Acidic/IQ motif regulator of calmodulin

The small IQ motif proteins PEP-19 (62 amino acids) and RC3 (78 amino acids) greatly accelerate the rates of Ca(2+) binding to sites III and IV in the C-domain of calmodulin (CaM). We show here that PEP-19 decreases the degree of cooperativity of Ca(2+) binding to sites III and IV, and we present a model showing that this could increase Ca(2+) binding rate constants. Comparative sequence analysis showed that residues 28 to 58 from PEP-19 are conserved in other proteins. This region includes the IQ motif (amino acids 39-62), and an adjacent acidic cluster of amino acids (amino acids 28-40). A synthetic peptide spanning residues 28-62 faithfully mimics intact PEP-19 with respect to increasing the rates of Ca(2+) association and dissociation, as well as binding preferentially to the C-domain of CaM. In contrast, a peptide encoding only the core IQ motif does not modulate Ca(2+) binding, and binds to multiple sites on CaM. A peptide that includes only the acidic region does not bind to CaM. These results show that PEP-19 has a novel acidic/IQ CaM regulatory motif in which the IQ sequence provides a targeting function that allows binding of PEP-19 to CaM, whereas the acidic residues modify the nature of this interaction, and are essential for modulating Ca(2+) binding to the C-domain of CaM.     

Fine-scale differentiation between sockeye salmon ecotypes and the effect of phenotype on straying

A long-standing goal of evolutionary biology is to understand the factors that drive population divergence, local adaptation and speciation. In particular, the effect of selection against dispersers on gene flow and local adaptation has attracted interest, although empirical data on phenotypic characters of dispersers are scarce. Here, we used genetic and phenotypic data from beach and creek ecotypes of sockeye salmon (Oncorhynchus nerka) in Little Togiak Lake, Alaska, to examine the relationship between gene flow and phenotypic and genetic differentiation. Despite close geographic proximity, both genetic and phenotypic differentiation between beach and creek fish was high and significant in all sampling years, with beach males having deeper bodies than creek males. Strays, or fish that did not return to their natal sites to spawn as determined by genetic assignment, tended to morphologically resemble the fish in the population that they joined. Male strays from beaches to creeks were shallower bodied than other beach fish, and male strays from creeks to beaches were deeper bodied than other creek males. Our results indicated that selection against strays may be moderated by the strays' phenotypic similarity to individuals in the recipient populations, but comparison of assignment results with long-term estimates of gene flow from F(ST) still suggested that strays had low reproductive success.     

Do group size and laying order influence maternal deposition of testosterone in smooth-billed ani eggs?

The avian egg contains maternal hormones that affect behavior, growth, morphology, and offspring survival. Evidence to date suggests that patterns of yolk androgen deposition could provide females with a means to manipulate sibling competition and, thereby, increase their fitness. We examined yolk testosterone (T) concentrations in eggs of the smooth-billed ani (Crotophaga ani) to understand patterns of androgen deposition in eggs of this plural-breeding joint-nesting cooperatively breeding species. We tested the hatching asynchrony adjustment hypothesis, which states that increases in yolk androgen levels over the laying sequence function to mitigate the disadvantage of being a later-hatched chick in species without adaptive brood reduction. We also investigated the effect of group size on yolk T deposition to test the hypothesis that females in multi-female groups could give a competitive edge to their own chicks by depositing higher T levels in their eggs. Predictions of the hatching asynchrony adjustment hypothesis were supported in both single- and multi-female groups as yolk testosterone levels increased from early- to late-laid eggs. This suggests that ani females can influence nestling competition and chick survival by within-clutch differential T allocation. Unexpectedly, we did not observe an effect of group size on yolk T deposition. Yolk testosterone concentrations may not be a mere reflection of a female's hormonal status as female plasma circulating levels of T did not vary in the same direction as yolk T levels. Results of this study therefore support the idea that females may adaptively manipulate chick behavior through hormonal deposition in eggs.     

