Evolution of the 28S ribosomal RNA gene in anurans: regions of variability and their phylogenetic implications

Fifteen restriction sites were mapped to the 28S ribosomal RNA gene of individuals representing 54 species of frogs, two species of salamanders, a caecilian, and a lungfish. Eight of these sites were present in all species examined, and two were found in all but one species. Alignment of these conserved restriction sites revealed, among anuran 28S rRNA genes, five regions of major length variation that correspond to four of 12 previously identified divergent domains of this gene. One of the divergent domains (DD8) consists of two regions of length variation separated by a short segment that is conserved at least throughout tetrapods. Most of the insertions, deletions, and restriction-site variations identified in the 28S gene will require sequence-level analysis for a detailed reconstruction of their history. However, an insertion in DD9 that is coextensive with frogs in the suborder Neobatrachia, a BstEII site that is limited to representatives of two leptodactylid subfamilies, and a deletion in DD10 that is found only in three ranoid genera are probably synapomorphies.      

Characterization of cross-bridge elasticity and kinetics of cross-bridge cycling during force development in single smooth muscle cells

Force development in smooth muscle, as in skeletal muscle, is believed to reflect recruitment of force-generating myosin cross-bridges. However, little is known about the events underlying cross-bridge recruitment as the muscle cell approaches peak isometric force and then enters a period of tension maintenance. In the present studies on single smooth muscle cells isolated from the toad (Bufo marinus) stomach muscularis, active muscle stiffness, calculated from the force response to small sinusoidal length changes (0.5% cell length, 250 Hz), was utilized to estimate the relative number of attached cross-bridges. By comparing stiffness during initial force development to stiffness during force redevelopment immediately after a quick release imposed at peak force, we propose that the instantaneous active stiffness of the cell reflects both a linearly elastic cross-bridge element having 1.5 times the compliance of the cross-bridge in frog skeletal muscle and a series elastic component having an exponential length-force relationship. At the onset of force development, the ratio of stiffness to force was 2.5 times greater than at peak isometric force. These data suggest that, upon activation, cross-bridges attach in at least two states (i.e., low-force-producing and high-force-producing) and redistribute to a steady state distribution at peak isometric force. The possibility that the cross-bridge cycling rate was modulated with time was also investigated by analyzing the time course of tension recovery to small, rapid step length changes (0.5% cell length in 2.5 ms) imposed during initial force development, at peak force, and after 15 s of tension maintenance. The rate of tension recovery slowed continuously throughout force development following activation and slowed further as force was maintained. Our results suggest that the kinetics of force production in smooth muscle may involve a redistribution of cross-bridge populations between two attached states and that the average cycling rate of these cross-bridges becomes slower with time during contraction.     

Excretion of superoxide by phagocytes measured with cytochrome c entrapped in resealed erythrocyte ghosts

Resealed erythrocyte membranes (ghosts) filled with (Fe3+)cytochrome c were used as an assay system to measure the release of superoxide (O-2) from human phagocytes into the incubation medium. Neutrophils, activated by either opsonized zymosan particles or the soluble stimulus phorbol myristate acetate, released O-2, which subsequently entered the ghosts and reduced (Fe3+)cytochrome c. This reaction was dependent on the time of incubation, the concentration of neutrophils, the concentration of stimulus, and the concentration of ghosts. The reaction was completely inhibited by superoxide dismutase and by 4,4'-diisothiocyano-2,2'-disulfonic acid, a specific blocker of anion channels in membranes. The reduction of (Fe3+)cytochrome c free in solution was about four times as fast as the reduction of (Fe3+)cytochrome c in the ghosts. Human eosinophils stimulated by phorbol myristate acetate reacted similarly to human neutrophils; the rate of O-2 production/cell was about twice as high for eosinophils as for neutrophils. In contrast, eosinophils stimulated with opsonized zymosan particles only reduced (Fe3+)cytochrome c free in solution, but not (Fe3+)cytochrome c in ghosts. This lack of reaction was not due to production of an inhibitor or below threshold generation of O-2 for the ghost assay. These results indicate: 1) activated human neutrophils and eosinophils can release O-2 or a similar product into the incubation medium; and 2) reduction of (Fe3+)cytochrome c free in solution is no proof for O-2 excretion by phagocytes.     

