Identification of disulfide-linked peptides by isotope profiles produced by peptic digestion of proteins in 50% (18)O water

Determination of the disulfide-bond arrangement of a protein by characterization of disulfide-linked peptides in proteolytic digests may be complicated by resistance of the protein to specific proteases, disulfide interchange, and/or production of extremely complex mixtures by less specific proteolysis. In this study, mass spectrometry has been used to show that incorporation of (18)O into peptides during peptic digestion of disulfide-linked proteins in 50% (18)O water resulted in isotope patterns and increases in average masses that facilitated identification and characterization of disulfide-linked peptides even in complex mixtures, without the need for reference digests in 100% (16)O water. This is exemplified by analysis of peptic digests of model proteins lysozyme and ribonuclease A (RNaseA) by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) and electrospray ionization (ESI) mass spectrometry (MS). Distinct isotope profiles were evident when two peptide chains were linked by disulfide bonds, provided one of the chains did not contain the C terminus of the protein. This latter class of peptide, and single-chain peptides containing an intrachain disulfide bond, could be identified and characterized by mass shifts produced by reduction. Reduction also served to confirm other assignments. Isotope profiling of peptic digests showed that disulfide-linked peptides were often enriched in the high molecular weight fractions produced by size exclusion chromatography (SEC) of the digests. Applicability of these procedures to analysis of a more complex disulfide-bond arrangement was shown with the hemagglutinin/neuraminidase of Newcastle disease virus.     

Eye size in birds and the timing of song at dawn

Why do different species of birds start their dawn choruses at different times? We test the hypothesis that the times at which different species start singing at dawn are related to their visual capability at low light intensities. Birds with large eyes can achieve greater pupil diameters and hence, all other things being equal, greater visual sensitivity and resolution than birds with small eyes. We estimated the maximum pupil diameter of passerine birds by measuring the diameter of the exposed eye surface, and measured the times of the first songs at dawn of songbirds present in different bird communities, and the light intensities at these times. Using phylogenetic comparative analyses, we found that songbirds with large eyes started to sing at lower light intensities (and therefore earlier) than species with smaller eyes. These relationships were stronger when differences in body size were controlled for statistically, and were consistent between two phylogenies and when species were treated as independent data points. Our results therefore provide robust support for the hypothesis that visual capability at low light levels influences the times at which birds start to sing at dawn.     

Systematic affinities of the lyrebirds (Passeriformes: Menura), with a novel classification of the major groups of passerine birds

Phylogenetic relationships of the lyrebirds are investigated using DNA sequence data. The aligned data matrix consists of 4027 bp obtained from three nuclear genes (c-myc, RAG-1 and myoglobin intron II) and two mitochondrial genes (cytochrome b and ND2). Both maximum-likelihood and parsimony analyses show that the lyrebirds unambiguously belong to the oscine radiation, and that they are the sister taxon to all other oscines. The results do not support the suggestion based on DNA-DNA hybridization data (Sibley and Ahlquist, 1990) that the treecreepers and bowerbirds are part of the lyrebird clade. Nevertheless, treecreepers and bowerbirds are sister taxa to all other oscines (except the lyrebirds) and may constitute a monophyletic group, although bootstrap support values for this clade are low. A major disagreement between the present analysis and that based on DNA-DNA hybridization data is that the Corvida (sensu Sibley and Ahlquist, 1990) and Passerida are not reciprocally monophyletic, as we find the latter group be nested within the Corvida. Also, the superfamilies Meliphagoidea and Corvoidea sensu, are not recovered as monophyletic in the present study. Within the oscine radiation, all taxa belonging to the earliest splits are confined to the Australo-Papuan region. This suggests strongly that the origins and early radiation of the oscines occurred in the southern supercontinent Gondwana. A new classification of the major groups of passerines is presented following from the results presented in the present study, as well as those published recently on analyses of sequence data from the nuclear c-myc and RAG-1 genes (Ericson et al., 2002; Irestedt et al., 2001).     

The effect of excesses and deficiencies in amino acids on the feeding behaviour of the common brushtail possum (Trichosurus vulpecula)

In this study of the amino acid nutrition of a marsupial we tested three hypotheses: (a) that brushtail possums eat less when diets contain excesses or deficiencies in essential amino acids, (b) that brushtail possums choose diets that do not contain amino acid excesses, and (c) that amino acid consumption is mediated partly by the 5HT3 receptor. Possums ate less when 0.2-1.0% methionine (wet matter) was added to the diet, but similar concentrations of lysine and threonine had little effect. However, when given a choice, possums always selected the basal ration over one with added lysine, methionine or threonine at concentrations between 0.05% and 0.9%. In contrast to the experiments with excess amino acids, possums did not eat less of a diet almost devoid of an essential amino acid. Instead, the possums ate less when their diets contained synthetic amino acids rather than similar amounts and proportions of amino acids as casein. Contrary to the third hypothesis, the 5HT3 receptor antagonist, ondansetron, did not affect feeding by possums given a diet containing 0.8% methionine, suggesting that post-ingestive feedback, via the 5HT3 receptor, does not regulate amino acid intake when diets contain amino acid excesses.     

