Involvement of Neisseria meningitidis Lipoprotein GNA2091 in the Assembly of a Subset of Outer Membrane Proteins

GNA2091 of Neisseria meningitidis is a lipoprotein of unknown function that is included in the novel 4CMenB vaccine. Here, we investigated the biological function and the subcellular localization of the protein. We demonstrate that GNA2091 functions in the assembly of outer membrane proteins (OMPs) because its absence resulted in the accumulation of misassembled OMPs. Cell fractionation and protease accessibility experiments showed that the protein is localized at the periplasmic side of the outer membrane. Pulldown experiments revealed that it is not stably associated with the β-barrel assembly machinery, the previously identified complex for OMP assembly. Thus, GNA2091 constitutes a novel outer membrane-based lipoprotein required for OMP assembly. Furthermore, its location at the inner side of the outer membrane indicates that protective immunity elicited by this antigen cannot be due to bactericidal or opsonic activity of antibodies.     

Characterization and quantification of the peroxidase in human monocytes.

1. The lifetime of thiamine pyrophosphate-Sepharose 2B affinity matrices synthesized according to Matsuura et al. (Matsuura, A., Iwashina, A. and Nose, Y. (1973) Biochem. Biophys. Res. Commun. 51, 241-246) has been improved. The matrix interacts with bacterial pyruvate dehydrogenase complexes. 2. The synthesis of a stable thiochrome-Sepharose 2B matrix is described. 3. Both matrices bind the pyruvate dehydrogenase complex of Escherichia coli in a 50 mM phosphate buffer, pH 7.0. Elution is possibly by an increase in ionic strength but not by the cofactor or metal-cofactor complexes. 4. The presence of Mg2+, reduces the capacity of the affinity matrices but leads to higher specificity for the multienzyme complex. 5. The pyruvate dehydrogenase complex of E. coli has been successfully purified by combining a classical purification step with these affinity chromatography systems. The method is less suitable for large scale operation.     

Plastics select for distinct early colonizing microbial populations with reproducible traits across environmental gradients

Little is known about early plastic biofilm assemblage dynamics and successional changes over time. By incubating virgin microplastics along oceanic transects and comparing adhered microbial communities with those of naturally occurring plastic litter at the same locations, we constructed gene catalogues to contrast the metabolic differences between early and mature biofilm communities. Early colonization incubations were reproducibly dominated by Alteromonadaceae and harboured significantly higher proportions of genes associated with adhesion, biofilm formation, chemotaxis, hydrocarbon degradation and motility. Comparative genomic analyses among the Alteromonadaceae metagenome assembled genomes (MAGs) highlighted the importance of the mannose-sensitive hemagglutinin (MSHA) operon, recognized as a key factor for intestinal colonization, for early colonization of hydrophobic plastic surfaces. Synteny alignments of MSHA also demonstrated positive selection for mshA alleles across all MAGs, suggesting that mshA provides a competitive advantage for surface colonization and nutrient acquisition. Large-scale genomic characteristics of early colonizers varied little, despite environmental variability. Mature plastic biofilms were composed of predominantly Rhodobacteraceae and displayed significantly higher proportions of carbohydrate hydrolysis enzymes and genes for photosynthesis and secondary metabolism. Our metagenomic analyses provide insight into early biofilm formation on plastics in the ocean and how early colonizers self-assemble, compared to mature, phylogenetically and metabolically diverse biofilms.     

Expression of biologically active human clotting factor IX in Drosophila S2 cells: γ-carboxylation of a human vitamin K-dependent protein by the insect enzyme

The Drosophila γ-glutamyl carboxylase (dγC) has substrate recognition properties similar to that of the vertebrate γ-carboxylase (γC), and its carboxylated product yield, in vitro, was shown to be more than that obtained with the human enzyme. However, whether the Drosophila enzyme is able to γ-carboxylate the human vitamin K-dependent (VKD) proteins, such as the human coagulation factor IX (hFIX), as synthesized in cultured Drosophila cells was not known. To examine this possibility, the Drosophila Schnider (S2) cell line was transfected with a metallothionein promoter-regulated hFIX-expressing plasmid. After induction with copper ion, expression efficiency of the active hFIX was analyzed by performing enzyme-linked immunosorbent assey (ELISA) and coagulation test on the culture supernatant of the transfected S2 cells during 72 h of postinduction. In comparison with Chinese hamster ovary cell line, S2 cells showed higher (≈ 12-fold) expression level of the hFIX. The γ-carboxylation of the Drosophila-derived hFIX was confirmed by evaluation of the expressed protein, after being precipitated with barium citrate. The biological activity of the S2 cell-derived hFIX indicated the capability of S2 cells to fulfill the required γ-carboxylation of the expressed hFIX. Coexpression of the human γ-glutamyl carboxylases (hγC) was also shown to improve both expression and γ-carboxylation of the hFIX. This is the first in vivo data to describe the ability of the dγC to recognize the human-based propeptide as substrate, which is an essential step for production of biologically active γ-carboxylated VKD proteins.     