Excess amino acid polymorphism in mitochondrial DNA: contrasts among genes from Drosophila, mice, and humans

Recent studies of mitochondrial DNA (mtDNA) variation in mammals and Drosophila have shown an excess of amino acid variation within species (replacement polymorphism) relative to the number of silent and replacement differences fixed between species. To examine further this pattern of nonneutral mtDNA evolution, we present sequence data for the ND3 and ND5 genes from 59 lines of Drosophila melanogaster and 29 lines of D. simulans. Of interest are the frequency spectra of silent and replacement polymorphisms, and potential variation among genes and taxa in the departures from neutral expectations. The Drosophila ND3 and ND5 data show no significant excess of replacement polymorphism using the McDonald-Kreitman test. These data are in contrast to significant departures from neutrality for the ND3 gene in mammals and other genes in Drosophila mtDNA (cytochrome b and ATPase 6). Pooled across genes, however, both Drosophila and human mtDNA show very significant excesses of amino acid polymorphism. Silent polymorphisms at ND5 show a significantly higher variance in frequency than replacement polymorphisms, and the latter show a significant skew toward low frequencies (Tajima's D = -1.954). These patterns are interpreted in light of the nearly neutral theory where mildly deleterious amino acid haplotypes are observed as ephemeral variants within species but do not contribute to divergence. The patterns of polymorphism and divergence at charge-altering amino acid sites are presented for the Drosophila ND5 gene to examine the evolution of functionally distinct mutations. Excess charge-altering polymorphism is observed at the carboxyl terminal and excess charge-altering divergence is detected at the amino terminal. While the mildly deleterious model fits as a net effect in the evolution of nonrecombining mitochondrial genomes, these data suggest that opposing evolutionary pressures may act on different regions of mitochondrial genes and genomes.      

Evidence for "twisted plane" undulations in golden hamster sperm tails

Motile spermatozoa from the golden hamster have been arrested by rapid freezing and then fixed with glutaraldehyde at low temperature after substitution with ethylene glycol. As far as can be judged, the flagellar waveforms thus stabilized are similar to those seen in living sperm; in contrast, fixation in glutaraldehyde, without prior freezing, induces agonal changes in flagellar conformation. The characteristics waveform after freeze substitution contains three bends. Approx. half of these flagella are entirely planar. The rest are three dimensional, with the third bend displaced in a regular way from the plane containing the proximal two bends. From the geometry of these flagella, it is concluded that the plane of action of a given bending cycle undergoes a clockwise twist (from a forward viewpoint) as the cycle is succeeded by new bending cycles. This "twisted plane" undulation is quite different from helical movement. The twisting seems to occur abruptly, between cycles, as if each bending cycle has a preferred plane of action. The mechanism underlying the twisting is uncertain. However, on the basis of the angular displacements between the preferred planes, and the findings from electron microscopy, the following idea is presented as a working hypothesis: that, if the most proximal plane of bending is topographically determined by peripheral doublet 1, then successive distal planes of action are influenced predominantly by doublets 2, 3, etc., in clockwise sequence. The merits and weaknesses of this hypothesis are discussed.     

Regulation of Rac1 and Reactive Oxygen Species Production in Response to Infection of Gastrointestinal Epithelia

