Development of recombinant OmpA and OmpB proteins as diagnostic antigens for rickettsial disease

In this study the diagnostic potential of Rickettsia conorii recombinant antigens was analyzed. For this, site-specific PCR primers were used to clone the OmpA and OmpB genes of R. conorii into pMAL-c2X plasmids. Six fragments of OmpA and four of OmpB were expressed as fusion proteins with maltose-binding protein in Escherichia coli. OmpA_1350-1784, OmpB_801-1269, and OmpB_1227-1634 regions from truncated proteins were selected as diagnostic candidate antigens by ELISA using control sera. ELISA results of three antigens were compared to the results obtained by using a commercial ELISA kit which contained whole OmpA and OmpB antigens from R. conorii . For this analysis, 40 serum samples taken from febrile patients and uninfected controls were tested. Of the 20 R. conorii test results which were positive with the commercial kit, 18 were shown to be positive by ELISA using OmpA_1350-1784 (a sensitivity of 90%). The specificity of the ELISA was 100%; all of the 20 samples shown to be negative using the commercial kit were also negative in our assay. The sensitivities of the ELISA using the OmpB_801-1269 and OmpB_1227-1634 were 90% and 95%, respectively. The specificities of the OmpB_801-1269 and the OmpB_1227-1634 were 100% and 95%, respectively. These results suggest that specific regions of OmpA and OmpB effectively detect antibodies against R. conorii , and the truncated recombinant antigens could be used for development of diagnostic tools for rickettsial disease.     

Genes of the major histocompatibility complex and the evolutionary genetics of lifespan

Mice that presumably differ just in the major histocompatibility complex (MHC) chromosomal region provide the best evidence that MHC genes affect lifespan. Further evidence is that MHC region genes in some cases are known to influence reproduction, growth, and development. Moreover, MHC genetic associations with disease are well documented. This paper summarizes and defines aspects of the molecular biology, cellular function, and evolution of MHC genes (with special emphasis on the polymorphic MHC class I and II genes) which are important in aging, and attempts to integrate these into an evolutionary genetic perspective of senescence. It is suggested that MHC genes provide a mammalian paradigm for the genetics of lifespan because of their intra- and interspecies diversification, evolutionary selection, and age-specific effects.     

Mosaic genomes of the six major primate lentivirus lineages revealed by phylogenetic analyses

To clarify the origin and evolution of the primate lentiviruses (PLVs), which include human immunodeficiency virus types 1 and 2 as well as their simian relatives, simian immunodeficiency viruses (SIVs), isolated from several host species, we investigated the phylogenetic relationships among the six supposedly nonrecombinant PLV lineages for which the full genome sequences are available. Employing bootscanning as an exploratory tool, we located several regions in the PLV genome that seem to have uncertain or conflicting phylogenetic histories. Phylogeny reconstruction based on distance and maximum-likelihood algorithms followed by a number of statistical tests confirms the existence of at least five putative recombinant fragments in the PLV genome with different clustering patterns. Split decomposition analysis also shows that phylogenetic relationships among PLVs may be better represented by network-based graphs, such as the ones produced by SplitsTree. Our findings not only imply that the six so-called pure PLV lineages have in fact mosaic genomes but also make more unlikely the hypothesis of cospeciation of SIVs and their simian hosts.     

Methionine adenosyltransferase as a useful molecular systematics tool revealed by phylogenetic and structural analyses

Structural and phylogenetic relationships among Bacteria and Eukaryota were analyzed by examining 292 methionine adenosyltransferase (MAT) amino acid sequences with respect to the crystal structure of this enzyme established for Escherichia coli and rat liver. Approximately 30% of MAT residues were found to be identical in all species. Five highly conserved amino acid sequence blocks did not vary in the MAT family. We detected specific structural features that correlated with sequence signatures for several clades, allowing taxonomical identification by sequence analysis. In addition, the number of amino acid residues in the loop connecting beta-strands A2 and A3 served to clearly distinguish sequences between eukaryotes and eubacteria. The molecular phylogeny of MAT genes in eukaryotes can be explained in terms of functional diversification coupled to gene duplication or alternative splicing and adaptation through strong structural constraints. Sequence analyses and intron/exon junction positions among nematodes, arthropods and vertebrates support the traditional Coelomata hypothesis. In vertebrates, the liver MAT I isoenzyme has gradually adapted its sequence towards one providing a more specific liver function. MAT phylogeny also served to cluster the major bacterial groups, demonstrating the superior phylogenetic performance of this ubiquitous, housekeeping gene in reconstructing the evolutionary history of distant relatives.     

