Parameterising a public good: how experiments on predation can be used to predict cheat frequencies

Chemical defence is superficially easy to understand as a means for individuals to protect themselves from enemies. The evolution of chemical defence is however potentially complex because such defences may cause the generation of a public good, protecting members of the population as a whole as well as individuals that deploy toxins defensively. If a public good of protection exists, it may be exploited and degraded by “cheats” that do not invest in defence. This can in turn lead to complex frequency (and density) dependent effects in toxin evolution. To investigate this we used ecologically relevant predators (Great tits, Parus major) and examined how individual and public benefits vary depending on the frequency of non-defended “cheating” prey and their spatial distribution. We found that the public benefit, of reduced attack probability, increased with increasing frequency of defended individuals. In contrast the individual benefit of chemical defence, measured as increased chance of rejection during an attack before injury, did not vary with the frequency of defended forms. Hence the selective dynamics of these two levels of benefits responded differently to the frequency of defended forms. Surprisingly, given the strong associations of chemical defences and grouping in animals, large aggregations did not help individuals in the group regardless of their defence status. The explanation for the result, may be that in our experiment birds did not have information about other potential aggregations (i.e. set up was sequential) and thus their giving up density was lower compared to the situations where set ups were simultaneous. We use behavioural data of our predators to construct a simple model of toxin evolution which can make quantitative predictions about the frequencies to which defence cheats evolve. We use this model to discuss how toxin evolution can be investigated in the wild and in laboratory settings.     

Genome-wide analysis to predict protein sequence variations that change phosphorylation sites or their corresponding kinases

We define phosphovariants as genetic variations that change phosphorylation sites or their interacting kinases. Considering the essential role of phosphorylation in protein functions, it is highly likely that phosphovariants change protein functions. Therefore, a comparison of phosphovariants between individuals or between species can give clues about phenotypic differences. We categorized phosphovariants into three subtypes and developed a system that predicts them. Our method can be used to screen important polymorphisms and help to identify the mechanisms of genetic diseases.     

Evidence that regenerative ability is an intrinsic property of limb cells in Xenopus

Xenopus laevis exhibits an ontogenetic decline in the ability to regenerate its limbs: Young tadpoles can completely regenerate an amputated limb, whereas post metamorphic froglets regenerate at most a cartilagenous "spike." We have tested the regenerative competence of normally regenerating limb buds of stage 52-53 Xenopus tadpoles grafted onto limb stumps of postmetamorphic froglets. The limb buds become vascularized and innervated by the host and, when amputated, regenerate limbs with normal or slightly less than normal numbers of tadpole hindlimb digits. Reciprocal grafts of froglet forelimb blastemas onto tadpole hindlimb stumps resulted in either autonomous development of tadpole hindlimb structures and/or formation of a cartilaginous spike typical of froglet forelimb regeneration. Our results suggest that the Xenopus froglet host environment is completely permissive for regeneration and that the ability to regenerate a complete limb pattern is an intrinsic property of young tadpole limb cells, a property that is lost during ontogenesis.     

The Rubella virus capsid is an anti-apoptotic protein that attenuates the pore-forming ability of Bax

Apoptosis is an important mechanism by which virus-infected cells are eliminated from the host. Accordingly, many viruses have evolved strategies to prevent or delay apoptosis in order to provide a window of opportunity in which virus replication, assembly and egress can take place. Interfering with apoptosis may also be important for establishment and/or maintenance of persistent infections. Whereas large DNA viruses have the luxury of encoding accessory proteins whose primary function is to undermine programmed cell death pathways, it is generally thought that most RNA viruses do not encode these types of proteins. Here we report that the multifunctional capsid protein of Rubella virus is a potent inhibitor of apoptosis. The main mechanism of action was specific for Bax as capsid bound Bax and prevented Bax-induced apoptosis but did not bind Bak nor inhibit Bak-induced apoptosis. Intriguingly, interaction with capsid protein resulted in activation of Bax in the absence of apoptotic stimuli, however, release of cytochrome c from mitochondria and concomitant activation of caspase 3 did not occur. Accordingly, we propose that binding of capsid to Bax induces the formation of hetero-oligomers that are incompetent for pore formation. Importantly, data from reverse genetic studies are consistent with a scenario in which the anti-apoptotic activity of capsid protein is important for virus replication. If so, this would be among the first demonstrations showing that blocking apoptosis is important for replication of an RNA virus. Finally, it is tempting to speculate that other slowly replicating RNA viruses employ similar mechanisms to avoid killing infected cells.     

