Genes and traits associated with chromosome 2H and 5H regions controlling sensitivity of reproductive tissues to frost in barley

Frost at flowering can cause significant damage to cereal crops. QTL for low temperature tolerance in reproductive tissues (LTR tolerance) were previously described on barley 2HL and 5HL chromosome arms. With the aim of identifying potential LTR tolerance mechanisms, barley Amagi Nijo × WI2585 and Haruna Nijo × Galleon populations were examined for flowering time and spike morphology traits associated with the LTR tolerance loci. In spring-type progeny of both crosses, winter alleles at the Vrn-H1 vernalization response locus on 5H were linked in coupling with LTR tolerance and were unexpectedly associated with earlier flowering. In contrast, tolerance on 2HL was coupled with late flowering alleles at a locus we named Flt-2L. Both chromosome regions influenced chasmogamy/cleistogamy (open/closed florets), although tolerance was associated with cleistogamy at the 2HL locus and chasmogamy at the 5HL locus. LTR tolerance controlled by both loci was accompanied by shorter spikes, which were due to fewer florets per spike on 5HL, but shorter rachis internodes on 2HL. The Eps-2S locus also segregated in both crosses and influenced spike length and flowering time but not LTR tolerance. Thus, none of the traits was consistently correlated with LTR tolerance, suggesting that the tolerance may be due to some other visible trait or an intrinsic (biochemical) property. Winter alleles at the Vrn-H1 locus and short rachis internodes may be of potential use in barley breeding, as markers for selection of LTR tolerance at 5HL and 2HL loci, respectively. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Assembly and intracellular trafficking of HLA-B*3501 and HLA-B*3503

Residue 116 of major histocompatibility complex (MHC) class I heavy chains is an important determinant of assembly, that can influence rates of ER-Golgi trafficking, binding to the transporter associated with antigen processing (TAP), tapasin dependence of assembly, and the efficiency and specificity of peptide binding. Here, we investigated assembly and peptide-binding differences between HLA-B*3501(S116) and HLA-B*3503(F116), two alleles differing only at position 116 of the MHC class I heavy chain, that are associated respectively with normal or rapid AIDS progression. A reduced intracellular maturation rate was observed for HLA-B*3503 in HIV-infected and uninfected cells, which correlated with enhanced binding of HLA-B*3503 to TAP. No significant differences in the intrinsic efficiency of in vitro peptide binding by HLA-B*3501 and HLA-B*3503 were measurable with several common peptides or peptide libraries, and both allotypes were relatively tapasin-independent for their assembly. However, thermostability differences between the two allotypes were measurable in a CD4⁺ T cell line. These findings suggest that compared to HLA-B*3501, a reduced intracellular peptide repertoire for HLA-B*3503 could contribute to its slower intracellular trafficking and stronger association with rapid AIDS progression.     

Transmission of different strains of Plasmodium cynomolgi to Aotus nancymaae monkeys and relapse

Forty-four splenectomized Aotus nancymaae monkeys were infected with 6 different strains of Plasmodium cynomolgi, 11 via trophozoites and 33 via sporozoites. Sporozoites from Anopheles dirus, Anopheles freeborni, Anopheles gambiae, Anopheles maculatus, and Anopheles stephensi resulted in prepatent periods ranging from 9 to 39 days (median of 15 days). Importantly, relapse was demonstrated in 5 of 5 sporozoite-induced infections with the Rossan strain following treatment with chloroquine.     

A Y526Q Mutation in the Newcastle Disease Virus HN Protein Reduces Its Functional Activities and Attenuates Virus Replication and Pathogenicity

The hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) is a multifunctional protein that plays a crucial role in virus infectivity. In this study, using the mesogenic strain Beaudette C (BC), we mutated three conserved amino acids thought to be part of the binding/catalytic active site in the HN protein. We also mutated five additional residues near the proposed active site that are nonconserved between BC and the avirulent strain LaSota. The eight recovered NDV HN mutants were assessed for effects on biological activities. While most of the mutations had surprisingly little effect, mutation at conserved residue Y526 reduced the neuraminidase, receptor binding, and fusion activities and attenuated viral virulence in eggs and young birds.Newcastle disease virus (NDV) is an avian pathogen of the genus Avulavirus in the family Paramyxoviridae (10). The envelope of NDV contains two surface glycoproteins, the fusion (F) protein and the HN (hemagglutinin-neuraminidase [NA]) protein. The F protein mediates viral penetration and requires cleavage-activation by host protease. Cleavability of the F protein is a major determinant of virulence. However, other viral proteins, including HN, also contribute to virulence (5). HN is a multifunctional glycoprotein. It recognizes sialic acid-containing receptors on cell surfaces; promotes the fusion activity of F protein, thereby allowing the virus to penetrate the cell surface; and acts as an NA that removes sialic acid from progeny virus particles to prevent viral self-aggregation (9).HN is a type II homotetrameric glycoprotein with a monomer length of 577 amino acids for most NDV strains (14). The ectodomain of the HN protein consists of a 95-amino-acid stalk region supporting a 428-amino-acid terminal globular head. Although mutations in the transmembrane and stalk regions of the HN protein can affect the structure and activities of the protein (11, 15), the antigenic, receptor recognition, and NA active sites are all localized in the globular head (12, 16). The X-ray crystal structure of the globular head of the NDV HN protein has identified residues that appear to contribute to receptor recognition, NA, and fusion activities (4). Previous studies have proposed that conserved residues R174, I175, D198, K236, R416, R498, Y526, and E547 are important in receptor recognition and NA activities and that residues R174 and E547 influence the fusion promotion activity of the HN protein (3, 4, 6). Although transfection studies using plasmids expressing HN mutants of NDV have highlighted the importance of these residues in different biological functions of the HN protein, their contribution to NDV biology and pathogenesis in the context of the complete virus was not known.In this study, we examined the roles of three of the above-named conserved residues, R416, R498, and Y526 (all located near the sialic acid binding site), in the biological activities and pathogenesis of the HN protein of NDV in the context of infectious virus. In addition, comparison of the HN protein sequence between the avirulent strain LaSota and the moderately virulent strain Beaudette C (BC) identified 12 amino acid differences in the globular head region of the HN protein (H203, T214, I219, S228, L269, A271, E293, G310, S494, E495, T502, and N568, named according to the BC amino acid assignment). We also examined five of these nonconserved residues, T214, I219, S494, E495, and N568, located in close proximity to residues identified earlier by crystal structure studies, to determine whether these might affect HN function and contribute to the difference in pathogenicity between the LaSota and BC strains (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Three-dimensional structure of the NDV HN protein showing the positions of amino acid residues that were substituted in the present study. The residues are shown in space-filling mode and represented in different colors. The MacPymol (DeLano Scientific) software was used to generate the model of the globular domain of the NDV HN monomer. The structure was derived from the crystal structure of the NDV HN protein reported by Crennell et al. (4).We used site-directed mutagenesis (2) to introduce individual amino acid substitutions into a cDNA of the HN gene of strain BC. For the conserved residues, we changed arginine at positions 416 and 498 and tyrosine at position 526 to polar glutamine. For the nonconserved residues, the assignments T214, I219, S494, E495, and N568 of strain BC were altered to the corresponding assignments of strain LaSota: S214, V219, G494, V495, and D568, respectively. Each mutagenized HN gene was then inserted into a full-length cDNA clone of the BC antigenome. These clones were transfected into HEp2 cells, and mutant viruses were recovered as previously described (8). These viruses were designated according to the substitutions introduced: T214S, I219V, R416Q, S494G, E495V, R498Q, Y526Q, and N568D. The HN genes from recovered viruses were sequenced. This confirmed the presence of each introduced mutation and the lack of adventitious mutations in the HN gene. To determine the stability of each HN mutation, the recovered viruses were passaged five times in 9-day-old embryonated chicken eggs and five times in chicken embryo fibroblast DF-1 cells. Sequence analysis of the HN gene of the mutant viruses at each passage showed that the introduced mutations were unaltered (data not shown). To rule out the possibility that change in the HN protein sequence could be compensated for by a mutation in the F protein, the F gene from each recovered virus was sequenced. No compensatory mutations in the F gene were observed (data not shown). The HN protein content of each mutant virus, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie staining, was very similar to that of the parental BC virus (pBC) (Table ​(Table1).1). The multicycle growth kinetics of the recombinant HN mutant viruses in DF-1 cells (Fig. ​(Fig.2)2) showed that the replication kinetics of all of the HN mutant viruses were similar to those of pBC, with the exception of the Y526Q mutant, which showed delayed growth and had a lower virus yield (1.5 to 2.0 log₁₀ PFU/ml) than the parental and other mutant viruses. In addition, the Y526Q mutant produced syncytia at 72 h, whereas the parental and other mutant viruses initiated syncytia at 24 h postinfection. These studies showed the importance of amino acid residue Y526 at the active site of the HN protein of NDV.Open in a separate windowFIG. 2.Multicycle growth kinetics of HN mutants of NDV in chicken embryo fibroblast (DF-1) cells. Cells were infected with the indicated parental or mutant virus at an multiplicity of infection of 0.01. Supernatant samples were collected at 8-h intervals until 64 h postinfection, and virus titers were determined at different time points by plaque assay. Values are averages from three independent experiments.