An integrated study of the temporal and spatial variation in the supply of propagules, recruitment and assemblages of intertidal macroalgae on a wave-exposed rocky coast, Victoria, Australia

Recruitment is known to influence distributions and abundances of benthic marine organisms. It is therefore important to document patterns of variability in recruitment and how these relate to patterns in established assemblages. This study provides an integrated assessment of the temporal and spatial variation in supply and recruitment of propagules and established populations of several macroalgae. Propagules in water samples from two stages of the incoming tide, recruitment to artificial substrata and percentage cover of species established on the shore were recorded every 2 months from December 1994 to October 1995, in two zones of an intertidal, wave-exposed rocky shore. Variability in recruitment was measured at three spatial scales: 10s cm, 100s cm and 100s m. Availability and recruitment of most taxa were greatest between April and August, although many species had available propagules and recruited throughout the year. Temporal variation in the established assemblages was, however, more species-specific. Differences in established assemblages between zones were reflected in differences in availability and recruitment of propagules between zones. Recruitment could not be predicted directly from supply of propagules, but the two processes were linked. For most species, the greatest variation in recruitment occurred at the smallest spatial scale of 10s cm, although there was also considerable large-scale (between site) variation in recruitment of several species. Results indicate that while pre-and post-settlement mortality are likely to influence macroalgal distribution and abundance, the temporal and spatial variability in supply and recruitment of propagules can explain much of the patchiness in macroalgal assemblages.     

Activity-based selection of a proteolytic species using ribosome display

We have examined the potential of displaying a protease species in vitro using ribosome display and demonstrate specific capture on the basis of its catalytic activity. Using a model bacterial cysteine protease, sortase A (SrtA), we show that this enzyme can be functionally expressed in vitro. By overlap PCR we constructed ribosome display templates with the SrtA open reading frame fused to a C terminal glycine-serine rich flexible linker and a tether derived from eGFP. Using the broad range cysteine protease irreversible inhibitor E-64 linked to acrylic beads, we show that we can isolate SrtA ribosome display ternary complexes, and recover their encoding mRNA by RT-PCR. This recovery was lost when applied to a SrtA catalytically inactive mutant, or could be alleviated by competition with free inhibitor. This sensitive technique could be further developed to allow the screening of proteases against putative inhibitors and/or the identification of novel proteolytic species.     

Pancreatic endocrine and exocrine cell ontogeny from renal capsule transplanted embryonic stem cells in streptozocin-injured mice.

In this study, we describe pancreatic cell ontogeny in renal capsule-transplanted embryonic stem cells (ES) after injury by streptozocin (STZ), showing pancreatogenesis in situ. Seven-week-old female BALB/c nude mice were treated with either a single 175- or 200-mg/kg STZ dose, a regimen that induces substantial beta-cell damage without overt hyperglycemia, and transplanted 24 hr later with 1 x 10(5) ES. Immunohistochemistry was performed on ES tissue at 15, 21, and 28 days after transplantation using antibodies against stage- and lineage-specific pancreatic markers. After 21 days, PDX-1+ pancreatic foci first appeared in the renal capsule and expressed both amylase and endocrine hormones (insulin, glucagon, and somatostatin). These foci increased in size by day 28 because of acinar and duct cell proliferation, whereas endocrine cells remained non-dividing, and made up 2-4% of ES tumor volume. PDX-1, Nkx6.1, Ngn3, and ISL-1 protein localization patterns in pancreatic foci were comparable with embryonic pancreatogenesis. A prevalence of multihormonal endocrine cells, a characteristic of adult beta-cell regeneration, indicated a possible divergence from embryonic islet cell development. The results indicate that beta-cell damage, without overt hyperglycemia, induces a process of fetal-like pancreatogenesis in renal capsule-transplanted ES, leading to beta-cell neogenesis.     