PLAC-24 is a cytoplasmic dynein-binding protein that is recruited to sites of cell-cell contact

We screened for polypeptides that interact specifically with dynein and identified a novel 24-kDa protein (PLAC-24) that binds directly to dynein intermediate chain (DIC). PLAC-24 is not a dynactin subunit, and the binding of PLAC-24 to the dynein intermediate chain is independent of the association between dynein and dynactin. Immunocytochemistry using PLAC-24-specific polyclonal antibodies revealed a punctate perinuclear distribution of the polypeptide in fibroblasts and isolated epithelial cells. However, as epithelial cells in culture make contact with adjacent cells, PLAC-24 is specifically recruited to the cortex at sites of contact, where the protein colocalizes with components of the adherens junction. Disruption of the cellular cytoskeleton with latrunculin or nocodazole indicates that the localization of PLAC-24 to the cortex is dependent on intact actin filaments but not on microtubules. Overexpression of beta-catenin also leads to a loss of PLAC-24 from sites of cell-cell contact. On the basis of these data and the recent observation that cytoplasmic dynein is also localized to sites of cell-cell contact in epithelial cells, we propose that PLAC-24 is part of a multiprotein complex localized to sites of intercellular contact that may function to tether microtubule plus ends to the actin-rich cellular cortex.     

Evidence that phospholipid turnover is the signal transducing system coupled to serotonin-S2 receptor sites

Upon stimulation with serotonin of washed human platelets prelabeled with [32P]orthophosphate, we found an approximately 250% increase in [32P]phosphatidic acid (PA) formation, a small decrease in [32P]phosphatidylinositol 4,5-bisphosphate, and a concomitant increase in [32P]phosphatidylinositol 4-phosphate. Using [3H]arachidonate for prelabeling, [3H]diacylglycerol accumulated transiently at 10 s after addition of the agonist, [3H]PA increased but to a lower extent compared to 32P-labeled lipid, and the formation of both [3H]polyphosphoinositides increased. The serotonin-induced dose-dependent changes in [32P]PA correlate with its effect on the changes in slope of aggregation of platelets. The potency of 13 drugs to antagonize the serotonin-induced PA formation closely corresponds to both their potency to inhibit platelet aggregation and their binding affinity for serotonin-S2 receptor sites. It is suggested that at least part of the signal transducing system following activation of the serotonin-S2 receptors involves phospholipase C catalyzed inositol lipid breakdown yielding diacylglycerol which is subsequently phosphorylated to PA.     

Phosphatidylethanolamine has an essential role in Saccharomyces cerevisiae that is independent of its ability to form hexagonal phase structures

Two yeast enzymes, Psd1p and Psd2p, catalyze the decarboxylation of phosphatidylserine to produce phosphatidylethanolamine (PtdEtn). Mitochondrial Psd1p provides approximately 90% of total cellular phosphatidylserine decarboxylase activity. When the PSD1 gene is deleted, the resultant strain (psd1Delta) grows normally at 30 degrees C in glucose and in the absence of exogenous choline or ethanolamine. However, at elevated temperature (37 degrees C) or on the nonfermentable carbon source lactate, the growth of psd1Delta strains is minimal without ethanolamine supplementation. The reduced growth and viability correlate with a PtdEtn content below 4% of total phospholipid. These results suggest that there is a critical level of PtdEtn required to support growth. This theory is supported by growth data revealing that a psd1Delta psd2Delta dpl1Delta strain can only grow in the presence of ethanolamine. In contrast, a psd1Delta psd2Delta strain, which makes low levels of PtdEtn from sphingolipid breakdown, can be rescued by ethanolamine, choline, or the ethanolamine analogue propanolamine. psd1Delta psd2Delta cells grown in 2 mm propanolamine accumulate a novel lipid, which was determined by mass spectrometry to be phosphatidylpropanolamine (PtdPrn). PtdPrn can comprise up to 40% of the total phospholipid content in supplemented cells at the expense of phosphatidylcholine and PtdEtn. The absolute level of PtdEtn required for growth when PtdPrn is present appears to be 1% of the total phospholipid content. The essential function of the PtdEtn in the presence of propanolamine does not appear to be the formation of hexagonal phase lipid, insofar as PtdPrn readily forms hexagonal phase structures detectable by (31)P NMR.     

Molecular motors: how to make models that can be used to convey the concept of molecular ratchets and thermal capture

A wide variety of cellular processes use molecular motors, including processive motors that move along some form of track (e.g., myosin with actin, kinesin or dynein with tubulin) and polymerases that move along a template (e.g., DNA and RNA polymerases, ribosomes). In trying to understand how these molecular motors actually move, many apply their understanding of how man-made motors work: the latter use some form of energy to exert a force or torque on its load. However, quite a different mechanism has been proposed to possibly account for the movement of molecular motors. Rather than hydrolyzing ATP to push or pull their load, they might use their own thermal vibrational energy as well as that of their load and their environment to move the load, capturing those movements that occur along a desired vector or axis and resisting others; ATP hydrolysis is required to make backward movements impossible. This intriguing thermal capture or Brownian ratchet model is relatively more difficult to convey to students. In this report, we describe several teaching aids that are very easily constructed using widely available household materials to convey the concept of a molecular ratchet.     

