Ten days of darkness causes temporary blindness during an early critical period in felines

Extended periods of darkness have long been used to study how the mammalian visual system develops in the absence of any instruction from vision. Because of the relative ease of implementation of darkness as a means to eliminate visually driven neural activity, it has usually been imposed earlier in life and for much longer periods than was the case for other manipulations of the early visual input used for study of their influences on visual system development. Recently, it was shown that following a very brief (10 days) period of darkness imposed at five weeks of age, kittens emerged blind. Although vision as assessed by measurements of visual acuity eventually recovered, the time course was very slow as it took seven weeks for visual acuity to attain normal levels. Here, we document the critical period of this remarkable vulnerability to the effects of short periods of darkness by imposing 10 days of darkness on nine normal kittens at progressively later ages. Results indicate that the period of susceptibility to darkness extends only to about 10 weeks of age, which is substantially shorter than the critical period for the effects of monocular deprivation in the primary visual cortex, which extends beyond six months of age.     

Assessing the alignment of sexual and natural selection using radiomutagenized seed beetles

A major unsolved question in evolutionary biology concerns the relationship between natural and sexual selection. Sexual selection might augment natural selection, for example if mutations that harm female fecundity also reduce male mating success. Conversely, sexual selection might favour traits that impair naturally selected fitness components. We induced detrimental mutations in Callosobruchus maculatus beetles using X‐ray irradiation and then experimentally measured the effect of precopulatory sexual selection on offspring number and survival rate. Sexual selection treatment had a negative effect on egg‐to‐adult survivorship, although the number of progeny reaching adulthood was unaffected, perhaps because eggs and juveniles that failed to develop lessened competition on the survivors. We hypothesize that the negative effect of sexual selection arose because sexually competitive males transmitted a smaller nuptial gift or carried alleles that conferred reduced survival. Although we found no evidence that sexual selection on males can purge alleles that are detrimental to naturally selected fitness components, such benefits might exist in other environmental or genetic contexts.     

Ru binding to RNA following treatment with the antimetastatic prodrug NAMI-A in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> and in vitro

[ImH][trans-Ru^IIICl₄(DMSO)(Im)] (where DMSO is dimethyl sulfoxide and Im is imidazole) (NAMI-A) is an antimetastatic prodrug currently in phase II clinical trials. The mechanisms of action of this and related Ru-based anticancer agents are not well understood, but several cellular targets have been suggested. Although Ru has been observed to bind to DNA following in vitro NAMI-A exposure, little is known about Ru–DNA interactions in vivo and even less is known about how this or related metallodrugs might influence cellular RNA. In this study, Ru accumulation in cellular RNA was measured following treatment of Saccharomyces cerevisiae with NAMI-A. Drug-dependent growth and cell viability indicate relatively high tolerance, with approximately 40% cell death occurring at 6 h for 450 μM NAMI-A. Significant dose-dependent accumulation of Ru in cellular RNA was observed by inductively coupled plasma mass spectrometry measurements on RNA extracted from yeast treated with NAMI-A. In vitro, binding of Ru species to drug-treated model DNA and RNA oligonucleotides at pH 6.0 and 7.4 was characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in the presence and absence of the reductant ascorbate. The extent of Ru–nucleotide interactions increases slightly with lower pH and significantly in the presence of ascorbate, with differences in observed species distribution. Taken together, these studies demonstrate the accumulation of aquated and reduced derivatives of NAMI-A on RNA in vitro and in cellulo, and enhanced binding with nucleic acid targets in a tumorlike acidic, reducing environment. To our knowledge, this is also the first study to characterize NAMI-A treatment of S. cerevisiae, a genetically tractable model organism.     

Global absolute quantification of a proteome: Challenges in the deployment of a QconCAT strategy

In this paper, we discuss the challenge of large-scale quantification of a proteome, referring to our programme that aims to define the absolute quantity, in copies per cell, of at least 4000 proteins in the yeast Saccharomyces cerevisiae. We have based our strategy on the well-established method of stable isotope dilution, generating isotopically labelled peptides using QconCAT technology, in which artificial genes, encoding concatenations of tryptic fragments as surrogate quantification standards, are designed, synthesised de novo and expressed in bacteria using stable isotopically enriched media. A known quantity of QconCAT is then co-digested with analyte proteins and the heavy:light isotopologues are analysed by mass spectrometry to yield absolute quantification. This workflow brings issues of optimal selection of quantotypic peptides, their assembly into QconCATs, expression, purification and deployment.     

Nodes in phylogenetic trees: the relation between imbalance and number of descendent species

The imbalance of a node in a phylogenetic tree can be defined in terms of the relative numbers of species (or higher taxa) on the branches that originate at the node. Empirically, imbalance also turns out to depend on the absolute total number of species on the branches: in a sample of large trees, nodes with more descendent species tend to be more unbalanced. Subsidiary analyses suggest that this pattern is not a result of errors in tree estimation. Instead, the increase in imbalance with species is consistent with a cumulative effect of differences in diversification rates between branches. [Equal-rates Markov model; imbalance; phylogeny shape; proportional-to-distinguishable-arrangements model.].     