TABLE 1.

Biological activities of HN mutants of NDV

Production of and applications for a polyclonal IgY diagnostic reagent specific for <Emphasis Type="Italic">Mycobacterium avium</Emphasis> subsp. <Emphasis Type="Italic">paratuberculosis</Emphasis>

Antibodies specific to the cell surface antigens of Mycobacterium avium subsp. paratuberculosis (MAP) have multiple useful applications, e.g. organism detection, immunoconcentration, and cell visualization. The aim of this study was to produce and compare polyclonal antibodies for such research and diagnostic purposes. Three polyclonal antibodies to MAP were produced using sera from immunized rabbits and chickens plus naturally infected cows. Cross-reactive antibodies in each MAP antibody preparation were removed by absorption with heterologous mycobacterial and non-mycobacterial cells. The specificity of each resulting polyclonal antibody preparation was evaluated by ELISA to multiple bacterial cell wall extract antigens. After absorption, chicken anti-MAP IgY had the highest specificity of the three antibody preparations. FITC-la-beled anti-MAP IgY was used to effectively locate MAP in macrophages 12 h post-infection. Also, immunomagnetic beads coated with anti-MAP IgY enhanced recovery of MAP from bacterial suspensions in comparison with non-antibody coated beads. Anti-MAP IgY provides a novel new reagent with broad diagnostic and research applications requiring specific concentration, detection, and quantification of MAP.     

Post-Fire Resource Redistribution in Desert Grasslands: A Possible Negative Feedback on Land Degradation

Desert grasslands, which are very sensitive to external drivers like climate change, are areas affected by rapid land degradation processes. In many regions of the world the common form of land degradation involves the rapid encroachment of woody plants into desert grasslands. This process, thought to be irreversible and sustained by biophysical feedbacks of global desertification, results in the heterogeneous distribution of vegetation and soil resources. Most of these shrub-grass transition systems at the desert margins are prone to disturbances such as fires, which affect the interactions between ecological, hydrological, and land surface processes. Here we investigate the effect of prescribed fires on the landscape heterogeneity associated with shrub encroachment. Replicated field manipulation experiments were conducted at a shrub-grass transition zone in the northern Chihuahuan desert (New Mexico, USA) using a combination of erosion monitoring techniques, microtopography measurements, infiltration experiments, and isotopic studies. The results indicate that soil erosion is more intense in burned shrub patches compared to burned grass patches and bare interspaces. This enhancement of erosion processes, mainly aeolian, is attributed to the soil–water repellency induced by the burning shrubs, which alters the physical and chemical properties of the soil surface. Further, we show that by enhancing soil erodibility fires allow erosion processes to redistribute resources accumulated by the shrub clumps, thereby leading to a more homogeneous distribution of soil resources. Thus fires counteract or diminish the heterogeneity-forming dynamics of land degradation associated with shrub encroachment by enhancing local-scale soil erodibility. Author Contributions  SR—Conceived of or designed study, performed research, analyzed data, wrote the paper; PD—Conceived of or designed study, performed research, wrote the paper; LW—Performed research, analyzed data; GO—Contributed new methods, analyzed data; SC—Conceived of or designed study; CW—Performed research, contributed new methods or models; and SM—Contributed new methods or models.     

Pigment organization in the photosynthetic apparatus of Roseiflexus castenholzii

The light-harvesting-reaction center (LHRC) complex from the chlorosome-lacking filamentous anoxygenic phototroph (FAP), Roseiflexus castenholzii (R. castenholzii) was purified and characterized for overall pigment organization. The LHRC is a single complex that is comprised of light harvesting (LH) and reaction center (RC) polypeptides as well as an attached c-type cytochrome. The dominant carotenoid found in the LHRC is keto-γ-carotene, which transfers excitation to the long wavelength antenna band with 35% efficiency. Linear dichroism and fluorescence polarization measurements indicate that the long wavelength antenna pigments absorbing around 880 nm are perpendicular to the membrane plane, with the corresponding Q_y transition dipoles in the plane of the membrane. The antenna pigments absorbing around 800 nm, as well as the bound carotenoid, are oriented at a large angle with respect to the membrane. The antenna pigments spectroscopically resemble the well-studied LH2 complex from purple bacteria, however the close association with the RC makes the light harvesting component of this complex functionally more like LH1.