Biochemical characterization of the first essential two-component signal transduction system from Staphylococcus aureus and Streptococcus pneumoniae

The YYCFG two-component signal transduction system (TCSTS) has been shown to be essential to the viability of several gram-positive bacteria. However, the function of the gene pair remains unknown. Interestingly, while both components are essential to Staphylococcus aureus and Bacillus subtilis, only the response regulator (YYCF) is essential to Streptococcus pneumoniae. To study this essential TCSTS further, the S. pneumoniae and S. aureus truncated YycG histidine kinase and full-length YycF response regulator proteins were characterized at a biochemical level. The recombinant proteins from both organisms were expressed in Escherichia coli and purified. The YycG autophosphorylation activities were activated by ammonium. The apparent K(m )(ATP) of S. aureus YycG autophosphorylation was 130 microM and S. pneumoniae was 3.0 microM. Each had similar K(cat )values of 0.036 and 0.024 min(-1), respectively. Cognate phosphotransfer was also investigated indicating different levels of the phosphorylated YycG intermediates during the reaction. The S. pneumoniae YycG phosphorylated intermediate was not detectable in the presence of its cognate YycF, while phosphorylated S. aureus YycG and YycF were detected concurrently. In addition, noncognate phosphotransfer was demonstrated between the two species. These studies thoroughly compare the essential YycFG TCSTS from the two species at the biochemical level and also establish methods for assaying the activities of these antibacterial targets.     

Inter-familial relationships of the shorebirds (Aves: Charadriiformes) based on nuclear DNA sequence data

Background  

Phylogenetic hypotheses of higher-level relationships in the order Charadriiformes based on morphological data, partly disagree with those based on DNA-DNA hybridisation data. So far, these relationships have not been tested by analysis of DNA sequence data. Herein we utilize 1692 bp of aligned, nuclear DNA sequences obtained from 23 charadriiform species, representing 15 families. We also test earlier suggestions that bustards and sandgrouses may be nested with the charadriiforms. The data is analysed with methods based on the parsimony and maximum-likelihood criteria.     

Crystal structure of the CUB1-EGF-CUB2 region of mannose-binding protein associated serine protease-2

Serum mannose-binding proteins (MBPs) are C-type lectins that recognize cell surface carbohydrate structures on pathogens, and trigger killing of these targets by activating the complement pathway. MBPs circulate as a complex with MBP-associated serine proteases (MASPs), which become activated upon engagement of a target cell surface. The minimal functional unit for complement activation is a MASP homodimer bound to two MBP trimeric subunits. MASPs have a modular structure consisting of an N-terminal CUB domain, a Ca(2+)-binding EGF-like domain, a second CUB domain, two complement control protein modules and a C-terminal serine protease domain. The CUB1-EGF-CUB2 region mediates homodimerization and binding to MBP. The crystal structure of the MASP-2 CUB1-EGF-CUB2 dimer reveals an elongated structure with a prominent concave surface that is proposed to be the MBP-binding site. A model of the full six-domain structure and its interaction with MBPs suggests mechanisms by which binding to a target cell transmits conformational changes from MBP to MASP that allow activation of its protease activity.     

Marker-assisted congenic screening (MACS): A database tool for the efficient production and characterization of congenic lines

Over the past decades, genetic studies in rodent models of human multifactorial disorders have led to the detection of numerous chromosomal regions associated with disease phenotypes. Owing to the complex control of these phenotypes and the size of the disease loci, identifying the underlying genes requires further analyses in new original models, including chromosome substitution (consomic) and congenic lines, derived to evaluate the phenotypic effects of disease susceptibility loci and fine-map the disease genes. We have developed a relational database (MACS) specifically designed for the genetic marker-assisted production of large series of rodent consomic and congenic lines (speed congenics), the organization of their genetic and phenotypic characterizations, and the acquisition and archiving of both genetic and phenotypic data. This database, originally optimized for the production of rat congenics, can also be applied to mouse mapping projects. MACS represents an essential system for significantly improving efficiency and accuracy in investigations of multiple consomic and congenic lines simultaneously derived for different disease loci, and ultimately cloning genes underlying complex phenotypes.     

The type III protein translocation system of enteropathogenic Escherichia coli involves EspA-EspB protein interactions

Enteropathogenic Escherichia coli (EPEC), like many bacterial pathogens, use a type III secretion system to deliver effector proteins across the bacterial cell wall. In EPEC, four proteins, EspA, EspB, EspD and Tir are known to be exported by a type III secretion system and to be essential for 'attaching and effacing' (A/E) lesion formation, the hallmark of EPEC pathogenicity. EspA was recently shown to be a structural protein and a major component of a large, transiently expressed, filamentous surface organelle which forms a direct link between the bacterium and the host cell. In contrast, EspB is translocated into the host cell where it is localized to both membrane and cytosolic cell fractions. EspA and EspB are required for translocation of Tir to the host cell membrane suggesting that they may both be components of the translocation apparatus. In this study, we show that EspB co-immunoprecipitates with the EspA filaments and that, during EPEC infection of HEp-2 cells, EspB localizes closely with EspA. Using a number of binding assays, we also show that EspB can bind and be copurified with EspA. Nevertheless, binding of EspA filaments to the host cell membranes occurred even in the absence of EspB. These results suggest that following initial attachment of the EspA filaments to the target cells, EspB is delivered into the host cell membrane and that the interaction between EspA and EspB may be important for protein translocation.     

Identification of SopE2, a Salmonella secreted protein which is highly homologous to SopE and involved in bacterial invasion of epithelial cells

Type III secreted Sop protein effectors are delivered into target eukaryotic cells and elicit cellular responses underlying Salmonella pathogenicity. In this work, we have identified another secreted protein, SopE2, and showed that SopE2 is an important invasion-associated effector. SopE2 is encoded by the sopE2 gene which is present and conserved in pathogenic strains of Salmonella. SopE2 is highly homologous to SopE, a protein encoded by a gene within a temperate bacteriophage and present in only some pathogenic strains.