Hatchery selection promotes boldness in newly hatched brown trout (Salmo trutta): implications for dominance

By using newly hatched (approximately 2 weeks old) brown trout(Salmo trutta) from six families of wild and six families ofsea-ranched origin (seventh generation), we tested the hypothesesthat (1) the hatchery environment selects for increased boldness,and (2) boldness predicts dominance status. Sea-ranched troutspend their first 2 years in the hatchery before being releasedinto the wild at the onset of seaward migration. Trout werepresented with a novel object (tack) and with food (brine shrimp),and their responses were measured and scored in terms of boldness.Siblings with increasing difference in boldness were then pairedin dyadic contests. Fish of sea-ranged origin were on averagebolder than were fish of wild origin, and bolder individualswere more likely to become dominant regardless of origin. Boldnesswas not related to RNA levels, indicating that bold behaviorwas not a consequence of higher metabolism or growth rate. Neitherwas size a predictor of bold behavior or the outcome of dyadiccontests. These results are consistent with studies on olderlife stages showing increased boldness toward predators in hatchery-selectedfish, which suggests that behavioral consequences of hatcheryselection are manifested very early in life. The concordancebetween boldness and dominance may suggest that these behaviorsare linked in a risk prone-aggressive phenotype, which may bepromoted by hatchery selection. However, we also found significantvariation in behavioral and growth-related traits among families,suggesting that heritable variation has not been exhausted bysea-ranching procedures.     

Selection of protein epitopes for antibody production

Protein functional analysis in the post-genomic era is a huge task that has to be approached by different methods in parallel. The use of protein-specific antibodies in conjunction with tissue microarrays has proven to be one important technology. In this study, we present a strategy for the optimized design of protein subfragments for subsequent antibody production. The fragments are selected based on a principle of lowest sequence similarity to other human proteins, optimally to generate antibodies with high selectivity. Furthermore, the fragments should have properties optimized for efficient protein production in Escherichia coli. The strategy has been implemented in Bishop, which is a Java-based software enabling the high-throughput production of protein fragments. Bishop allows for the avoidance of certain restriction enzyme sites, transmembrane regions, and signal peptides. A Basic Local Alignment Search Tool (BLAST) scanning procedure permits the selection of fragments of a selected size with a minimal sequence similarity to other proteins. The software and the strategy were evaluated on a human test data set and verified to fulfill the requested criteria.     

Chromogranin A-derived peptides: interaction with the rat posterior cerebral artery

Chromogranin A (CgA), an acidic granule protein of the regulated secretory pathway in the diffuse neuroendocrine system, is postulated to serve as a prohormone for regulatory peptides. Betagranin (rCgA(1-128)), the first N-terminal cleavage product of rat CgA, is 87% homologous to the bovine vasostatin I (bCgA(1-76)), previously shown to be vasoinhibitory in bovine resistance arteries. In this study the vasoactivity of homologous rat and bovine peptides was investigated in the rat posterior cerebral artery. Firstly, we examined the interaction of rhodamine (Rh)-labelled bCgA(7-40) and bCgA(47-70) with elements of the arterial wall by fluorescence microscopy. Secondly, rCgA(7-57), bCgA(1-40), bCgA(7-40) and bCgA(47-66) (chromofungin) were studied for effects on arterial tone and intracellular calcium as function of pressure in an arteriograph. Although without dilator or constrictor responses at 60-150 mm Hg, the rat peptide (rCgA(7-57)) evoked a significant delay in the onset of forced dilatation at 170 mm Hg, in contrast to the bovine peptides bCgA(1-40), bCgA(7-40) and bCgA(47-66) (chromofungin). Neither Rh-bCgA(7-40) nor Rh-bCgA(47-70) stained the endothelial layer, while Rh-bCgA(47-70) but not Rh-bCgA(7-40) stained the smooth muscle compartment. Analogously, bCgA(47-66) but not bCgA(7-40) reduced intracellular calcium, however without modifying the myogenic response. Thus, the betagranin peptide rCgA(7-57) and the two bovine chromofungin-containing peptides, highly homologous to the corresponding sequence (rCgA(47-66)), affected the rat cerebral artery without vasodilator effects, indicating significant species differences in vasoactivity of the N-terminal domain of CgA.     

