Characterization of disulfide bond position in proteins and sequence analysis of cystine-bridged peptides by tandem mass spectrometry.

Tandem mass spectrometry employing high-energy, collisionally activated dissociation (CAD) is shown to be a useful method for sequencing through the cystine bridge of intermolecularly disulfide-bonded peptides. A characteristic triplet of intense fragment ions is observed corresponding to cleavage through and to either side of the disulfide bridge. These fragments define the masses of the linked peptides. Fragments due to peptide chain cleavage are also observed at lower abundance in the product-ion spectra and can be sufficient to sequence both of the disulfide-linked peptides without any prior knowledge of the peptide or protein sequence. Even in cases where the peptide sequence-related product-ion yields are poor, the intensities of the disulfide cleavage ions are usually sufficient to determine the molecular weights of the component cystine-bridged peptides. In this paper we demonstrate that the high-energy CAD tandem MS approach may be used to characterize disulfide-bonded peptides directly in complex enzymatic or chemical digests of native proteins. This obviates the need for individual purification of intermolecularly disulfide-linked peptides prior to analysis. The techniques are illustrated here for synthetic, inter- and intramolecularly disulfide-linked peptides and for human transforming growth factor-alpha (des-Val-Val-TGF-alpha), a compact protein containing 48 residues and three disulfides.     

CD8 T cell-initiated blood-brain barrier disruption is independent of neutrophil support

Blood-brain barrier (BBB) disruption is a common feature of numerous neurologic disorders. A fundamental question in these diseases is the extent inflammatory immune cells contribute to CNS vascular permeability. We have previously shown that CD8 T cells play a critical role in initiating BBB disruption in the peptide-induced fatal syndrome model developed by our laboratory. However, myelomonocytic cells such as neutrophils have also been implicated in promoting CNS vascular permeability and functional deficit in murine models of neuroinflammatory disease. For this reason, we evaluated neutrophil depletion in a murine model of CD8 T cell-initiated BBB disruption by employing traditionally used anti-granulocyte receptor-1 mAb RB6-8C5 and Ly-6G-specific mAb 1A8. We report that CNS-infiltrating antiviral CD8 T cells express high levels of granulocyte receptor-1 protein and are depleted by treatment with RB6-8C5. Mice treated with RB6-8C5, but not 1A8, display: 1) intact BBB tight junction proteins; 2) reduced CNS vascular permeability visible by gadolinium-enhanced T1-weighted magnetic resonance imaging; and 3) preservation of motor function. These studies demonstrate that traditional methods of neutrophil depletion with RB6-8C5 are broadly immune ablating. Our data also provide evidence that CD8 T cells initiate disruption of BBB tight junction proteins and CNS vascular permeability in the absence of neutrophil support.     

Salivary enzymes are injected into xylem by the glassy-winged sharpshooter, a vector of Xylella fastidiosa

A few phytophagous hemipteran species such as the glassy-winged sharpshooter, Homalodisca vitripennis, (Germar), subsist entirely on xylem fluid. Although poorly understood, aspects of the insect's salivary physiology may facilitate both xylem-feeding and transmission of plant pathogens. Xylella fastidiosa is a xylem-limited bacterium that causes Pierce's disease of grape and other scorch diseases in many important crops. X. fastidiosa colonizes the anterior foregut (precibarium and cibarium) of H. vitripennis and other xylem-feeding vectors. Bacteria form a dense biofilm anchored in part by an exopolysaccharide (EPS) matrix that is reported to have a β-1,4-glucan backbone. Recently published evidence supports the following, salivation-egestion hypothesis for the inoculation of X. fastidiosa during vector feeding. The insect secretes saliva into the plant and then rapidly takes up a mixture of saliva and plant constituents. During turbulent fluid movements in the precibarium, the bacteria may become mechanically and enzymatically dislodged; the mixture is then egested back out through the stylets into plant cells, possibly including xylem vessels. The present study found that proteins extracted from dissected H. vitripennis salivary glands contain several enzyme activities capable of hydrolyzing glycosidic linkages in polysaccharides such as those found in EPS and plant cell walls, based on current information about the structures of those polysaccharides. One of these enzymes, a β-1,4-endoglucanase (EGase) was enriched in the salivary gland protein extract by subjecting the extract to a few, simple purification steps. The EGase-enriched extract was then used to generate a polyclonal antiserum that was used for immunohistochemical imaging of enzymes in sharpshooter salivary sheaths in grape. Results showed that enzyme-containing gelling saliva is injected into xylem vessels during sharpshooter feeding, in one case being carried by the transpiration stream away from the injection site. Thus, the present study provides support for the salivation-egestion hypothesis.     