Generation of reactive oxygen species (ROS) during infection is an immediate host defense leading to microbial killing. APE1 is a multifunctional protein induced by ROS and after induction, protects against ROS-mediated DNA damage. Rac1 and NAPDH oxidase (Nox1) are important contributors of ROS generation following infection and associated with gastrointestinal epithelial injury. The purpose of this study was to determine if APE1 regulates the function of Rac1 and Nox1 during oxidative stress. Gastric or colonic epithelial cells (wild-type or with suppressed APE1) were infected with Helicobacter pylori or Salmonella enterica and assessed for Rac1 and NADPH oxidase-dependent superoxide production. Rac1 and APE1 interactions were measured by co-immunoprecipitation, confocal microscopy and proximity ligation assay (PLA) in cell lines or in biopsy specimens. Significantly greater levels of ROS were produced by APE1-deficient human gastric and colonic cell lines and primary gastric epithelial cells compared to control cells after infection with either gastric or enteric pathogens. H. pylori activated Rac1 and Nox1 in all cell types, but activation was higher in APE1 suppressed cells. APE1 overexpression decreased H. pylori-induced ROS generation, Rac1 activation, and Nox1 expression. We determined that the effects of APE1 were mediated through its N-terminal lysine residues interacting with Rac1, leading to inhibition of Nox1 expression and ROS generation. APE1 is a negative regulator of oxidative stress in the gastrointestinal epithelium during bacterial infection by modulating Rac1 and Nox1. Our results implicate APE1 in novel molecular interactions that regulate early stress responses elicited by microbial infections.     

Impact of human activities on the reproduction of Hooded Vultures Necrosyrtes monachus in Burkina Faso

During the last decades, the critically endangered Hooded Vulture Necrosyrtes monachus has strongly declined across its African range. Although direct persecution has been suggested as a major cause of this decline, little is known about the impact of humans on reproductive output in West Africa. We studied the impact of human activities on the reproductive output of Hooded Vultures in the Garango area of Burkina Faso. Twenty and 56 nesting attempts were monitored, respectively, during the breeding season in 2013/14 and 2014/15, to determine reproductive success and identify causes of nest failure. Annual breeding success varied between 0.68 and 0.71 chicks fledged per breeding pair per year and productivity was assessed at 0.57 chicks fledged per territorial pair in 2014/15. The main threats imposed by humans were poaching of eggs, chicks and collection of nest materials, leading to 20% (13 out of 64 breeding attempts) of nest failures over the two years. An additional important reason for nest failure was the pruning and (partial) cutting of nest trees. Despite this high level of human interference, we found that Hooded Vulture nest success increased with proximity to human settlements, probably because breeding vultures benefit from protection by people against persecution and disturbance.     

Conserved metabolic enzymes as vaccine antigens for giardiasis

Giardia lamblia is a leading protozoal cause of diarrheal disease worldwide. Infection is associated with abdominal pain, malabsorption and weight loss, and protracted post-infectious syndromes. A human vaccine is not available against G. lamblia. Prior studies with human and murine immune sera have identified several parasite antigens, including surface proteins and metabolic enzymes with intracellular functions. While surface proteins have demonstrated vaccine potential, they can exhibit significant variation between G. lamblia strains. By comparison, metabolic enzymes show greater conservation but their vaccine potential has not been established. To determine whether such proteins can serve as vaccine candidates, we focused on two enzymes, α-enolase (ENO) and ornithine carbamoyl transferase (OCT), which are involved in glycolysis and arginine metabolism, respectively. We show in a cohort of patients with confirmed giardiasis that both enzymes are immunogenic. Intranasal immunization with either enzyme antigen in mice induced strong systemic IgG1 and IgG2b responses and modest mucosal IgA responses, and a marked 100- to 1,000-fold reduction in peak trophozoite load upon oral G. lamblia challenge. ENO immunization also reduced the extent and duration of cyst excretion. Examination of 44 cytokines showed only minimal intestinal changes in immunized mice, although a modest increase of CCL22 was observed in ENO-immunized mice. Spectral flow cytometry revealed increased numbers and activation state of CD4 T cells in the small intestine and an increase in α4β7-expressing CD4 T cells in mesenteric lymph nodes of ENO-immunized mice. Consistent with a key role of CD4 T cells, immunization of CD4-deficient and Rag-2 deficient mice failed to induce protection, whereas mice lacking IgA were fully protected by immunization, indicating that immunity was CD4 T cell-dependent but IgA-independent. These results demonstrate that conserved metabolic enzymes can be effective vaccine antigens for protection against G. lamblia infection, thereby expanding the repertoire of candidate antigens beyond primary surface proteins.