Exploring the relationship between parental relatedness and male reproductive success in the Antarctic fur seal Arctocephalus gazella

Recent genetic studies of natural populations have shown that heterozygosity and other genetic estimates of parental relatedness correlate with a wide variety of fitness traits, from juvenile survival and parasite resistance to male reproductive success. Many of these traits involve health and survival, where the underlying mechanism may involve changes in the effectiveness of the immune system. However, for traits such as reproductive success, the likely mechanisms remain less obvious. In this paper, we examine the relationship between heterozygosity and a range of traits that contribute to male reproductive success, including time spent on territories and competitiveness. Our analysis is based on observational and genetic data from eight consecutive breeding seasons at a colony of the Antarctic fur seal, Arctocephalus gazella. Overall, male reproductive success was found to correlate strongly with internal relatedness (IR, a form of heterozygosity). When different components of success were analyzed, we found that IR correlates independently with reproductive longevity, time spent ashore, and competitive ability per unit mating opportunity on the study beach, with more heterozygous males being more successful. Behavioral observations were sufficiently detailed to allow examination of how daily mean IR values for males present on the beach varied within seasons and from year to year. Again, significant variation was found both among and within seasons, with more homozygous males appearing less able to hold territories in poor seasons when pup production is low and, within a season, at both the start of the season and to some extent around the peak of female estrus. Finally, we tested whether the benefits of high heterozygosity are due mainly to a genomewide effect (e.g. inbreeding depression) or to single locus heterosis by asking whether the relationship between IR and male success was robust to the removal of any single locus or to any pair of loci. Since the relationship remained significant in all cases, we favor a multilocus explanation for the effects we report.     

Using comparative genomic data to test for fast-X evolution

Genes may acquire nonsynonymous substitutions more rapidly when X-linked than when autosomal, but evidence for "fast-X evolution" has been elusive. Fast-X evolution could explain the disproportionate contribution of X-linked genes to hybrid sterility and other traits. Here, we use a comparative genomic approach, with sequences of 30-110 genes in four Drosophila species, to test for fast-X evolution. Specifically, the 3L autosome arm in D. melanogaster and D. simulans is homologous to the right arm of the X chromosome in D. pseudoobscura and D. miranda. We executed two paired comparisons to determine how often genes on this chromosome arm exhibit higher rates of nonsynonymous substitution in the D. pseudoobscura species group, as predicted by fast-X evolution. We found a statistically significant pattern consistent with fast-X evolution in one comparison and a similar trend in the other comparison. Variation in functional constraints across genes may have masked the signature of fast-X evolution in some previous studies, and we conclude paired comparisons are more powerful for examining rates of evolution of genes when X-linked over autosomal.     

Exchange of serine residues 263 and 266 reduces the function of mouse gap junction protein connexin31 and exhibits a dominant-negative effect on the wild-type protein in HeLa cells

To characterize the role of Cx31 phosphorylation, serine residues 263 and 266 (Cx31Delta263,266) or 266 (Cx31Delta266) alone were exchanged for amino acids that cannot be phosphorylated. HeLa cells, which were stably transfected with wild type and the two different mutant Cx31-cDNA constructs, were analyzed for expression, phosphorylation, localization, formation of functional gap junction channels, and degradation of mutant Cx31 protein. Both mutant proteins showed similar reduced phosphorylation levels compared to Cx31 wild type, indicating a pivotal role of serine residue 266 for Cx31 phosphorylation. None of these mutations did interfere with correct intracellular trafficking of gap junction proteins. Pulse chase experiments with the different transfectants revealed an increased turnover of both mutated Cx31 proteins. They showed decreased intercellular communication as shown by dye transfer to neighboring cells and measurement of total conductance (mutant Cx31Delta263,266). Mutated Cx31 protein (Cx31Delta263,266) diminished the function of the Cx31 wild-type protein dependent on the amount of the mutated protein, indicating a dominant-negative effect of the mutated protein in HeLa cells.     