Comparison of Bayesian models to estimate direct genomic values in multi-breed commercial beef cattle

Background

While several studies have examined the accuracy of direct genomic breeding values (DGV) within and across purebred cattle populations, the accuracy of DGV in crossbred or multi-breed cattle populations has been less well examined. Interest in the use of genomic tools for both selection and management has increased within the hybrid seedstock and commercial cattle sectors and research is needed to determine their efficacy. We predicted DGV for six traits using training populations of various sizes and alternative Bayesian models for a population of 3240 crossbred animals. Our objective was to compare alternate models with different assumptions regarding the distributions of single nucleotide polymorphism (SNP) effects to determine the optimal model for enhancing feasibility of multi-breed DGV prediction for the commercial beef industry.

Results

Realized accuracies ranged from 0.40 to 0.78. Randomly assigning 60 to 70% of animals to training (n ≈ 2000 records) yielded DGV accuracies with the smallest coefficients of variation. Mixture models (BayesB95, BayesCπ) and models that allow SNP effects to be sampled from distributions with unequal variances (BayesA, BayesB95) were advantageous for traits that appear or are known to be influenced by large-effect genes. For other traits, models differed little in prediction accuracy (~0.3 to 0.6%), suggesting that they are mainly controlled by small-effect loci.

Conclusions

The proportion (60 to 70%) of data allocated to training that optimized DGV accuracy and minimized the coefficient of variation of accuracy was similar to large dairy populations. Larger effects were estimated for some SNPs using BayesA and BayesB95 models because they allow unequal SNP variances. This substantially increased DGV accuracy for Warner-Bratzler Shear Force, for which large-effect quantitative trait loci (QTL) are known, while no loss in accuracy was observed for traits that appear to follow the infinitesimal model. Large decreases in accuracy (up to 0.07) occurred when SNPs that presumably tag large-effect QTL were over-regressed towards the mean in BayesC0 analyses. The DGV accuracies achieved here indicate that genomic selection has predictive utility in the commercial beef industry and that using models that reflect the genomic architecture of the trait can have predictive advantages in multi-breed populations.

Electronic supplementary material

The online version of this article (doi:10.1186/s12711-015-0106-8) contains supplementary material, which is available to authorized users.     

Liver-infiltrating lymphocytes in chronic human hepatitis C virus infection display an exhausted phenotype with high levels of PD-1 and low levels of CD127 expression

The majority of people infected with hepatitis C virus (HCV) fail to generate or maintain a T-cell response effective for viral clearance. Evidence from murine chronic viral infections shows that expression of the coinhibitory molecule PD-1 predicts CD8+ antiviral T-cell exhaustion and may contribute to inadequate pathogen control. To investigate whether human CD8+ T cells express PD-1 and demonstrate a dysfunctional phenotype during chronic HCV infection, peripheral and intrahepatic HCV-specific CD8+ T cells were examined. We found that in chronic HCV infection, peripheral HCV-specific T cells express high levels of PD-1 and that blockade of the PD-1/PD-L1 interaction led to an enhanced proliferative capacity. Importantly, intrahepatic HCV-specific T cells, in contrast to those in the periphery, express not only high levels of PD-1 but also decreased interleukin-7 receptor alpha (CD127), an exhausted phenotype that was HCV antigen specific and compartmentalized to the liver, the site of viral replication.     

Methanogens with pseudomurein use diaminopimelate aminotransferase in lysine biosynthesis

Methanothermobacter thermautotrophicus uses lysine for both protein synthesis and cross-linking pseudomurein in its cell wall. A diaminopimelate aminotransferase enzyme from this methanogen (MTH0052) converts tetrahydrodipicolinate to l,l-diaminopimelate, a lysine precursor. This gene complemented an Escherichia coli diaminopimelate auxotrophy, and the purified protein catalyzed the transamination of diaminopimelate to tetrahydrodipicolinate. Phylogenetic analysis indicated this gene was recruited from anaerobic Gram-positive bacteria. These results expand the family of diaminopimelate aminotransferases to a diverse set of plant, bacterial and archaeal homologs. In contrast marine methanogens from the Methanococcales, which lack pseudomurein, appear to use a different diaminopimelate pathway for lysine biosynthesis.