Evidence that positional information is used to establish the prestalk-prespore pattern in Dictyostelium discoideum aggregates

Two contrasting mechanisms have been proposed for the establishment of the prestalk-prespore pattern in the multicellular aggregate of the simple eukaryote Dictyostelium discoideum. One involves intermingled, non-position-dependent cell differentiation followed by sorting out which produces the pattern of prestalk cells in the anterior region and prespore cells posteriorly. The second mechanism involves patterning according to the position of cells within the aggregate, in which case intermingled cell types are not expected. Here we use a monoclonal antibody (MUD1), recognising a prespore cell surface antigen, to study the initial appearance of prespore cells in aggregates. Quantitative studies were made with a flow cytometer and frozen sections were used to localise the cells expressing the prespore antigen. This antigen first appeared at the onset of tip formation in the centre of aggregates in a position-dependent fashion. The prespore antigen was not detected in the tip region or in streams of cells entering the aggregate. We re-examined the evidence on which the non-position-dependent differentiation model is based. Our results support the positional model for pattern formation.     

Evaluating the ability of regional models to predict local avian abundance

Spatial modeling over broad scales can potentially direct conservation efforts to areas with high species-specific abundances. We examined the performance of regional models for predicting bird abundance at spatial scales typically addressed in conservation planning. Specifically, we used point count data on wood thrush (Hylocichla mustelina) and blue-winged warbler (Vermivora cyanoptera) from 2 time periods (1995–1998 and 2006–2007) to evaluate the ability of regional models derived via Bayesian hierarchical techniques to predict bird abundance. We developed models for each species within Bird Conservation Region (BCR) 23 in the upper midwestern United States at 800-ha, 8,000-ha, and approximately 80,000-ha scales. We obtained count data from the Breeding Bird Survey and land cover data from the National Land Cover Dataset (1992). We evaluated predictions from the best models, as defined by an information-theoretic criterion, using point count data collected within an ecological subregion of BCR 23 at 131 count stations in the 1990s and again in 2006–2007. Competing (Deviance Information Criteria <5) blue-winged warbler models accounted for 67% of the variability and suggested positive associations with forest edge and proportion of forest at the 8,000-ha scale, and negative associations with forest patch area (800 ha) and wetness (800 ha and 80,000 ha). The regional model performed best for blue-winged warbler predicted abundances from point counts conducted in Iowa during 1995–1996 (r_s = 0.57; P = 0.14), the survey period that most closely aligned with the time period of data used for regional model construction. Wood thrush models exhibited positive correlations with point count data for all survey areas and years combined (r_s = 0.58, P ≤ 0.001). In comparison, blue-winged warbler models performed worse as time increased between the point count surveys and vintage of the model building data (r_s = 0.03, P = 0.92 for Iowa and r_s = 0.13, P = 0.51 for all areas, 2006–2007), likely related to the ephemeral nature of their preferred early successional habitat. Species abundance and sensitivity to changing habitat conditions seems to be an important factor in determining the predictive ability of regional models. Hierarchical models can be a useful tool for concentrating efforts at the scale of management units and should be one of many tools used by land managers, but we caution that the utility of such models may decrease over time for species preferring relatively ephemeral habitats if model inputs are not updated accordingly. © 2012 The Wildlife Society.     

Landmark registering waveform data improves the ability to predict performance measures

The purpose of this study was to investigate the benefit of landmark registration when applied to waveform data. We compared the ability of data reduced from time-normalised and landmark registered vertical ground reaction force (vGRF) waveforms captured during maximal countermovement jumps (CMJ) of 53 active male subjects to predict jump height. vGRF waveforms were landmark registered using different landmarks resulting in four registration conditions: (i) end of the eccentric phase, (ii) adding maximum centre of mass (CoM) power, (iii) adding minimum CoM power, (iv) adding minimum vGRF. In addition to the four registration conditions, the non-registered vGRF and concentric phase only were time-normalised and used in subsequent analysis. Analysis of characterising phases was performed to reduce the vGRF data to features that captured the behaviour of each waveform. These features were extracted from each condition’s vGRF waveform, time-domain (time taken to complete the movement), and warping functions (generated from landmark registration). The identified features were used as predictor features to fit a step-wise multilinear regression to jump height. Features generated from the best performing registration condition were able to predict jump height to a similar extent as the concentric phase (86–87%), while all registration conditions could explain jump height to a greater extent than time-normalisation alone (65%). This suggests waveform variability was reduced as phases of the CMJ were aligned. However, findings suggest that over-registration can occur when applying landmark registration. Overall, landmark registration can improve prediction power to performance measures as waveform data can be reduced to more appropriate performance related features.     

