Isotopic evidence for the occurrence of biological nitrification and nitrogen deposition processing in forest canopies

This study examines the role of tree canopies in processing atmospheric nitrogen (N_dep) for four forests in the United Kingdom subjected to different N_dep: Scots pine and beech stands under high N_dep (HN, 13–19 kg N ha^?1 yr^?1), compared to Scots pine and beech stands under low N_dep (LN, 9 kg N ha^?1 yr^?1). Changes of NO₃‐N and NH₄‐N concentrations in rainfall (RF) and throughfall (TF) together with a quadruple isotope approach, which combines δ¹⁸O, Δ¹⁷O and δ¹⁵N in NO₃^? and δ¹⁵N in NH₄⁺, were used to assess N transformations by the canopies. Generally, HN sites showed higher NH₄‐N and NO₃‐N concentrations in RF compared to the LN sites. Similar values of δ¹⁵N‐NO₃^? and δ¹⁸O in RF suggested similar source of atmospheric NO₃^? (i.e. local traffic), while more positive values for δ¹⁵N‐NH₄⁺ at HN compared to LN likely reflected the contribution of dry NH_x deposition from intensive local farming. The isotopic signatures of the N‐forms changed after interacting with tree canopies. Indeed, ¹⁵N‐enriched NH₄⁺ in TF compared to RF at all sites suggested that canopies played an important role in buffering dry N_dep also at the low N_dep site. Using two independent methods, based on δ¹⁸O and Δ¹⁷O, we quantified for the first time the proportion of NO₃^? in TF, which derived from nitrification occurring in tree canopies at the HN site. Specifically, for Scots pine, all the considered isotope approaches detected biological nitrification. By contrast for the beech, only using the mixing model with Δ¹⁷O, we were able to depict the occurrence of nitrification within canopies. Our study suggests that tree canopies play an active role in the N cycling within forest ecosystems. Processing of N_dep within canopies should not be neglected and needs further exploration, with the combination of multiple isotope tracers, with particular reference to Δ¹⁷O.     

Identification of prolyl hydroxylation modifications in mammalian cell proteins

Prolyl hydroxylation is a PTM that plays an important role in the formation of collagen fibrils and in the oxygen‐dependent regulation of hypoxia inducible factor‐α (HIF‐α). While this modification has been well characterized in the context of these proteins, it remains unclear to what extent it occurs in the remaining mammalian proteome. We explored this question using MS to analyze cellular extracts subjected to various fractionation strategies. In one strategy, we employed the von Hippel Lindau tumor suppressor protein, which recognizes prolyl hydroxylated HIF‐α, as a scaffold for generating hydroxyproline capture reagents. We report novel sites of prolyl hydroxylation within five proteins: FK506‐binding protein 10, myosin heavy chain 10, hexokinase 2, pyruvate kinase, and C‐1 Tetrahydrofolate synthase. Furthermore, we show that identification of prolyl hydroxylation presents a significant technical challenge owing to widespread isobaric methionine oxidation, and that manual inspection of spectra of modified peptides in this context is critical for validation.     

Ivermectin is a specific inhibitor of importin α/β-mediated nuclear import able to inhibit replication of HIV-1 and dengue virus

The movement of proteins between the cytoplasm and nucleus mediated by the importin superfamily of proteins is essential to many cellular processes, including differentiation and development, and is critical to disease states such as viral disease and oncogenesis. We recently developed a high-throughput screen to identify specific and general inhibitors of protein nuclear import, from which ivermectin was identified as a potential inhibitor of importin α/β-mediated transport. In the present study, we characterized in detail the nuclear transport inhibitory properties of ivermectin, demonstrating that it is a broad-spectrum inhibitor of importin α/β nuclear import, with no effect on a range of other nuclear import pathways, including that mediated by importin β1 alone. Importantly, we establish for the first time that ivermectin has potent antiviral activity towards both HIV-1 and dengue virus, both of which are strongly reliant on importin α/β nuclear import, with respect to the HIV-1 integrase and NS5 (non-structural protein 5) polymerase proteins respectively. Ivermectin would appear to be an invaluable tool for the study of protein nuclear import, as well as the basis for future development of antiviral agents.     