Molecular cloning of a phospholipid-hydroperoxide glutathione peroxidase gene from the tick, Boophilus microplus (Acari: Ixodidae)

Phospholipid-hydroperoxide glutathione peroxidase (PHGPx) enzymes are associated with cellular protection by the role they play in reducing hydroperoxides of phospholipids, thereby preventing membrane lipoperoxidation. As part of their toxic effect, some pesticides stimulate peroxidation of cellular membranes. We isolated and sequenced a PHGPx gene from the cattle tick Boophilus microplus that encodes a protein of 169 amino acids, including a TGA-encoded selenocysteine at residue 46 and active site residues Gln⁸² and Trp¹³⁵ that interact with the selenocysteine. The motif that directs the insertion of selenocysteine at the opal codon is found in the 3′-untranslated region. PHGPx sequences from pesticide-resistant and susceptible B. microplus ticks show nucleotide differences at eight positions among the strains, with five resulting in amino acid substitutions in the deduced protein sequence. Two distinct PHGPx alleles were identified in an organophosphate-resistant tick strain. Real-time PCR quantification of gene expression revealed increased PHGPx in two strains resistant to a single acaricide class. Strains resistant to two or more classes showed a reduction in PHGPx.     

Modulation of sexual signalling by immune challenged male mealworm beetles (Tenebrio molitor, L.): evidence for terminal investment and dishonesty

Organisms partition resources into life-history traits in order to maximise fitness over their expected lifespan. For the males of many species fitness is determined by qualitative and quantitative aspects of costly sexual signals: The notion that epigamic traits are costly forms the cornerstone of those theories that propose parasites drive sexual selection. Consequently studies examining this notion assume sexual signalling is honest (i.e. driven by cost) when they seek to identify correlations or causal links between male immune function and attractiveness. We demonstrate that immune challenged males of the mealworm beetle, Tenebrio molitor, increased their investment in epigamic pheromone signals: these males became significantly more attractive to females whilst increasing the activity of a key immune effector system. In other words males increase terminal reproductive effort (invest in attractiveness) in response to a survival threat (immune insult). Consequently the signal preferred by the female is dishonest when considering the male's condition.     

Immune defense reduces respiratory fitness in Callinectes sapidus, the Atlantic blue crab

Crustacean gills function in gas exchange, ion transport, and immune defense against microbial pathogens. Hemocyte aggregates that form in response to microbial pathogens become trapped in the fine vasculature of the gill, leading to the suggestion by others that respiration and ion regulation might by impaired during the course of an immune response. In the present study, injection of the pathogenic bacterium Vibrio campbellii into Callinectes sapidus, the Atlantic blue crab, caused a dramatic decline in oxygen uptake from 4.53 to 2.56 micromol g-1 h-1. This decline in oxygen uptake is associated with a large decrease in postbranchial PO2, from 16.2 (+/-0.46 SEM, n=7) to 13.1 kPa (+/-0.77 SEM, n=9), while prebranchial PO2 remains unchanged. In addition, injection of Vibrio results in the disappearance of a pH change across the gills, an indication of reduced CO2 excretion. The hemolymph hydrostatic pressure change across the gill circulation increases nearly 2-fold in Vibrio-injected crabs compared with a negligible change in pressure across the gill circulation in saline-injected, control crabs. This change, in combination with stability of heart rate and branchial chamber pressure, is indicative of a significant increase in vascular resistance across the gills that is induced by hemocyte nodule formation. A healthy, active blue crab can eliminate most invading bacteria, but the respiratory function of the gills is impaired. Thus, when blue crabs are engaged in the immune response, they are less equipped to engage in oxygen-fueled activities such as predator avoidance, prey capture, and migration. Furthermore, crabs are less fit to invade environments that are hypoxic.     

Kinetic, spectroscopic, and structural investigations of the soybean lipoxygenase-1 first-coordination sphere mutant, Asn694Gly

In wild-type soybean LO-1 (WT sLO-1), Asn694 is a weak sixth ligand that is thought to be critical for enzymatic catalysis. In this investigation, N694G sLO-1 was studied to probe its contribution at this sixth ligand position to the kinetic and spectroscopic properties. The k(cat) value of N694G is approximately 230 times lower than that of WT sLO-1 at 25 degrees C, which can be partially explained by a lowered reduction potential of the iron as seen as a shift in the visible ligand-to-metal charge-transfer band (lambda(max) = 410 nm for N694G and lambda(max) = 425 nm for WT sLO-1). This conclusion was supported by a faster rate of oxidation of N694G by the product than that of WT sLO-1 (k(2) = 606 s(-)(1) for N694G and k(2) = 349 s(-)(1) for WT sLO-1). These results suggest a stronger ligand at the active site iron than the native Asn694, which is confirmed to be a water bound to the Fe(II) in the crystal structure. This produces a six-coordinate circular dichroism/magnetic circular dichroism (CD/MCD) spectra for ferrous N694G and an intermediate rhombic electron paramagnetic resonance (EPR) signal for ferric N694G. The EPR spectrum and its pH dependence suggest that the coordination environment of ferric N694G contains one hydroxide and one water. On the basis of both kinetic and structural factors, we propose that the Asn694 water-derived ligand would likely be a hydroxide and the active site, water-derived ligand a water in the ferric state, hence lowering the reaction rate of N694G more than would be expected from the lowered reduction potential alone.