Directed evolution to low nanomolar affinity of a tumor-targeting epidermal growth factor receptor-binding affibody molecule

The epidermal growth factor receptor 1 (EGFR) is overexpressed in various malignancies and is associated with a poor patient prognosis. A small, receptor-specific, high-affinity imaging agent would be a useful tool in diagnosing malignant tumors and in deciding upon treatment and assessing the response to treatment. We describe here the affinity maturation procedure for the generation of Affibody molecules binding with high affinity and specificity to EGFR. A library for affinity maturation was constructed by rerandomization of selected positions after the alignment of first-generation binding variants. New binders were selected with phage display technology, using a single oligonucleotide in a single-library effort, and the best second-generation binders had an approximately 30-fold improvement in affinity (K_d = 5-10 nM) for the soluble extracellular domain of EGFR in biospecific interaction analysis using Biacore. The dissociation equilibrium constant, K_d, was also determined for the Affibody with highest affinity using EGFR-expressing A431 cells in flow cytometric analysis (K_d = 2.8 nM). A retained high specificity for EGFR was verified by a dot blot assay showing staining only of EGFR proteins among a panel of serum proteins and other EGFR family member proteins (HER2, HER3, and HER4). The EGFR-binding Affibody molecules were radiolabeled with indium-111, showing specific binding to EGFR-expressing A431 cells and successful targeting of the A431 tumor xenografts with 4-6% injected activity per gram accumulated in the tumor 4 h postinjection.     

Gib2, a novel Gbeta-like/RACK1 homolog, functions as a Gbeta subunit in cAMP signaling and is essential in Cryptococcus neoformans

Canonical G proteins are heterotrimeric, consisting of alpha, beta, and gamma subunits. Despite multiple Galpha subunits functioning in fungi, only a single Gbeta subunit per species has been identified, suggesting that non-conventional G protein signaling exists in this diverse group of eukaryotic organisms. Using the Galpha subunit Gpa1 that functions in cAMP signaling as bait in a two-hybrid screen, we have identified a novel Gbeta-like/RACK1 protein homolog, Gib2, from the human pathogenic fungus Cryptococcus neoformans. Gib2 contains a seven WD-40 repeat motif and is predicted to form a seven-bladed beta propeller structure characteristic of beta transducins. Gib2 is also shown to interact, respectively, with two Ggamma subunit homologs, Gpg1 and Gpg2, similar to the conventional Gbeta subunit Gpb1. In contrast to Gpb1 whose overexpression promotes mating response, overproduction of Gib2 suppresses defects of gpa1 mutation in both melanization and capsule formation, the phenotypes regulated by cAMP signaling and associated with virulence. Furthermore, depletion of Gib2 by antisense suppression results in a severe growth defect, suggesting that Gib2 is essential. Finally, Gib2 is shown to also physically interact with a downstream target of Gpa1-cAMP signaling, Smg1, and the protein kinase C homolog Pkc1, indicating that Gib2 is also a multifunctional RACK1-like protein.     

On the origin of Z-band material and myofilaments in myoblasts from the human atrial wall

Summary The origin of cardiac myofibrils in cells from the atrial wall in human embryos was studied. Z-band substance appears throughout the cytoplasm as irregular electron dense patches in a network of thin filaments. The thin and thick filaments are synthesized as separate units in the sarcoplasm and are later aggregated into myofibrils. Complexes of Z substance and thin filaments occur numerously at different stages of myofibrillar organisation. Thick filaments are formed in close proximity to free ribosomes and are later incorporated in an hexagonal pattern into the Z-band/thin filament complex.This work was supported by grants from The Norwegian Council on Cardiovascular Disease and from The Norwegian Research Council for Science and the Humanities