Linkage disequilibrium across six prion gene regions spanning 20 kbp in U.S. sheep

Single nucleotide polymorphisms (SNPs) and haplotype alleles within the prion gene (PRNP) coding sequence of domestic sheep (Ovis aries) are associated with genetic predisposition to scrapie, a transmissible spongiform encephalopathy disease of sheep. This report describes regions of linkage disequilibrium (LD) throughout the PRNP gene region in U.S. sheep and provides a genetic framework for identifying additional PRNP determinants associated with scrapie resistance. Four sequence tagged sites (i.e., STS or amplicons) totaling 3869 bp and spanning 20 kbp of genomic PRNP sequence were sequenced in a diverse panel of 90 sires representing ten popular U.S. breeds of sheep. Analysis of these sequences identified 36 previously unreported polymorphisms. In combination with two previously characterized STS, 62 polymorphisms were analyzed in a 20-kbp PRNP region in this panel of U.S. sheep. Two regions of strong LD and ten common haplotypes were identified. The haplotype encoding amino acid residues A, R, and Q at codons 136, 154, and 171, respectively, was observed on nine larger haplotypes spanning PRNP from the promoter region to the 3′ untranslated region. The haplotype encoding VRQ was observed on two larger haplotypes, whereas ARR, ARH, and AHQ were each present on a single haplotype. The existence of multiple haplotypes encoding ARQ raises the question of whether sheep bearing these different haplotypes are equally susceptible to scrapie. The haplotype structure within the 20-kbp region of PRNP identified in this study is important for higher-resolution analysis of genetics contributions to scrapie susceptibility. Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession number DQ077504.     

Newcastle Disease Virus-Like Particles Containing Respiratory Syncytial Virus G Protein Induced Protection in BALB/c Mice,with No Evidence of Immunopathology