A novel mechanism of TRAF signaling revealed by structural and functional analyses of the TRADD-TRAF2 interaction

TRAF proteins are major mediators for the cell activation, cell survival, and antiapoptotic functions of the TNF receptor superfamily. They can be recruited to activated TNF receptors either by direct interactions with the receptors or indirectly via the adaptor protein TRADD. We now report the structure of the TRADD-TRAF2 complex, which is highly distinct from receptor-TRAF2 interactions. This interaction is significantly stronger and we show by an in vivo signaling assay that TRAF2 signaling is more readily initiated by TRADD than by direct receptor-TRAF2 interactions. TRADD is specific for TRAF1 and TRAF2, which ensures the recruitment of clAPs for the direct inhibition of caspase activation in the signaling complex. The stronger affinity and unique specificity of the TRADD-TRAF2 interaction are crucial for the suppression of apoptosis and provide a mechanistic basis for the perturbation of TRAF recruitment in sensitizing cell death induction.     

Significant natural product biosynthetic potential of actinorhizal symbionts of the genus frankia, as revealed by comparative genomic and proteomic analyses

Bacteria of the genus Frankia are mycelium-forming actinomycetes that are found as nitrogen-fixing facultative symbionts of actinorhizal plants. Although soil-dwelling actinomycetes are well-known producers of bioactive compounds, the genus Frankia has largely gone uninvestigated for this potential. Bioinformatic analysis of the genome sequences of Frankia strains ACN14a, CcI3, and EAN1pec revealed an unexpected number of secondary metabolic biosynthesis gene clusters. Our analysis led to the identification of at least 65 biosynthetic gene clusters, the vast majority of which appear to be unique and for which products have not been observed or characterized. More than 25 secondary metabolite structures or structure fragments were predicted, and these are expected to include cyclic peptides, siderophores, pigments, signaling molecules, and specialized lipids. Outside the hopanoid gene locus, no cluster could be convincingly demonstrated to be responsible for the few secondary metabolites previously isolated from other Frankia strains. Few clusters were shared among the three species, demonstrating species-specific biosynthetic diversity. Proteomic analysis of Frankia sp. strains CcI3 and EAN1pec showed that significant and diverse secondary metabolic activity was expressed in laboratory cultures. In addition, several prominent signals in the mass range of peptide natural products were observed in Frankia sp. CcI3 by intact-cell matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS). This work supports the value of bioinformatic investigation in natural products biosynthesis using genomic information and presents a clear roadmap for natural products discovery in the Frankia genus.     

Phylogeny of galactolipid synthase homologs together with their enzymatic analyses revealed a possible origin and divergence time for photosynthetic membrane biogenesis

The photosynthetic membranes of cyanobacteria and chloroplasts of higher plants have remarkably similar lipid compositions. In particular, thylakoid membranes of both cyanobacteria and chloroplasts are composed of galactolipids, of which monogalactosyldiacylglycerol (MGDG) is the most abundant, although MGDG biosynthetic pathways are different in these organisms. Comprehensive phylogenetic analysis revealed that MGDG synthase (MGD) homologs of filamentous anoxygenic phototrophs Chloroflexi have a close relationship with MGDs of Viridiplantae (green algae and land plants). Furthermore, analyses for the sugar specificity and anomeric configuration of the sugar head groups revealed that one of the MGD homologs exhibited a true MGDG synthetic activity. We therefore presumed that higher plant MGDs are derived from this ancestral type of MGD genes, and genes involved in membrane biogenesis and photosystems have been already functionally associated at least at the time of Chloroflexi divergence. As MGD gene duplication is an important event during plastid evolution, we also estimated the divergence time of type A and B MGDs. Our analysis indicated that these genes diverged -323 million years ago, when Spermatophyta (seed plants) were appearing. Galactolipid synthesis is required to produce photosynthetic membranes; based on MGD gene sequences and activities, we have proposed a novel evolutionary model that has increased our understanding of photosynthesis evolution.     

