Evolution after gene duplication: models, mechanisms, sequences, systems, and organisms

Gene duplication is postulated to have played a major role in the evolution of biological novelty. Here, gene duplication is examined across levels of biological organization in an attempt to create a unified picture of the mechanistic process by which gene duplication can have played a role in generating biodiversity. Neofunctionalization and subfunctionalization have been proposed as important processes driving the retention of duplicate genes. These models have foundations in population genetic theory, which is now being refined by explicit consideration of the structural constraints placed upon genes encoding proteins through physical chemistry. Further, such models can be examined in the context of comparative genomics, where an integration of gene-level evolution and species-level evolution allows an assessment of the frequency of duplication and the fate of duplicate genes. This process, of course, is dependent upon the biochemical role that duplicated genes play in biological systems, which is in turn dependent upon the mechanism of duplication: whole genome duplication involving a co-duplication of interacting partners vs. single gene duplication. Lastly, the role that these processes may have played in driving speciation is examined.     

Genetic characterization of the Guinea-Bissau family of Mycobacterium tuberculosis complex strains

In a previous study of Mycobacterium tuberculosis complex isolates from Guinea-Bissau in West Africa, we identified a unique group of strains, designated here as the Guinea-Bissau family of strains, which, although genotypically closely related, phenotypically demonstrated a considerable heterogeneity. We conducted here a detailed genotypic analysis of a subset (n = 35) of these isolates. Based on the data obtained, and by comparison of known corresponding genes in mycobacteria outside the M. tuberculosis complex, we propose that the Guinea-Bissau strains belong to a unique branch of the M. tuberculosis complex tree in between classical M. tuberculosis and classical M. bovis. These observations are discussed in their significance in M. tuberculosis complex classification.     

Phylogeography of Cavernularia hultenii: evidence of slow genetic drift in a widely disjunct lichen

Population structure and history is poorly known in most lichenized ascomycetes. Many species display large-scale infraspecific disjunctions, which have been explained alternately by range fragmentation in species of high age and widespread long-distance dispersal. Using the lichen Cavernularia hultenii, which is widely disjunct across North America and Europe, Pleistocene and Holocene population history was inferred. The internal transcribed spacer (ITS) and part of the the intergenic spacer (IGS) region of the nuclear ribosomal DNA were sequenced in 300 individuals representing 62 populations across the range of the species. While four ancestral haplotypes are found in all areas, none of the observed tip haplotypes is present in more than one of the three part ranges. Although this is evidence for a past fragmentation event, nested clade analysis (NCA) remains equivocal in the choice between allopatric fragmentation and long-distance dispersal. Mismatch distributions indicate exponential population growth, probably during postglacial invasion of C. hultenii into formerly glaciated areas of western North America. The presence of one southern and at least one northern glacial refugium in South Central Alaska is inferred. Evidence for another refugium in the Queen Charlotte Islands or Alexander Archipelago is inconclusive because of sparse sampling. However, a range expansion was not confirmed unambiguously by NCA. The limited power of NCA to infer past range fragmentations and expansions is due apparently to the shallow haplotype network and widespread ancestral haplotypes. This can be explained by slow genetic drift causing incomplete removal of ancestral haplotypes from the postfragmentation and postexpansion areas.     

Strong differential regulation of serum and mucosal IgA responses as revealed in CD28-deficient mice using cholera toxin adjuvant

In this study, we show that costimulation required for mucosal IgA responses is strikingly different from that needed for systemic responses, including serum IgA. Following oral immunization with cholera toxin (CT) adjuvant we found that whereas CTLA4-H1 transgenic mice largely failed to respond, CD28-/- mice developed near normal gut mucosal IgA responses but poor serum Ab responses. The local IgA response was functional in that strong antitoxic protection developed in CT-immunized CD28-/- mice. This was in spite of the fact that no germinal centers (GC) were observed in the Peyer's patches, spleen, or other peripheral lymph nodes. Moreover, significant somatic hypermutation was found in isolated IgA plasma cells from gut lamina propria of CD28-/- mice. Thus, differentiation to functional gut mucosal IgA responses against T cell-dependent Ags does not require signaling through CD28 and can be independent of GC formations and isotype-switching in Peyer's patches. By contrast, serum IgA responses, similar to IgG-responses, are dependent on GC and CD28. However, both local and systemic responses are impaired in CTLA4-Hgamma1 transgenic mice, indicating that mucosal IgA responses are dependent on the B7-family ligands, but require signaling via CTLA4 or more likely a third related receptor. Therefore, T-B cell interactions leading to mucosal as opposed to serum IgA responses are uniquely regulated and appear to represent separate events. Although CT is known to strongly up-regulate B7-molecules, we have demonstrated that it acts as a potent mucosal adjuvant in the absence of CD28, suggesting that alternative costimulatory pathways are involved.     