Respiratory syncytial virus (RSV) is the leading cause of serious respiratory infections in children as well as a serious cause of disease in elderly and immunosuppressed populations. There are no licensed vaccines available to prevent RSV disease. We have developed a virus-like particle (VLP) vaccine candidate for protection from RSV. The VLP is composed of the NP and M proteins of Newcastle disease virus (NDV) and a chimeric protein containing the cytoplasmic and transmembrane domains of the NDV HN protein and the ectodomain of the human RSV G protein (H/G). Immunization of mice with 10 or 40 μg total VLP-H/G protein by intraperitoneal or intramuscular inoculation stimulated antibody responses to G protein which were as good as or better than those stimulated by comparable amounts of UV-inactivated RSV. Immunization of mice with two doses or even a single dose of these particles resulted in the complete protection of mice from RSV replication in the lungs. Immunization with these particles induced neutralizing antibodies with modest titers. Upon RSV challenge of VLP-H/G-immunized mice, no enhanced pathology in the lungs was observed, although lungs of mice immunized in parallel with formalin-inactivated RSV (FI-RSV) showed the significant pathology that has previously been documented after immunization with FI-RSV. Thus, the VLP-H/G candidate vaccine was immunogenic in BALB/c mice and prevented replication of RSV in murine lungs, with no evidence of immunopathology. These data support further development of virus-like particle vaccine candidates for protection against RSV.Human respiratory syncytial virus (RSV), a member of the Paramyxoviridae family, is the primary cause of serious lower respiratory tract infections in infants and young children and is an important pathogen in elderly and immunocompromised populations worldwide (15, 16, 23, 42). RSV infections can induce a wide spectrum of respiratory diseases, ranging from common cold-like symptoms to more serious disease, such as bronchiolitis or pneumonia (16, 57). Despite the significance of this pathogen, no vaccine is available. Strategies utilizing traditional subunit vaccines or attenuated virus preparations as well as live virus vectors and DNA vaccines have not resulted in a licensed vaccine (reviewed in reference 42). Complicating RSV vaccine development are previous vaccine trials of a formalin-inactivated vaccine (FI-RSV), which predisposed infants to more severe disease upon natural exposure to live virus. These studies have raised concerns about the safety of all subsequently developed RSV vaccines (reviewed in references 15 and 42).Both soluble and cell-mediated immune responses have been proposed to be important for protection from RSV infections (3, 13-15, 29, 42, 67). The RSV F protein, one of the two major antigens expressed on virion surfaces (15), is thought to be the most important target of neutralizing and protective antibodies (15, 25, 72). Indeed, monoclonal antibodies specific for the RSV F protein are used clinically for RSV disease prophylaxis in high-risk infants (4, 61). The F protein is also a major target of CD8 T cells in mice (12), but the association between cell-mediated immunity and protection from RSV disease has not been established (62). The role of the G protein, the other major antigen on virion surfaces, in stimulating protective immune responses is less clear, although it is thought that antibodies to this molecule do have a role in protection (54, 68). No CD8 T-cell epitopes have been reported for this protein. The G protein is unlike other paramyxovirus glycoproteins. Its ectodomain is heavily glycosylated by N-linked and, primarily, O-linked carbohydrates (77). The estimated 24 or 25 O-linked carbohydrate side chains and 4 N-linked side chains increase the molecular mass of the protein, as synthesized in Vero cells, from 32.5 kDa to approximately 90 kDa (15, 16). This extensive glycosylation may help to mask the underlying polypeptide backbone from immune recognition (15).A previous RSV vaccine, FI-RSV, resulted not in protection but in disease enhancement upon subsequent live virus infection (37, 38). Many subsequent studies have attempted to define the reasons for this response. These studies have consistently shown that enhanced disease is characterized by unbalanced Th2-biased cytokine responses, weak CD8 T-cell responses, pronounced eosinophilia, and induction of low-affinity and nonneutralizing antibodies (20, 21, 63, 64, 75). It is less clear which precise properties of the FI-RSV vaccine led to these results (reviewed in reference 42). The absence of these characteristics of enhanced disease is now one of the benchmarks for development of a successful RSV vaccine. Thus far, no vaccine approach reported has resulted in both the absence of enhanced disease upon RSV challenge and adequate, long-lasting protective responses in animal models (42).A virus-like particle (VLP) vaccine strategy has not been reported for RSV. VLPs are large particles, the size of viruses, composed of repeating structural arrays on their surfaces and in their cores, and these structures mimic those of infectious viruses (reviewed in references 36 and 56). VLPs are formed by the assembly of the structural proteins and lipids into particles, but without the incorporation of the viral genome. Thus, VLPs are incapable of the multiple rounds of infection typical of an infectious virus, yet they retain the superb antigenicity of virus particles. Native viral antigens arrayed on VLP surfaces and in their cores likely contribute to potent humoral responses, CD4 T-cell proliferation, and expansion of cytotoxic CD8 T cells, unlike less immunogenic subunit vaccines, which are often comprised of individual purified viral proteins (9-11, 27, 41, 43, 66, 70). The potential of VLPs as safe, effective vaccines for viral disease is increasingly being recognized. Indeed, two VLP vaccines are now licensed for use in humans, namely, the papillomavirus vaccine and the hepatitis B virus vaccine, and a number of other VLP vaccines are being evaluated in preclinical and clinical trials (reviewed in reference 36). Therefore, VLPs expressing one or both RSV glycoproteins may be an attractive strategy for designing an effective RSV vaccine.There is only one report of VLPs formed with RSV proteins (73). These particles have not been well characterized, nor is their efficiency of release known. Furthermore, their detection requires incorporation of a minigenome. However, we have previously reported that the expression of the four major structural proteins of Newcastle disease virus (NDV), an avian paramyxovirus, results in the very efficient release of particles that structurally and functionally resemble virus particles (60; L. W. McGinnes et al., unpublished data). Furthermore, we have found that these particles (ND VLPs) stimulate potent anti-NDV immune responses in mice, including neutralizing antibody responses (McGinnes et al., unpublished data). These results led us to test the hypothesis that ND VLPs could serve as a platform for the expression of antigens from human viruses, including RSV G and F proteins, and that these particles could serve as an effective RSV vaccine.In this study, we report that the ectodomain of the RSV G protein, fused to the cytoplasmic tail (CT) and the transmembrane (TM) domain of the NDV hemagglutinin-neuraminidase (HN) protein, can be incorporated efficiently into VLPs containing the NDV NP and M proteins and that these particles can be prepared quantitatively and used as an immunogen. We demonstrate that immunization with these particles stimulated robust soluble immune responses. Furthermore, these particles conferred protection in BALB/c mice, characterized by increased viral clearance in lung tissue, after live RSV challenge. Importantly, infectious RSV challenge of mice following VLP-H/G immunization did not result in the enhanced lung pathology typified by FI-RSV immunization (17, 18, 55).     

