Modulation of glucan-enzyme interactions by domain V in GTF-SI from Streptococcus mutans

Glucansucrase GTF-SI from Streptococcus mutans is a multidomain enzyme that catalyzes the synthesis of glucan polymers. Domain V locates 100 Å from the catalytic site and is required for an optimal activity. Nevertheless, the mechanism governing its functional role remains elusive. In this work, homology modeling and molecular dynamics simulations were employed to examine the effect of domain V in the structure and glucan-binding ability of GTF-SI in full and truncated enzyme models. Our results showed that domain V increases the flexibility of the α4′-loop-α4″ motif near the catalytic site resulting in a higher surface for glucan association, and modulates the orientation of a growing oligosaccharide (N=8-23) in glucan-enzyme complexes towards engaging in favorable contacts throughout the protein, whereas in the truncated model the glucan protrudes randomly from domain B towards the solvent. These results are valuable to increase understanding about the functional role of domain V in GH70 glucansucrases.     

Cuttlefish see shape from shading,fine-tuning coloration in response to pictorial depth cues and directional illumination

Humans use shading as a cue to three-dimensional form by combining low-level information about light intensity with high-level knowledge about objects and the environment. Here, we examine how cuttlefish Sepia officinalis respond to light and shadow to shade the white square (WS) feature in their body pattern. Cuttlefish display the WS in the presence of pebble-like objects, and they can shade it to render the appearance of surface curvature to a human observer, which might benefit camouflage. Here we test how they colour the WS on visual backgrounds containing two-dimensional circular stimuli, some of which were shaded to suggest surface curvature, whereas others were uniformly coloured or divided into dark and light semicircles. WS shading, measured by lateral asymmetry, was greatest when the animal rested on a background of shaded circles and three-dimensional hemispheres, and less on plain white circles or black/white semicircles. In addition, shading was enhanced when light fell from the lighter side of the shaded stimulus, as expected for real convex surfaces. Thus, the cuttlefish acts as if it perceives surface curvature from shading, and takes account of the direction of illumination. However, the direction of WS shading is insensitive to the directions of background shading and illumination; instead the cuttlefish tend to turn to face the light source.     

Influence of N-linked glycosylation of porcine reproductive and respiratory syndrome virus GP5 on virus infectivity, antigenicity, and ability to induce neutralizing antibodies

Porcine reproductive and respiratory syndrome virus (PRRSV) glycoprotein 5 (GP5) is the most abundant envelope glycoprotein and a major inducer of neutralizing antibodies in vivo. Three putative N-linked glycosylation sites (N34, N44, and N51) are located on the GP5 ectodomain, where a major neutralization epitope also exists. To determine which of these putative sites are used for glycosylation and the role of the glycan moieties in the neutralizing antibody response, we generated a panel of GP5 mutants containing amino acid substitutions at these sites. Biochemical studies with expressed wild-type (wt) and mutant proteins revealed that the mature GP5 contains high-mannose-type sugar moieties at all three sites. These mutations were subsequently incorporated into a full-length cDNA clone. Our data demonstrate that mutations involving residue N44 did not result in infectious progeny production, indicating that N44 is the most critical amino acid residue for infectivity. Viruses carrying mutations at N34, N51, and N34/51 grew to lower titers than the wt PRRSV. In serum neutralization assays, the mutant viruses exhibited enhanced sensitivity to neutralization by wt PRRSV-specific antibodies. Furthermore, inoculation of pigs with the mutant viruses induced significantly higher levels of neutralizing antibodies against the mutant as well as the wt PRRSV, suggesting that the loss of glycan residues in the ectodomain of GP5 enhances both the sensitivity of these viruses to in vitro neutralization and the immunogenicity of the nearby neutralization epitope. These results should have great significance for development of PRRSV vaccines of enhanced protective efficacy.     

Novel Nuclear Protein Complexes of Dystrophin 71 Isoforms in Rat Cultured Hippocampal GABAergic and Glutamatergic Neurons

The precise functional role of the dystrophin 71 in neurons is still elusive. Previously, we reported that dystrophin 71d and dystrophin 71f are present in nuclei from cultured neurons. In the present work, we performed a detailed analysis of the intranuclear distribution of dystrophin 71 isoforms (Dp71d and Dp71f), during the temporal course of 7-day postnatal rats hippocampal neurons culture for 1h, 2, 4, 10, 15 and 21 days in vitro (DIV). By immunofluorescence assays, we detected the highest level of nuclear expression of both dystrophin Dp71 isoforms at 10 DIV, during the temporal course of primary culture. Dp71d and Dp71f were detected mainly in bipolar GABAergic (≥60%) and multipolar Glutamatergic (≤40%) neurons, respectively. We also characterized the existence of two nuclear dystrophin-associated protein complexes (DAPC): dystrophin 71d or dystrophin 71f bound to β-dystroglycan, α1-, β-, α2-dystrobrevins, α-syntrophin, and syntrophin-associated protein nNOS (Dp71d-DAPC or Dp71f-DAPC, respectively), in the hippocampal neurons. Furthermore, both complexes were localized in interchromatin granule cluster structures (nuclear speckles) of neuronal nucleoskeleton preparations. The present study evinces that each Dp71’s complexes differ slightly in dystrobrevins composition. The results demonstrated that Dp71d-DAPC was mainly localized in bipolar GABAergic and Dp71f-DAPC in multipolar Glutamatergic hippocampal neurons. Taken together, our results show that dystrophin 71d, dystrophin 71f and DAP integrate protein complexes, and both complexes were associated to nuclear speckles structures.     