Coordination behavior of phosphino-phosphaferrocenes: monodentate versus bidentate coordination to divalent palladium

Two novel phosphino-phosphaferrocenes [η⁵-C₅H₄(CH₂)_nPPh₂]Fe(η⁵-PC₄H₂-2,5-Cy₂) (PP1: n=1; PP2: n=2) have been designed and prepared in order to clarify weak chelate effect in the previously reported (η⁵-C₅H₄CH₂PPh₂)Fe[η⁵-PC₄H₂-2,5-((-)-menthyl)₂] (1). ³¹P NMR studies of reactions of PP1 with PdCl₂(cod) (6) revealed that PP1 showed stronger tendency to coordinate to the Pd^II center in bidentate fashion compared to 1. On the other hand, chelate effect in PP2 was negligibly weak and a reaction of PP2 with 6 in a PP2/6 = 2/1 molar ratio gave a complex PdCl₂(PP2)₂ (10) cleanly in which PP2 coordinated to the palladium center at the PPh₂ moiety as a monodentate ligand. X-ray crystal structure studies of chelate complexes PdCl₂(PP1) (7) and PdCl₂(PP2) (9) showed that 9 had deviations from an idealized geometry in the square planar complex which could be attributed to a larger chelate ring of PP2, while PP1 in 7 constructed nearly ideal geometry for the square planar complex.From comparison of the coordination behavior between 1, PP1, and PP2, it is concluded that steric bulk of (-)-menthyl groups in 1 is the main factor of the weak chelate coordination of 1.     

The case-only odds ratio as a causal parameter

In the simplest case-only design, cases of a disease are cross-classified into a 2 x 2 table describing a genotype attribute and exposure to some environmental agent. In some instances, the genetic attribute has described inherited genes; in other instances, it has described mutations, for instance, damage to proto-oncogenes or tumor suppressor genes leading to cancer. Here, the population case-only odds ratio is written as a causal parameter in terms of potential outcomes with and without exposure to the agent. It is shown that the case-only odds ratio makes sense as a causal parameter with inherited genes, but its magnitude does not have a causal interpretation with mutations, although deviations from 1 do provide information. The difference is that the environmental agent certainly did not cause an individual to inherit particular genes, but it may have caused the mutation.     

The reaction of indole with the aminoacrylate intermediate of Salmonella typhimurium tryptophan synthase: observation of a primary kinetic isotope effect with 3-[(2)H]indole

The bacterial tryptophan synthase alpha(2)beta(2) complex catalyzes the final reactions in the biosynthesis of L-tryptophan. Indole is produced at the active site of the alpha-subunit and is transferred through a 25-30 A tunnel to the beta-active site, where it reacts with an aminoacrylate intermediate. Lane and Kirschner proposed a two-step nucleophilic addition-tautomerization mechanism for the reaction of indole with the aminoacrylate intermediate, based on the absence of an observed kinetic isotope effect (KIE) when 3-[(2)H]indole reacts with the aminoacrylate intermediate. We have now observed a KIE of 1.4-2.0 in the reaction of 3-[(2)H]indole with the aminoacrylate intermediate in the presence of monovalent cations, but not when an alpha-subunit ligand, disodium alpha-glycerophosphate (Na(2)GP), is present. Rapid-scanning stopped flow kinetic studies were performed of the reaction of indole and 3-[(2)H]indole with tryptophan synthase preincubated with L-serine, following the decay of the aminoacrylate intermediate at 350 nm, the formation of the quinonoid intermediate at 476 nm, and the formation of the L-Trp external aldimine at 423 nm. The addition of Na(2)GP dramatically slows the rate of reaction of indole with the alpha-aminoacrylate intermediate. A primary KIE is not observed in the reaction of 3-[(2)H]indole with the aminoacrylate complex of tryptophan synthase in the presence of Na(2)GP, suggesting binding of indole with tryptophan synthase is rate limiting under these conditions. The reaction of 2-methylindole does not show a KIE, either in the presence of Na(+) or Na(2)GP. These results support the previously proposed mechanism for the beta-reaction of tryptophan synthase, but suggest that the rate limiting step in quinonoid intermediate formation from indole and the aminoacrylate intermediate is deprotonation.     

Acute effect of corticosterone on N-methyl-D-aspartate receptor-mediated Ca2+ elevation in mouse hippocampal slices

We examined the rapid effects of corticosterone (CORT) on N-methyl-D-aspartate (NMDA) receptor-mediated Ca2+ signals in adult mouse hippocampal slices by using Ca2+ imaging technique. Application of NMDA caused a transient elevation of intracellular Ca2+ concentration followed by a decay to a plateau within 150s. The 30min preincubation of CORT induced a significant decrease of the peak amplitude of NMDA-induced Ca2+ elevation in the CA1 region. The rapid effect of CORT was induced at a stress-induced level (0.4-10microM). Because the membrane non-permeable bovine serum albumin-conjugated CORT also induced a similar rapid effect, the rapid effect of CORT might be induced via putative surface CORT receptors. In contrast, CORT induced no significant effects on NMDA-induced Ca2+ elevation in the dentate gyrus. In the CA3 region, CORT effects were not evaluated, because the marked elevation of NMDA-induced Ca2+ signals was not observed there.     

