs), 803 EST-SSRs were identified over 19,709 ESTs. Characteristics, polymorphism and cross-species transferability of these microsatellites were compared. Based on these markers, a high density genetic map was constructed using 92 F₂ individuals from a cross between a sugar and a table beet. The map contains 284 markers, spans over 555 cM and covers the nine chromosomes of the species with an average markers density of one marker every 2.2 cM. A set of markers for assignation to the nine chromosomes of sugar beet is provided.     

Characterization of the <Emphasis Type="Italic">Trichomonas vaginalis</Emphasis> surface-associated AP65 and binding domain interacting with trichomonads and host cells

Background  

AP65 is a prominent adhesin of Trichomonas vaginalis that mediates binding of parasites to host vaginal epithelial cells (VECs). AP65 with no secretion signal sequence, membrane targeting peptide, and anchoring motif was recently found to be secreted.