Comparison of bacterial diversity in full scale anammox bioreactors operated under different conditions

Bacterial community structure of full‐scale anammox bioreactor is still mainly unknown. It has never been analyzed whether different anammox bioreactor configurations might result in the development of different bacterial community structures among these systems. In this work, the bacterial community structure of six full‐scale autotrophic nitrogen removal bioreactors located in The Netherlands and China operating under three different technologies and with different influent wastewater characteristics was studied by the means of pyrotag sequencing evaluation of the bacterial assemblage yielded a great diversity in all systems. The most represented phyla were the Bacteroidetes and the Proteobacteria, followed by the Planctomycetes. 14 OTUs were shared by all bioreactors, but none of them belonged to the Brocadiales order. Statistical analysis at OTU level showed that differences in the microbial communities were high, and that the main driver of the bacterial assemblage composition was different for the distinct phyla identified in the six bioreactors, depending on bioreactor technology or influent wastewater characteristics. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1464–1472, 2015     

Comparison of adjuvant efficacy of herpes simplex virus type 1 recombinant viruses expressing TH1 and TH2 cytokine genes

The adjuvant effects of cytokines in humoral and cell-mediated immunity to herpes simplex virus type 1 (HSV-1) have been examined in mice using HSV-1 recombinant viruses expressing murine interleukin-2 (IL-2), IL-4, or gamma interferon (IFN-gamma) gene. Groups of naive BALB/c mice were immunized intraperitoneally with one or three doses of the HSV-1 recombinant viruses expressing IL-2, IL-4, or IFN-gamma or with parental control virus. Despite similar replication kinetics, these three recombinant viruses elicited different immune responses to HSV-1 on immunization. Immunization with the recombinant virus expressing IL-4 elicited a humoral response of greater magnitude than immunization with the recombinant viruses expressing IL-2 or IFN-gamma or with parental virus. In contrast, immunization with recombinant virus expressing IL-2 elicited a higher cytotoxic T-cell response than immunization with viruses expressing IL-4 or IFN-gamma. Stimulation in vitro of splenocytes obtained from the mice immunized with UV-inactivated HSV-1 McKrae resulted in a T(H)1 pattern of cytokine expression irrespective of the recombinant virus used in the immunization. As observed for the parental virus, both CD4(+) and CD8(+) T cells contributed equally to the production of IL-2 by the splenocytes of mice immunized with any of the three recombinant viruses. However, the pattern of IFN-gamma production by CD4(+) and CD8(+) T cells differed according to the recombinant virus used. After lethal ocular challenge, all immunized mice were protected completely against death and manifestations of eye disease caused by HSV-1, which are typical responses in unimmunized mice. Mice immunized with IL-4-expressing virus cleared the virus from their eyes more rapidly than mice immunized with IL-2- or IFN-gamma-expressing virus. Taken together, our results suggest that, in contrast to IFN-gamma which did not exhibit an adjuvant effect, both IL-4 and IL-2 act as adjuvants in immunization with HSV, with IL-4 showing greater efficacy.     

Spectral sensitivities of photoreceptors and lamina monopolar cells in the dragonfly,Hemicordulia tau

Summary Five spectral types of photoreceptors with peak sensitivities at 330 nm, 410 nm, 460 nm, 525 nm and 630 nm were recorded from the ventral eye of the dragonfly, Hemicordulia tau. Often the 525 nm photoreceptors presented broader, and the 630 nm photoreceptors narrower, spectral sensitivities than would be excepted of a photopigment with the same peak sensitivity. Four types of lamina monopolar cells (cell types 1–4) were recognised from their dark-adapted spectral sensitivities and their anatomy. The anatomical identification allows tentative assignation to the monopolar cell classification from Sympetrum rubicundulum obtained using Golgi staining (Meinertzhagen and Armett-Kibel 1982). When dark-adapted, the monopolar cells had peak spectral sensitivities that were similar to single photoreceptors or appeared to pool receptor outputs, but in some cases spectral sensitivity changed markedly upon adaptation to white and to chromatic light, in one case (cell type 2) apparently switching off a UV-sensitive input.     

Recovery of Mycobacterium bovis from Soft Fresh Cheese Originating in Mexico

Recent outbreaks of human tuberculosis in the United States caused by Mycobacterium bovis have implicated cheese originating in Mexico as a source of these infections. A total of 203 samples of cheese originating in Mexico were cultured, and M. bovis was recovered from one specimen. Therefore, M. bovis can be recovered from cheese and may be a source of human infections.     

Germination and soil seed bank traits of Podocarpus angustifolius (Podocarpaceae): an endemic tree species from Cuban rain forests

Podocarpus angustifolius is an endangered recalcitrant-seeded small tree, endemic to mountain rain forests in the central and Pinar del Río regions in Cuba. In this study, the germination patterns of P. angustifolius seeds were evaluated and the nature of the soil seed bank was determined. Using a weighted two-factor design, we analyzed the combined germination response to seed source (i.e. freshly matured seeds directly collected from trees versus seeds extracted from soil samples) and pretreatment (i.e. seed water-immersion for 48h at room temperature). Germination was delayed for four weeks (= 30 days) in all cases, regardless of both factors analyzed. Moreover, nine additional days were necessary to achieve high germination values (in the case of fresh, pretreated seeds). These results overall may indicate the existence of a non-deep simple morphophysiological dormancy in P. angustifolius seeds. The water-immersion significantly enhanced seed germination, probably as a result of the hydration of recalcitrant seeds. Although germination of seeds extracted from soil samples was low, probably due to aging and pathogen effects throughout the time of burial, the study revealed the existence of a persistent soil seed bank (at least short-termed) of approximately 42 viable seeds per m2 in the upper 10cm of soil. Such a record is noteworthy since references to persistent soil seed banks in recalcitrant-seeded species are scarce in the literature. The population consequences derived from the formation of persistent soil seed banks in this endangered species are discussed.