Strain-specific markers for the major structural proteins of highly oncogenic murine mammary tumor viruses by tryptic peptide analyses.

Tryptic peptide analyses were performed on the major structural 52,000- and 36,000-dalton glycoproteins (gp52 and gp36-38) and the nonglycosylated 28,000-, 14,000-, and 10,000-dalton proteins (p28, p14, and p10) of the highly oncogenic murine mammary tumor viruses (MMTVs) of C3H, RIII, and GR mice, i.e., MMTV(C3H), MMTV(RIII), and MMTV(GR), respectively. Each virus was grown in both murine and feline cells to ensure the virus-coded nature of each peptide analyzed. The gp36-38 peptide maps of all three MMTVs were indistinguishable, as were the p14 maps of the different MMTVs. Both the p28 and the gp52 of MMTV(C3H), however, could be clearly distinguished from the corresponding proteins of MMTV(RIII) and MMTV(GR), regardless of whether the viruses were grown in feline or murine cells. The p1o of MMTV(RIII) was clearly different from that of MMTV(C3H) and MMTV(GR). Therefore, tryptic peptide analysis of three proteins, gp52, p28, and p10, can serve to distinguish these three viruses from one another. These studies further characterize the heterogeneity in polypeptides among MMTVs.     

Genes coding for the selenocysteine-inserting tRNA species from Desulfomicrobium baculatum and Clostridium thermoaceticum: structural and evolutionary implications.

The genes (selC) coding for the selenocysteine-inserting tRNA species (tRNA(Sec)) from Clostridium thermoaceticum and Desulfomicrobium baculatum were cloned and sequenced. Although they differ in numerous positions from the sequence of the Escherichia coli selC gene, they were able to complement the selC lesion of an E. coli mutant and to promote selenoprotein formation in the heterologous host. The tRNA(Sec) species from both organisms possess all of the unique primary, secondary, and tertiary structural features exhibited by E. coli tRNA(Sec) (C. Baron, E. Westhof, A. Böck, and R. Giegé, J. Mol. Biol. 231:274-292, 1993). The structural and functional properties of the tRNA(Sec) species from prokaryotes analyzed thus far support the notion that tRNA(Sec) may be an evolutionarily conserved structure whose function in the primordial genetic code was to decode UGA with selenocysteine.     

Two structural states of complexes of peptide and class II major histocompatibility complex revealed by photoaffinity-labeled peptides.

The complex of the murine class II histocompatibility molecules I-A(k) with high affinity binding peptides were resistant to denaturation when examined by SDS-polyacrylamide gel electrophoresis at various pH levels. In contrast, complexes made with low affinity binding peptides were highly sensitive to denaturation by SDS. This effect was more pronounced at low pH. Placing a photoactivatable probe at the amino terminus of the peptides resulted in their covalent linkage to soluble I-A(k) molecules. We found an inverse relationship between the capacity of peptides to form SDS-stable complexes with I-A(k) and their extent of covalent association with either the alpha or beta chain. The relationship held true for three different peptides in which the main anchor residues were changed so as to affect their binding affinity for I-A(k) molecules. Thus, high affinity peptides generate a complex in which the motion of their amino termini was restricted, whereas complexes of low affinity peptides are more flexible. In agreement with this observation, complexes of I-A(k) with high affinity peptides were highly resistant to proteolysis, in contrast to those formed with weakly binding peptides, which were more likely to be cleaved. Complexes with low affinity peptides generate a structure with enhanced flexibility as compared with complexes with high affinity peptides.     