IL-23 inhibits osteoclastogenesis indirectly through lymphocytes and is required for the maintenance of bone mass in mice

IL-23 stimulates the differentiation and function of the Th17 subset of CD4(+) T cells and plays a critical role in chronic inflammation. The IL-23 receptor-encoding gene is also an inflammatory disease susceptibility gene. IL-23 shares a common subunit with IL-12, a T cell-dependent osteoclast formation inhibitor, and we found that IL-23 also dose-dependently inhibited osteoclastogenesis in a CD4(+) T lymphocyte-dependent manner. When sufficiently enriched, gammadelta T cells also mediated IL-23 inhibition. Like IL-12, IL-23 acted synergistically with IL-18 to block osteoclastogenesis but, unlike IL-12, IL-23 action depended on T cell GM-CSF production. IL-23 did not mediate IL-12 action although IL-12 induced its expression. Male mice lacking IL-23 (IL-23p19(-/-)) had approximately 30% lower bone mineral density and tibial trabecular bone mass (bone volume (BV)/total volume (TV)) than wild-type littermates at 12 wk and 40% lower BV/TV at 26 wk of age; male heterozygotes also had lower bone mass. Female IL-23p19(-/-) mice also had reduced BV/TV. IL-23p19(-/-) mice had no detectable osteoclast defect in trabecular bone but IL-23p19(-/-) had thinner growth plate hypertrophic and primary spongiosa zones (and, in females, less cartilage remnants) compared with wild type. This suggests increased osteoclast action at and below the growth plate, leading to reduced amounts of mature trabecular bone. Thus, IL-23 inhibits osteoclast formation indirectly via T cells in vitro. Under nonpathological conditions (unlike inflammatory conditions), IL-23 favors higher bone mass in long bones by limiting resorption of immature bone forming below the growth plate.     

Development of an in vitro culture method for cells and tissues from the zebra mussel (<Emphasis Type="Italic">Dreissena polymorpha</Emphasis>)

Despite the successful transfer of mammalian in vitro techniques for use with fish and other vertebrates, little progress has been made in the area of invertebrate tissue culture. This paper describes the development of an in vitro technique for the culture of both cells in suspension and tissue explants from the gill, digestive gland and mantle of the zebra mussel (Dreissena polymorpha) and their successful maintenance in culture for up to 14 days. Cell suspensions from the gills and digestive gland were the most successful technique developed with viability >80% maintained for up to 8 days in culture, suitable for use in short term toxicity tests. Tissue explants from the mantle were also maintained in culture for up to 14 days. This paper describes the challenges involved in the development of a novel in vitro culture technique for aquatic invertebrates.     

Monitoring conformational dynamics with solid-state R 1ρ experiments

A new application of solid-state rotating frame (R _1ρ) relaxation experiments to observe conformational dynamics is presented. Studies on a model compound, dimethyl sulfone (DMS), show that R _1ρ relaxation due to reorientation of a chemical shift anisotropy (CSA) tensor undergoing chemical exchange can be used to monitor slow-to-intermediate timescale conformational exchange processes. Control experiments used d ₆ -DMS and alanine to confirm that the technique is monitoring reorientation of the CSA tensor rather than dipolar interactions or methyl group rotation. The application of this method to proteins could represent a new site-specific probe of conformational dynamics.     

Comparing alternative systematic conservation planning strategies against a politically driven conservation plan

Decisions regarding the level of detail included in conservation planning and the importance given to feasibility considerations can greatly influence management in terms of total area required, achievement of conservation targets and costs. This work had two main objectives: (1) to compare priority sites proposed by the Chilean commission for the environment in a politically driven process to the results of alternative systematic conservation planning scenarios; and (2) to compare the efficacy of systematic conservation planning based on different types of conservation targets (forest types and bird species) and minimum area thresholds. To address these issues, we used vegetation cover as well as field data on forest birds in central Chile. Bird species distributions were modeled using a variety of climatic and environmental layers, allowing for the integration of environmental heterogeneity into the planning process. We then ran several conservation planning scenarios considering conservation targets based on vegetation types alone, birds alone, or a combination of vegetation and birds. Collectively these results show that conservation planning results differ significantly when considering birds or vegetation types, and that minimum area requirements for each conservation feature has a great influence on the final results. Moreover, important conservation sites are not represented in the current government plan, and these sites are related to the small representation of rare vegetation types. This study suggests that using appropriate minimum area requirements can greatly affect the results of a conservation planning exercise and therefore represents a key knowledge gap in the region.