Adaptive evolution has targeted the C-terminal domain of the RXLR effectors of plant pathogenic oomycetes

Oomycete plant pathogens deliver effector proteins inside host cells to modulate plant defense circuitry and to enable parasitic colonization. These effectors are defined by a conserved motif, termed RXLR (for Arg, any amino acid, Leu, Arg), that is located downstream of the signal peptide and that has been implicated in host translocation. Because the phenotypes of RXLR effectors extend to plant cells, their genes are expected to be the direct target of the evolutionary forces that drive the antagonistic interplay between pathogen and host. We used the draft genome sequences of three oomycete plant pathogens, Phytophthora sojae, Phytophthora ramorum, and Hyaloperonospora parasitica, to generate genome-wide catalogs of RXLR effector genes and determine the extent to which these genes are under positive selection. These analyses revealed that the RXLR sequence is overrepresented and positionally constrained in the secretome of Phytophthora relative to other eukaryotes. The three examined plant pathogenic oomycetes carry complex and diverse sets of RXLR effector genes that have undergone relatively rapid birth and death evolution. We obtained robust evidence of positive selection in more than two-thirds of the examined paralog families of RXLR effectors. Positive selection has acted for the most part on the C-terminal region, consistent with the view that RXLR effectors are modular, with the N terminus involved in secretion and host translocation and the C-terminal domain dedicated to modulating host defenses inside plant cells.     

Down-regulation of the ubiquitin�Cproteasome proteolysis system by amino acids and insulin involves the adenosine monophosphate-activated protein kinase and mammalian target of rapamycin pathways in rat hepatocytes

The purpose of this work was to examine whether changes in dietary protein levels could elicit differential responses of tissue proteolysis and the pathway involved in this response. In rats fed with a high protein diet (55%) for 14?days, the liver was the main organ where adaptations occurred, characterized by an increased protein pool and a strong, meal-induced inhibition of the protein breakdown rate when compared to the normal protein diet (14%). This was associated with a decrease in the key-proteins involved in expression of the ubiquitin-proteasome and autophagy pathway gene and a reduction in the level of hepatic ubiquitinated protein. In hepatocytes, we demonstrated that the increase in amino acid (AA) levels was sufficient to down-regulate the ubiquitin proteasome pathway, but this inhibition was more potent in the presence of insulin. Interestingly, AICAR, an adenosine monophosphate-activated protein kinase (AMPK) activator, reversed the inhibition of protein ubiquination induced by insulin at high AA concentrations. Rapamycin, an mammalian target of rapamycin (mTOR) inhibitor, reversed the inhibition of protein ubiquination induced by a rise in insulin levels with both high and low AA concentrations. Moreover, in both low and high AA concentrations in the presence of insulin, AICAR decreased the mTOR phosphorylation, and in the presence of both AICAR and rapamycin, AICAR reversed the effects of rapamycin. These results demonstrate that the inhibition of AMPK and the activation of mTOR transduction pathways, are required for the down-regulation of protein ubiquitination in response to high amino acid and insulin concentrations.     

A novel model of cryoinjury-induced myocardial infarction in the mouse: a comparison with coronary artery ligation

Mouse myocardial infarction (MI) models are frequently used research tools. The most commonly applied model is coronary artery ligation. However, coronary ligation often gives rise to apical aneurysmatic infarcts of variable size. Other infarct models include cryoinfarction, which produces reproducible infarcts of the anterior wall. Thus far, this model has not been extensively described in mice. Therefore, we developed a murine cryoinfarction model and compared it with coronary ligation. Studies were performed under isoflurane anesthesia with a follow-up of 4 and 8 wk. Cryoinfarction was induced using a 2- or 3-mm cryoprobe. Two-dimensional guided M-mode echocardiography was used to assess fractional shortening and left ventricular (LV) dimensions at baseline and end point. At end point, hemodynamics were assessed using a 1.4-Fr Millar catheter. Pressure-diameter relations were constructed by combining echocardiography and hemodynamic data. Histological and morphometric analyses of infarct and remote areas were performed. At 4 wk, 3-mm cryoinfarction resulted in decreased LV fractional shortening as well as decreased global LV contractility and relaxation, which was comparable with coronary ligation. No adverse remodeling was observed at this time point, in contrast with the ligation model. However, progressive LV remodeling occured between 4 and 8 wk after cryoinfarction with a further decline in hemodynamic parameters and LV pump function. Histologically, cryoinfarction resulted in highly reproducible, transmural, cone-shaped infarcts with reperfusion at the macrovascular level. These results indicate that the cryoinfarction model represents the anterior myocardial infarct with modest adverse remodeling and may thus be representative for infarcts encountered in clinical practice.     

Fine structure of the 21S ribosomal RNA region on yeast mitochondrial DNA

Summary The omega locus controls the polarity of recombination and transmission of genetic markers in the 21S ribosomal RNA region in yeast mtDNA. Polarity is observed in crosses between omega⁺ and omega^- strains. These two strains differ by the presence of an intervening sequence in the 21S ribosomal RNA gene of omega⁺ strains. Mutations of the omega^- allele, omega neutral (omegaⁿ), can eliminate the polarity effect. We have made DNA:RNA hybrids containing ribosomal RNA from an omegaⁿ strain and mtDNA from Saccharomyces carlsbergensis (identical to omega^- in the nucleotide sequence of the omega region). These hybrids contain no mismatch at the omega region detectable by digestion with S₁ nuclease. We conclude that omegaⁿ differs from omega^- only in a point mutation or analogous small alteration and that the omegaⁿ mutation can result either m a C^r phenotype (omegaⁿC^r) or in the phenotypic suppression of pre-existing C^r mutations (omegaⁿC^s). All results can be explained by a model which postulates interaction in the ribosome between the C^r and omegaⁿ regions of the ribosomal RNA and interference of the omegaⁿ mutation with splicing of the precursor ribosomal RNA in omega⁺ strains. The mechanism of omega-directed polarity is discussed.Abbreviations rRNA ribosomal RNA - bp base pair(s) - kb kilo base pair(s)