A proteomic approach for identification and localization of the pericellular components of chondrocytes

Although the pericellular matrix (PCM) plays a central role in the communication between chondrocytes and extracellular matrix, its composition is largely unknown. In this study, the PCM was investigated with a proteomic approach using chondrons, which are enzymatically isolated constructs including the chondrocyte and its surrounding PCM. Chondrons and chondrocytes alone were isolated from human articular cartilage. Proteins extracted from chondrons and chondrocytes were used for two-dimensional electrophoresis. Protein spots were quantitatively compared between chondron and chondrocyte gels. Cellular proteins, which had similar density between chondron and chondrocyte gels, did not proceed for analysis. Since chondrons only differ from chondrocytes in association of the PCM, protein spots in the chondron gels that had higher quantity than that in the chondrocyte gels were selected as candidates of the PCM components and processed for mass spectrometry. Among 15 identified peptides, several were fragments of the three type VI collagen chains (α-1, α-2, and α-3). Other identified PCM proteins included triosephosphate isomerase, transforming growth factor-β induced protein, peroxiredoxin-4, ADAM (A disintegrin and metalloproteinases) 28, and latent-transforming growth factor beta-binding protein-2. These PCM components were verified with immunohisto(cyto)chemistry for localization in the PCM region of articular cartilage. The abundance of type VI collagen in the PCM emphasizes its importance to the microenvironment of chondrocytes. Several proteins were localized in the PCM of chondrocytes for the first time and that warrants further investigation for their functions in cartilage biology.     

A differential proteomic approach identifies structural and functional components that contribute to the differentiation of brain capillary endothelial cells

When in the vicinity of astrocytes, brain capillary endothelial cells (BCECs) develop the characteristic structural and functional features of the blood-brain barrier (BBB). The latter has low cellular permeability and restricts various compounds from entering the brain. We recently reported that the cytoskeleton-related proteins actin, gelsolin and filamin-A undergo the largest quantitative changes in bovine BCECs after re-induction of BBB functions by co-culture with glial cells. In the present study, we used an in-depth, proteomic approach to quantitatively compare differences in Triton-X-100-solubilized proteins from bovine BCECs with limited or re-induced BBB functions (i.e. cultured in the absence or presence of glial cells, respectively). The 81 protein spots of differing abundance were linked to 55 distinct genes. According to the Protein ANalysis THrough Evolutionary Relationships classification system and an Ingenuity Pathway Analysis, these quantitative changes mainly affected proteins involved in (i) cell structure and motility and (ii) protein metabolism and modification. The fold-changes affecting HSPB1, moesin and ANXA5 protein levels were confirmed by western blot analysis but were not accompanied by changes in the corresponding mRNA expression levels. Our results reveal that the bovine BCECs' phenotype adaptation to variations in their environment involves the reorganization of the actin cytoskeleton.     

Molecular characterization of the gene coding for major outer membrane protein OmpA from Enterobacter aerogenes

The ompA gene from Enterobacter aerogenes was subcloned into a low-copy-number plasmid vector and the resultant plasmid, pTU7En, used to study its expression in Escherichia coli K12. Although the gene was strongly expressed and large amounts of OmpA protein were present in the outer membrane its product was not functionally identical to the E. coli polypeptide. In particular, the E. aerogenes OmpA protein was unable to confer sensitivity to OmpA-specific phages of E. coli. When the primary structure of the protein was deduced from the nucleotide sequence of its gene it was found that three domains differed extensively from the corresponding regions of the E. coli protein. As two of these are known to be exposed on the cell surface we inferred that these alterations are responsible for differences in the biological activity of the two proteins.     

The evolutionary dynamics of the Helena retrotransposon revealed by sequenced Drosophila genomes

Background  

Several studies have shown that genomes contain a mixture of transposable elements, some of which are still active and others ancient relics that have degenerated. This is true for the non-LTR retrotransposon Helena, of which only degenerate sequences have been shown to be present in some species (Drosophila melanogaster), whereas putatively active sequences are present in others (D. simulans). Combining experimental and population analyses with the sequence analysis of the 12 Drosophila genomes, we have investigated the evolution of Helena, and propose a possible scenario for the evolution of this element.