Physical and functional interaction of the active zone proteins, CAST, RIM1, and Bassoon, in neurotransmitter release

We have recently isolated a novel cytomatrix at the active zone (CAZ)-associated protein, CAST, and found it directly binds another CAZ protein RIM1 and indirectly binds Munc13-1 through RIM1; RIM1 and Munc13-1 directly bind to each other and are implicated in priming of synaptic vesicles. Here, we show that all the CAZ proteins thus far known form a large molecular complex in the brain, including CAST, RIM1, Munc13-1, Bassoon, and Piccolo. RIM1 and Bassoon directly bind to the COOH terminus and central region of CAST, respectively, forming a ternary complex. Piccolo, which is structurally related to Bassoon, also binds to the Bassoon-binding region of CAST. Moreover, the microinjected RIM1- or Bassoon-binding region of CAST impairs synaptic transmission in cultured superior cervical ganglion neurons. Furthermore, the CAST-binding domain of RIM1 or Bassoon also impairs synaptic transmission in the cultured neurons. These results indicate that CAST serves as a key component of the CAZ structure and is involved in neurotransmitter release by binding these CAZ proteins.     

Comparative analysis of the production of insecticidal and melanizing macromolecules by strains of Beauveria spp.: in vivo studies

Eleven strains of Beauveria bassiana, and a further five species of Beauveria sp., were tested by injection of 8x10(2) conidia into the haemocoel of the larvae of the lepidopteran Galleria mellonella with the aim of analysing their toxin producing activity in vivo. Although the virulent strains killed 100% of the insects at slightly different rates (4-6 days) there were significant differences in the pattern and intensity of host melanization caused by isolates. The majority of the isolates of Beauveria spp. induced a fast and intense melanization of the cuticle of the integument and of tracheal wall, which followed one of three patterns. Another small group of two B. bassiana strains, isolated from Ostrinia nubilalis, induced very weak or no melanization. Strains 618 and 101 of B. bassiana, were selected as models of "melanizing" and "non-melanizing" strains, respectively. Ultrastructural alterations of cells of hypodermal and tracheal epithelium and of haemocytes, assumed to be at least partially caused by fungal toxins, were revealed in larvae infected by both isolates. However, their effects on the fine structure of the hypodermis were different. Injection of sera obtained from haemolymph of insects infected with B. bassiana 618 showed that they have insecticidal, melanizing, and cytotoxic effects similar to those occurring during mycosis. Chromatographic studies and bioassays with fractions prepared from crude serum have allowed a partial identification of the toxic molecules secreted by the fungus in vivo. They are proteinaceous, as shown by protease treatments, thermolabile, negatively charged, and not glycosylated with alpha-d-mannose or alpha-d-glucose. If strain B. bassiana 618 produces melanizing macromolecules which are vivotoxins secreted during the mycosis, the mode of action of isolate 101 is different. Its capacity to kill the host depends on active mycelial development, and on the production of low molecular weight toxins.     

Electrostatics of ion stabilization in a ClC chloride channel homologue from Escherichia coli

The structural determinants of electrostatics of ion stabilization within EcClC, a ClC-type chloride channel homologue from Escherichia coli, are studied using a continuum dielectric approximation. Specifically, the ion occupancy is investigated in the wild-type protein and a mutant thereof, and the contribution to the electrostatic binding free energy of local and non-local interactions is characterized at the single-residue level. This analysis shows that, in spite of the desolvation cost and the strong ion-ion repulsion, all previously reported binding sites can be occupied simultaneously. The stabilizing effect of the protein arises from hydrogen bonding as well as from longer-range favorable interactions, such as with the strictly conserved Lys131 side-chain. The latter is involved in the stabilization of the conserved GSGIP motif that delimits two of the binding sites. Interestingly, an additional low-affinity binding site, mediated by a structurally analogous motif including the side-chain of Arg340, can be identified on the extracellular side of the permeation pathway. Finally, it is shown that, in contrast to K-channels, and in analogy to the SBP/PBP sulfate/phosphate-binding proteins, the contribution of helix macrodipoles to chloride binding in EcClC is only marginal.