Cox17 is functional when tethered to the mitochondrial inner membrane

Cox17 is an essential protein in the assembly of cytochrome c oxidase within the mitochondrion. Cox17 is implicated in providing copper ions for formation of CuA and CuB sites in the oxidase complex. To address whether Cox17 is functional in shuttling copper ions to the mitochondrion, Cox17 was tethered to the mitochondrial inner membrane by a fusion to the transmembrane domain of the inner membrane protein, Sco2. The copper-binding domain of Sco2 that projects into the inter-mitochondrial membrane space was replaced with Cox17. The Sco2/Cox17 fusion protein containing the mitochondrial import sequence and transmembrane segment of Sco2 is exclusively localized within the mitochondrion. The Sco2/Cox17 protein restores respiratory growth and normal cytochrome oxidase activity in cox17Delta cells. These studies suggest that the function of Cox17 is confined to the mitochondrial intermembrane space. Domain mapping of yeast Cox17 reveals that the carboxyl-terminal segment of the protein has a function within the intermembrane space that is independent of copper ion binding. The essential C-terminal function of Cox17 maps to a candidate amphipathic helix that is important for mitochondrial uptake and retention of the Cox17 protein. This motif can be spatially separated from the N-terminal copper-binding functional motif. Possible roles of the C-terminal motif are discussed.     

Oxygen-mediated regulation of tumor cell invasiveness. Involvement of a nitric oxide signaling pathway

Tumor hypoxia is associated with a poor prognosis for patients with various cancers, often resulting in an increase in metastasis. Moreover, exposure to hypoxia increases the ability of breast carcinoma cells to invade the extracellular matrix, an important aspect of metastasis. Here, we demonstrate that the hypoxic up-regulation of invasiveness is linked to reduced nitric oxide signaling. Incubation of human breast carcinoma cells in 0.5% versus 20% oxygen increased their in vitro invasiveness and their expression of the urokinase receptor, an invasion-associated molecule. These effects of hypoxia were inhibited by nitric oxide-mimetic drugs; and in a manner similar to hypoxia, pharmacological inhibition of nitric oxide synthesis increased urokinase receptor expression. The nitric oxide signaling pathway involves activation of soluble guanylyl cyclase (sGC) and the subsequent activation of protein kinase G (PKG). Culture of tumor cells under hypoxic conditions (0.5% versus 20% oxygen) resulted in lower cGMP levels, an effect that could be prevented by incubation with glyceryl trinitrate. Inhibition of sGC activity with a selective blocker or with the heme biosynthesis inhibitor desferrioxamine increased urokinase receptor expression. These compounds also prevented the glyceryl trinitrate-mediated suppression of urokinase receptor expression in cells incubated under hypoxic conditions. In contrast, direct activation of PKG using 8-bromo-cGMP prevented the hypoxia- and desferrioxamine-induced increases in urokinase receptor expression as well as the hypoxia-mediated enhanced invasiveness. Further involvement of PKG in the regulation of invasion-associated phenotypes was established using a selective PKG inhibitor, which alone increased urokinase receptor expression. These findings reveal that an important mechanism by which hypoxia increases tumor cell invasiveness (and possibly metastasis) requires inhibition of the nitric oxide signaling pathway involving sGC and PKG activation.     

Education as policy: the impact of education on marriage, contraception, and fertility in Colombia, Peru, and Bolivia

Using data from the World Fertility and Demographic and Health Surveys of Colombia, Peru, and Bolivia, we model the effects of education on three demographic outcomes: the timing of first sexual union, contraceptive use, and fertility. These effects are examined over time and across geographic areas using a multivariate framework. We find substantial improvements in female educational attainment over the last fifty years and a strong relationship between education and the demographic outcomes. Each successive increment in education is associated with declines in the marriage rate, increased contraceptive use, and lower fertility. Education accounts for some of the changes over time in the demographic outcomes, but the pattern varies by outcome, time period, and geographic area. In support of the social diffusion hypothesis, our results indicate that educational differences in reproductive behavior are reduced as the level of development increases and societies pass through their demographic transition.     