The ability of plant genotoxicity assays to predict carcinogenicity

A number of assays have been developed which use higher plants for measuring mutagenic or cytogenetic effects of chemicals, as an indication of carcinogenicity. Plant assays require less extensive equipment, materials and personnel than most other genotoxicity tests, which is a potential advantage, particularly in less developed parts of the world. We have analyzed data on 9 plant genotoxicity assays evaluated by the Gene-Tox program of the U.S. Environmental Protection Agency, using methodologies we have recently developed to assess the capability of assays to predict carcinogenicity and carcinogenic potency. All 9 of the plant assays appear to have high sensitivity (few false negatives). Specificity (rate of true negatives) was more difficult to evaluate because of limited testing on non-carcinogens; however, available data indicate that only the Arabidopsis mutagenicity (ArM) test appears to have high specificity. Based upon their high sensitivity, plant genotoxicity tests are most appropriate for a risk-averse testing program, because although many false positives will be generated, the relatively few negative results will be quite reliable.     

Caspase-15 is autoprocessed at two sites that contain an aspartate residue in the P1' position

Our recent characterization of porcine caspase-15 suggested that the amino terminus of the small catalytic subunit is formed by proteolytic processing between the consecutive aspartate residues D277 and D278. Since a charged residue (D278) is highly unusual in the P1' position of a caspase cleavage site, we further characterized the mechanism of caspase-15 autoproteolysis. Amino acid sequence alignments showed that D277 and D278 as well as another pair of aspartates, D270 and D271, were evolutionarily conserved among species of the mammalian clade Laurasiatheria. Site-directed mutations of these four residues and analysis of recombinant proteins showed that D270 was crucial for autoproteolysis whereas the three other aspartates were dispensable for separation of the catalytic subunits. Mutation of D270 prevented catalytic activation and abolished subsequent processing at D277. Together with previous reports, our results show that caspase-15, unlike all other caspases, efficiently cleaves sites with an aspartate in the P1' position.     

FMRFamide tagging—how an ionotropic receptor can be used to measure peptide secretion

In this chapter, we describe a technique, FMRFamide tagging, that in principle can be used to measure the release of any sequenced neuropeptide. The method relies upon the addition of an “electrophysiologically active” tag to the prohormone that encodes the neuropeptide of interest. Secretion of the electrophysiological tag (and thus the peptide of interest) is detected by activation of the ionotropic “tag receptor.” Both the tagged prohormone and the tag receptor are expressed in the cell type under investigation. Since the tag and the neuropeptide of interest are on the same prohormone they are co-secreted and thus secretion of the tag reflects the co-secretion of the neuropeptide of interest. This method can be used to detect neuropeptide secretion on a millisecond timescale.     

The ability to develop an activity that transfers histones onto sperm chromatin is acquired with meiotic competence during oocyte growth.

Following fertilization, the oocyte remodels the sperm chromatin into the male pronucleus. As a component of this process, during meiotic maturation, oocytes develop an activity that transfers histones onto sperm DNA. To further characterize this activity, we tested whether oocytes at different stages of growth could, upon entry into metaphase of maturation, transfer histones onto sperm DNA, as judged by chromatin morphology and immunocytochemistry. Meiotically competent growing oocytes, which spontaneously enter metaphase upon culture, transferred histones onto sperm chromatin, whereas incompetent oocytes did not, even when treated with okadaic acid to induce germinal vesicle breakdown (GVBD) and chromosome condensation. When incompetent oocytes were cultured until they acquired the ability to undergo GVBD, only a small proportion also developed histone-transfer activity during maturation. However, this proportion significantly increased when the oocytes were cultured as granulosa-oocyte complexes. The failure of histone-transfer activity to develop in incompetent oocytes treated with okadaic acid was not linked to low H1 kinase activity nor rescued by injected histones. Because competent, but not incompetent, oocytes produce natural calcium oscillations, incompetent oocytes were exposed to SrCl2. One-third of treated oocytes produced at least one Ca2+ oscillation and, following insemination, the same proportion transferred histones onto sperm DNA. Histone transfer did not occur in oocytes pretreated with the Ca2+ chelator, BAPTA-AM. These results indicate that the ability to develop histone-transfer activity is acquired by growing oocytes near the time of meiotic competence, that it is separable from this event, and that it may be regulated through a Ca2+-dependent process.