Cadmium-copper interaction in intestinal mucosal cell cytosol of mice

In vivo as well as in vitro protein-metal interaction was studied in cytosolic fractions from intestinal mucosal cells. Female Swiss-Webster mice wre pretreated with cadmium (25 ppm) or copper (100 ppm) in drinking water for 3 weeks. Treatment groups were divided into subgroups receiving Cd or Cd+Cu for an additional 6 weeks. In the in vitro study, mucosal cytosol obtained from pretreated animals was incubated with Cd-109 or Cd-109+Cu. Proteins were separated by gel filtration chromatography and metals determined by furnace AAS or gamma-spectrometry. Cadmium-induced synthesis of metallothionein-like proteins (MTP) in cytosol was indicated by increased Cd in those eluted fractions corresponding to the molecular weight of purified equine renal metallothionein. This cadmium level reached a plateau after 3 weeks of cadmium treatment. In addition, an increased amount of cadmium bound to MTP was noted when copper was added to cadmium in drinking water of mice pretreated with copper. This was not the case for Cd-pretreated animals. The in vitro experiments produced similar results, in that MTP fractions retained a greater percentage of Cd when animals were pretreated with copper compared to controls. Cadmium pretreatment resulted in even higher amounts of cadmium bound to MTP. The existence of a Cd as well as a separate Cu MTP, each with specific metal-binding properties, is suggested.     

Residency and movement patterns of adult Port Jackson sharks (Heterodontus portusjacksoni) at a breeding aggregation site

Examining the movement ecology of mesopredators is fundamental to developing an understanding of their biology, ecology and behaviour, as well as the communities and ecosystems they influence. The limited research on the residency and movements of benthic marine mesopredators has primarily used visual tags, which do not allow for the efficient and accurate monitoring of individual space use. In this study, the authors investigated the residency and movement patterns of Port Jackson sharks Heterodontus portusjacksoni (Meyer 1793) at a breeding aggregation site in Jervis Bay, south-eastern Australia, using passive acoustic telemetry to further our understanding of the movement ecology of these important mesopredators. Between 2012 and 2014, individuals were tagged with acoustic transmitters, and their residency and movements within the bay were monitored for up to 4 years. H. portusjacksoni showed strong preferences for particular reefs within and between breeding seasons. Males had significantly higher residency indices at their favoured sites relative to females, suggesting that males may be engaging in territorial behaviour. Conversely, female H. portusjacksoni exhibited higher roaming indices relative to males indicating that females may move between sites to assess males. Finally, H. portusjacksoni showed temporal variation in movements between reefs with individuals typically visiting more reefs at night relative to the day, dusk and dawn corresponding with their nocturnal habits.     

Are our phylomorphospace plots so terribly tangled? An investigation of disorder in data simulated under adaptive and nonadaptive models

Contemporary methods for visualizing phenotypic evolution, such as phylomorphospaces, often reveal patterns which depart strongly from a naïve expectation of consistently divergent branching and expansion. Instead, branches regularly crisscross as convergence, reversals, or other forms of homoplasy occur, forming patterns described as “birds’ nests”, “flies in vials”, or less elegantly, “a mess”. In other words, the phenotypic tree of life often appears highly tangled. Various explanations are given for this, such as differential degrees of developmental constraint, adaptation, or lack of adaptation. However, null expectations for the magnitude of disorder or “tangling” have never been established, so it is unclear which or even whether various evolutionary factors are required to explain messy patterns of evolution. I simulated evolution along phylogenies under a number of varying parameters (number of taxa and number of traits) and models (Brownian motion, Ornstein–Uhlenbeck (OU)-based, early burst, and character displacement (CD)] and quantified disorder using 2 measures. All models produce substantial amounts of disorder. Disorder increases with tree size and the number of phenotypic traits. OU models produced the largest amounts of disorder—adaptive peaks influence lineages to evolve within restricted areas, with concomitant increases in crossing of branches and density of evolution. Large early changes in trait values can be important in minimizing disorder. CD consistently produced trees with low (but not absent) disorder. Overall, neither constraints nor a lack of adaptation is required to explain messy phylomorphospaces—both stochastic and deterministic processes can act to produce the tantalizingly tangled phenotypic tree of life.     