Etk/Bmx transactivates vascular endothelial growth factor 2 and recruits phosphatidylinositol 3-kinase to mediate the tumor necrosis factor-induced angiogenic pathway

Tumor necrosis factor (TNF), via its receptor 2 (TNFR2), induces Etk (or Bmx) activation and Etk-dependent endothelial cell (EC) migration and tube formation. Because TNF receptor 2 lacks an intrinsic kinase activity, we examined the kinase(s) mediating TNF-induced Etk activation. TNF induces a coordinated phosphorylation of vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) and Etk, which is blocked by VEGFR2-specific inhibitors. In response to TNF, Etk and VEGFR2 form a complex resulting in a reciprocal activation between the two kinases. Subsequently, the downstream phosphatidylinositol 3-kinase (PI3K)-Akt signaling (but not signaling through phospholipase C-gamma) was initiated and directly led to TNF-induced EC migration, which was significantly inhibited by VEGFR2-, PI3K-, or Akt-specific inhibitors. Phosphorylation of VEGFR2 at Tyr-801 and Tyr-1175, the critical sites for VEGF-induced PI3K-Akt signaling, was not involved in TNF-mediated Akt activation. However, TNF induces phosphorylation of Etk at Tyr-566, directly mediating the recruitment of the p85 subunit of PI3K. Furthermore, TNF- but not VEGF-induced activation of VEGFR2, Akt, and EC migration are blunted in EC genetically deficient with Etk. Taken together, our data demonstrated that TNF induces transactivation between Etk and VEGFR2, and Etk directly activates PI3K-Akt angiogenic signaling independent of VEGF-induced VEGFR2-PI3K-Akt signaling pathway.     

Induction of innate immune responses by Escherichia coli and purified lipopolysaccharide correlate with organ- and cell-specific expression of Toll-like receptors within the human urinary tract

Mucosal epithelial linings function as physical barriers against microbes. In addition, they participate in the first line of host defence by production of a variety of proinflammatory mediators when exposed to microbes and microbial agents. Here, we use a human urinary tract infection model to demonstrate that organ- and cell-specific innate responses induced by lipopolysaccharides (LPS) present on Gram-negative bacteria correlates with the expression of Toll-like receptor 4 (TLR4). The presence of TLR4 on human bladder epithelial cells enables them to rapidly respond to bacterial infections in vitro and in vivo . In contrast, TLR4 is not expressed on human proximal tubule cells isolated from the renal cortex, which may explain the cortical localization of bacteria in pyelonephritis. TLR4-negative renal epithelial cells, A498, are non-responsive to purified LPS, however, they respond to viable bacteria via a mannose-sensitive attachment-mediated pathway. To identify LPS components recognised by bladder epithelial cells, a bacterial lipid A mutant and LPS of different chemotypes were tested. Full interleukin 8 induction required hexa-acylated lipid A and was decreased by between 50% and 70% in the presence of O-antigen. Taken together, we propose that multiple independent pathways, which are organ- and cell-specifically expressed, mediate bacterial recognition and determine the outcome of innate responses to infection.     

Nanoinjection as a tool to mimic vertical transmission of Flavobacterium psychrophilum in rainbow trout Oncorhynchus mykiss

Newly fertilised eggs of rainbow trout Oncorhynchus mykiss were nanoinjected with Flavobacterium psychrophilum in order to mimic vertical transmission. Two bacterial isolates with different elastin-degrading capacity were used. All infected groups (10, 100 and 1000 colony forming units egg(-1)) showed significantly higher cumulative mortalities than the control groups at the end of the experiment, 70 d post-hatching. The total mortalities in the control groups were below 2.5%. In the high-dose groups, 95 to 100% of the eggs died during the eyed stage. In the intermediate group infected with the elastin-negative isolate, the major mortality occurred during the eyed stage of the egg, with a total cumulative mortality of 83% at the end of the experiment. In the intermediate group infected with the elastin-positive isolate, a total mortality of 63% was recorded. In this group, diseased fry showed clinical signs of disease and morphological changes similar to those described in connection with rainbow trout fry syndrome (RTFS) shortly after the beginning of feeding. In the low dose groups, the mortality in the elastin-negative group was 14% and in the elastin-positive group 11%. The bacterium was isolated from dead eggs and fry in infected groups and demonstrated in internal organs of dead and moribund fry by immunohistochemistry. The nanoinjection method used in this study may be a useful method to study pathogens, like F. psychrophilum, that can be vertically transmitted.     