Rapid and sensitive GC/MS characterization of glycolipid released Galalpha1,3Gal-terminated oligosaccharides from small organ specimens of a single pig

Pig to human xenotransplantation is considered a possible solution to the prevailing chronic lack of human donor organs for allotransplantation. The Galalpha1,3Gal determinant is the major porcine xenogeneic epitope causing hyperacute rejection following human antibody binding and complement activation. In order to characterize the tissue distribution of Galalpha1,3Gal-containing and blood group- type glycosphingolipids in pig, acid and nonacid glycosphingolipids were isolated from the kidney, small intestine, spleen, salivary gland, liver, and heart of a single pig obtained from a semi-inbred strain homozygous at the SLA locus. Glycolipids were analyzed by thin-layer immunostaining using monoclonal antibodies, and following ceramide glycanase cleavage as permethylated oligosaccharides by gas chromatography, gas chromatography-mass spectrometry, and matrix- assisted laser desorption/ionization mass spectrometry. The kidney contained large amounts of Galalpha1,3Gal-containing penta- and hexasaccharides having carbohydrate sequences consistent with the Galalpha1,3nLc4and Galalpha1,3Lexstructures, respectively. The former structure was tentatively identified in all organs by GC/MS. The presence of extended Galalpha1,3Gal-terminated structures in the kidney and heart was suggested by antibody binding, and GC/MS indicated the presence of a Galalpha1,3nLc6structure in the heart. The kidney, spleen, and heart contained blood group H pentaglycosylceramides based on type 1 (H-5-1) and type 2 (H-5-2) chains, and H hexaglycosylceramides based on the type 4 chain (H-6-4). In the intestine H-5-1 and H-6-4 were expressed, in the salivary gland H-5-1 and H-5-2, whereas only the H-5-1 structure was identified in the liver. Blood group A structures were identified in the salivary gland and the heart by antibody binding and GC/MS, indicating an organ- specific expression of blood group AH antigens in the pig.      

Role of the D-alanyl carrier protein in the biosynthesis of D-alanyl-lipoteichoic acid.

D-Alanyl-lipoteichoic acid (D-alanyl-LTA) is a widespread macroamphiphile which plays a vital role in the growth and development of gram-positive organisms. The biosynthesis of this polymer requires the enzymic activation of D-alanine for its transfer to the membrane-associated LTA (mLTA). A small, heat-stable, and acidic protein that is required for this transfer was purified to greater than 98% homogeneity from Lactobacillus casei ATCC 7469. This protein, previously named the D-alanine-membrane acceptor ligase (V. M. Reusch, Jr., and F. C. Neuhaus, J. Biol. Chem. 246:6136-6143, 1971), functions as the D-alanyl carrier protein (Dcp). The amino acid composition, beta-alanine content, and N-terminal sequence of this protein are similar to those of the acyl carrier proteins (ACPs) of fatty acid biosynthesis. The isolation of Dcp and its derivative, D-alanyl approximately Dcp, has allowed the characterization of two novel reactions in the pathway for D-alanyl-mLTA biosynthesis: (i) the ligation of Dcp with D-alanine and (ii) the transfer of D-alanine from D-alanyl approximately Dcp to a membrane acceptor. It has not been established whether the membrane acceptor is mLTA or another intermediate in the pathway for D-alanyl-mLTA biosynthesis. Since the D-alanine-activating enzyme (EC 6.1.1.13) catalyzes the ligation reaction, this enzyme functions as the D-alanine-Dcp ligase (Dcl). Dcl also ligated the ACPs from Escherichia coli, Vibrio harveyi, and Saccharopolyspora erythraea with D-alanine. In contrast to the relaxed specificity of Dcl in the ligation reaction, the transfer of D-alanine to the membrane acceptor was highly specific for Dcp and did not occur with other ACPs. This transfer was observed by using only D-[14C]alanyl approximately Dcp and purified L. casei membranes. Thus, D-alanyl approximately Dcp is an essential intermediate in the transfer of D-alanine from Dcl to the membrane acceptor. The formation of D-alanine esters of mLTA provides a mechanism for modulating the net anionic charge in the cell wall.