Affinity maturation of a novel antagonistic human monoclonal antibody with a long VH CDR3 targeting the Class A GPCR formyl-peptide receptor 1

Therapeutic monoclonal antibodies targeting G-protein-coupled receptors (GPCRs) are desirable for intervention in a wide range of disease processes. The discovery of such antibodies is challenging due to a lack of stability of many GPCRs as purified proteins. We describe here the generation of Fpro0165, a human anti-formyl peptide receptor 1 (FPR1) antibody generated by variable domain engineering of an antibody derived by immunization of transgenic mice expressing human variable region genes. Antibody isolation and subsequent engineering of affinity, potency and species cross-reactivity using phage display were achieved using FPR1 expressed on HEK cells for immunization and selection, along with calcium release cellular assays for antibody screening. Fpro0165 shows full neutralization of formyl peptide-mediated activation of primary human neutrophils. A crystal structure of the Fpro0165 Fab shows a long, protruding V_H CDR3 of 24 amino acids and in silico docking with a homology model of FPR1 suggests that this long V_H CDR3 is critical to the predicted binding mode of the antibody. Antibody mutation studies identify the apex of the long V_H CDR3 as key to mediating the species cross-reactivity profile of the antibody. This study illustrates an approach for antibody discovery and affinity engineering to typically intractable membrane proteins such as GPCRs.     

Structure and Function of a Fungal Adhesin that Binds Heparin and Mimics Thrombospondin-1 by Blocking T Cell Activation and Effector Function

Blastomyces adhesin-1 (BAD-1) is a 120-kD surface protein on B. dermatitidis yeast. We show here that BAD-1 contains 41 tandem repeats and that deleting even half of them impairs fungal pathogenicity. According to NMR, the repeats form tightly folded 17-amino acid loops constrained by a disulfide bond linking conserved cysteines. Each loop contains a highly conserved WxxWxxW motif found in thrombospondin-1 (TSP-1) type 1 heparin-binding repeats. BAD-1 binds heparin specifically and saturably, and is competitively inhibited by soluble heparin, but not related glycosaminoglycans. According to SPR analysis, the affinity of BAD-1 for heparin is 33 nM±14 nM. Putative heparin-binding motifs are found both at the N-terminus and within each tandem repeat loop. Like TSP-1, BAD-1 blocks activation of T cells in a manner requiring the heparan sulfate-modified surface molecule CD47, and impairs effector functions. The tandem repeats of BAD-1 thus confer pathogenicity, harbor motifs that bind heparin, and suppress T-cell activation via a CD47-dependent mechanism, mimicking mammalian TSP-1.     

Canalization and adaptation in a landscape of sources and sinks

Landscapes are often spatially heterogeneous, and many species frequently confront novel environments to which they are not adapted. Whether a species becomes adapted to a novel environment, and thus undergoes niche evolution, may depend not only on the genetic architecture of the traits under selection, but also on the structure of the ecological landscape. Different models of gene architecture are used to show that complex genetic architectures tends to produce genetic canalization that slows adaptation to novel environments compared to simpler additive polygenic architectures, but that the topology of the landscape interacts with genetic architecture to influence the probability of adaptation. This interaction can lead to unexpected results, such as a greater probability of adaptation to a novel environment for a population of more highly canalized individuals than a population of less canalized individuals. The interplay between landscape structure and genetic architecture may influence the balance of evolutionary forces acting on a population, and thus whether a species is likely to adapt to the novel environments it confronts.     

Effects of gastric digestive products from casein on CCK release by intestinal cells in rat

We investigated the ability of gastric digestive products from casein to stimulate cholecystokinin release by intestinal cells using the isolated vascularly perfused rat duodenojejunum. Casein digests were prepared with an in vitro system simulating gastric digestion and emptying.

The luminal infusion of the digesta emptied from the artificial stomach for the first 10 minutes produced a sharp rise of portal cholecystokinin-like immunoreactivity to 300% of basal, followed by a well-sustained plateau secretion until the end of the infusion. The residual casein fraction of this digest brought about a modest cholecystokinin secretion, while the peptide component was as strong a stimulant as total digest. The peptide responsible for this effect was the glycomacropeptide that is a glycosylated fragment (106–169) of κ-casein. Only the slightly glycosylated forms of the peptide originating from variant A of κ-casein were active. The carbohydrate-free peptide did not alter basal cholecystokinin. The highly glycosylated forms of the peptide and the slightly glycosylated peptide from κ-casein variant B induced only a transient and low rise of portal cholecystokinin. The removal of N-acetylneuraminic acid from the active peptide suppressed its effect, while the infusion of an N-acetylneuraminic acid solution induced only a very low response.

It is concluded that the glycomacropeptide released from dietary casein during gastric digestion can stimulate cholecystokinin release by intestinal cells in the rat. A well-defined structure is required for the peptide activity. A part of the peptide chain and some glycosidic chains containing N-acetylneuraminic acid, especially those bound to the amino acid residue threonyl 31 of caseinomacropeptide variant A, would be involved in this structure.