Distributions of Li+, Na+, K+, Rb+, and Cs+ tracer ions in erythrocytes at 38 °C in relation to entry rates of these ions into cells at 0 °C

Forces that are able to transport Na+ and K+ into two compartments were investigated. A modified Nernst-Planck equation for coupled flows of electric current, water, and ions was integrated. The result shows that if alkali ions in the ion channel of the cell membrane are separated by their electric-current-induced inward flows against an electro-osmotic outward flow of water, the logarithms of the stationary cell/medium distributions of these ions should be proportional to the inverse of their diffusion mobilities. The relationship was tested in human erythrocytes. From inward and outward movements of tracer alkali ions, calculations were made to obtain their stationary distributions at infinite time. The cell/medium distributions determined in this way at 38 degrees C are Li+ = 0.59, 22Na+ = 0.044, 42K+ = 10.0, 86Rb+ = 11.9, and 137Cs+ = 3.07. The entry rates of ions into the cell at 0 degrees C are understood to represent their diffusion mobilities in the pump channel. The entry rates are Li+ = 1.44, 2Na+ = 1, 42K+ = 2.22, 86Rb+ = 2.39, and 137Cs+ = 1.72 relative to that of 22Na+. There is an expected negative correlation between the logarithms of the stationary cell/ medium distributions at 38 degrees C and the inverse of the entry rates into the cell at 0 degrees C for the five ions. It is suggested that the proposed physical forces cause the separation of alkali ions in the channel of Na,K-ATPase.     

The ADP-ribosylating CTA1-DD adjuvant enhances T cell-dependent and independent responses by direct action on B cells involving anti-apoptotic Bcl-2- and germinal center-promoting effects

We recently developed a novel immunomodulating gene fusion protein, CTA1-DD, that combines the ADP-ribosylating ability of cholera toxin (CT) with a dimer of an Ig-binding fragment, D, of Staphylococcus aureus protein A. The CTA1-DD adjuvant was found to be nontoxic and greatly augmented T cell-dependent responses to soluble protein Ags after systemic as well as mucosal immunizations. Here we show that CTA1-DD does not appear to form immune complexes or bind to soluble Ig following injections, but, rather, it binds directly to B cells of all isotypes, including naive IgD+ cells. No binding was observed to macrophages or dendritic cells. Immunizations in FcepsilonR (common FcRgamma-chain)- and FcgammaRII-deficient mice demonstrated that CTA1-DD exerted unaltered enhancing effects, indicating that FcgammaR-expressing cells are not required for the adjuvant function. Whereas CT failed to augment Ab responses to high m.w. dextran B512 in athymic mice, CTA1-DD was highly efficient, demonstrating that T cell-independent responses were also enhanced by this adjuvant. In normal mice both CT and CTA1-DD, but not the enzymatically inactive CTA1-R7K-DD mutant, were efficient enhancers of T cell-dependent as well as T cell-independent responses, and both promoted germinal center formation following immunizations. Although CT augmented apoptosis in Ag receptor-activated B cells, CTA1-DD strongly counteracted apoptosis by inducing Bcl-2 in a dose-dependent manner, a mechanism that was independent of the CD19 coreceptor. However, in the presence of CD40 stimulation, apoptosis was low and unaffected by CT, suggesting that the adjuvant effect of CT is dependent on the presence of activated CD40 ligand-expressing T cells.     

Dystroglycan overexpression in vivo alters acetylcholine receptor aggregation at the neuromuscular junction

Dystroglycan is a member of the transmembrane dystrophin glycoprotein complex in muscle that binds to the synapse-organizing molecule agrin. Dystroglycan binding and AChR aggregation are mediated by two separate domains of agrin. To test whether dystroglycan plays a role in receptor aggregation at the neuromuscular junction, we overexpressed it by injecting rabbit dystroglycan RNA into one- or two-celled Xenopus embryos. We measured AChR aggregation in myotomes by labeling them with rhodamine-alpha-bungarotoxin followed by confocal microscopy and image analysis. Dystroglycan overexpression decreased AChR aggregation at the neuromuscular junction. This result is consistent with dystroglycan competition for agrin without signaling AChR aggregation. It also supports the hypothesis that dystroglycan is not the myotube-associated specificity component, (MASC) a putative coreceptor needed for agrin to activate muscle-specific kinase (MuSK) and signal AChR aggregation. Dystroglycan was distributed along the surface of muscle membranes, but was concentrated at the ends of myotomes, where AChRs normally aggregate at synapses. Overexpressed dystroglycan altered AChR aggregation in a rostral-caudal gradient, consistent with the sequential development of neuromuscular synapses along the embryo. Increasing concentrations of dystroglycan RNA did not further decrease AChR aggregation, but decreased embryo survival. Development often stopped during gastrulation, suggesting an essential, nonsynaptic role of dystroglycan during this early period of development.