Evidence of positive selection on the Atlantic salmon CD3γδ gene

Atlantic salmon are typically anadromous, spending the majority of their lifetime in oceans and returning to fresh water to breed. This diversity of environments likely results in strong selective forces shaping their genome. In this paper, we present the first genomics approach to detect positive selection operating on the Salmo salar (salmon) lineage, an important aquaculture species. We identify a panel of candidate genes that may have been subject to adaptive evolution in this species. In particular, we identify a robust signature of positive selection operating on the salmon CD3γδ gene, which encodes one of the protein chains essential for formation of the T-cell receptor complex and for T-cell activation. Furthermore, we identified the particular codon sites that have been subject to positive selection in fish and highlight two sites flanking an important N-glycosylation site in this molecule. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

ERRβ splice variants differentially regulate cell cycle progression

Orphan receptors comprise nearly half of all members of the nuclear receptor superfamily. Despite having broad structural similarities to the classical estrogen receptors, estrogen-related receptors (ERRs) have their own unique DNA response elements and functions. In this study, we focus on 2 ERRβ splice variants, short form ERRβ (ERRβsf) and ERRβ2, and identify their differing roles in cell cycle regulation. Using DY131 (a synthetic agonist of ERRβ), splice-variant selective shRNA, and exogenous ERRβsf and ERRβ2 cDNAs, we demonstrate the role of ERRβsf in mediating the G1 checkpoint through p21. We also show ERRβsf is required for DY131-induced cellular senescence. A key novel finding of this study is that ERRβ2 can mediate a G2/M arrest in response to DY131. In the absence of ERRβ2, the DY131-induced G2/M arrest is reversed, and this is accompanied by p21 induction and a G1 arrest. This study illustrates novel functions for ERRβ splice variants and provides evidence for splice variant interaction.     

Identification of distinct physiochemical properties of toxic prefibrillar species formed by Aβ peptide variants

The formation of amyloid-β peptide (Aβ) aggregates at an early stage during the self-assembly process is an important factor in the development of Alzheimer's disease. The toxic effect is believed to be exerted by prefibrillar species of Aβ. It is therefore important to identify which prefibrillar species are toxic and characterize their distinct properties. In the present study, we investigated the in vitro aggregation behavior of Aβ-derived peptides possessing different levels of neurotoxic activity, using fluorescence spectroscopy in combination with transmission electron microscopy. The toxicity of various Aβ aggregates was assessed by using cultures of human neuroblastoma cells. Through combined use of the fluorescence probe 8-anilino-1-napthalenesulfonate (ANS) and the novel luminescent probe pentamer formyl thiophene acetic acid (p-FTAA), we were able to identify those Aβ peptide-derived prefibrillar species which exhibited cellular toxicity. In particular, species, which formed early during the aggregation process and showed strong p-FTAA and ANS fluorescence, were the species that possessed toxic activities. Moreover, by manipulating the aggregation conditions, it was possible to change the capacity of the Aβ peptide to form nontoxic versus toxic species.     

μ-Calpain-mediated deregulation of cardiac, brain, and kidney NCX1 splice variants

μ-Calpain is a Ca(2+)-activated protease abundant in mammalian tissues. Here, we examined the effects of μ-calpain on three alternatively spliced variants of NCX1 using the giant, excised patch technique. Membrane patches from Xenopus oocytes expressing either heart (NCX1.1), kidney (NCX1.3), or brain (NCX1.4) variants of NCX1 were exposed to μ-calpain and their Na(+)-dependent (I(1)) and Ca(2+)-dependent (I(2)) regulatory phenotypes were assessed. For these exchangers, I(1) inactivation is evident as a Na(+)(i)-dependent decay of peak outward currents whereas I(2) regulation manifests as outward current activation by micromolar Ca(2+)(i) concentrations. Notably, with NCX1.1 and NCX1.4 but not in NCX1.3, higher Ca(2+)(i) levels alleviate I(1) inactivation. Our results show that (i) μ-calpain selectively ablates Ca(2+)-dependent (I(2)) regulation leading to a constitutive activation of exchange current, (ii) μ-calpain has much smaller effects on Na(+)-dependent (I(1)) regulation, produced by a slight destabilization of the I(1) state, and (iii) Ca(2+)-dependent regulation (I(2)) and Ca(2+)-mediated alleviation of I(1) appear to be functionally distinct mechanisms, the latter of which is left largely intact after μ-calpain treatment. The ability of μ-calpain to selectively and constitutively activate Na(+)-Ca(2+) exchange currents may have important pathophysiological implications in tissue where these splice variants are expressed.     

Specificity, location and function of βTrCP isoforms and their splice variants

SCF^βTrCP is the ubiquitin ligase for a wide variety of substrates and functions in many cellular processes. βTrCP, the substrate binding factor of the SCF complex, has two isoforms, produced from different genes, and several splice variants. Despite a certain level of redundancy, knock-out studies show different phenotypes indicating different preferential substrates for the two isoforms. However, until now functional differences between βTrCP1 and 2 were not studied at the endogenous protein level. We generated isoform-specific antibodies against βTrCP to characterise endogenous βTrCP isoforms and splice variants. We show that endogenous βTrCP1 and 2 localise to both nucleus and cytosol. Interestingly, we find that one splice variant of βTrCP2 localises exclusively to the nucleus and another only to the cytosol. In addition, we show that the substrate binding domain of βTrCP is the dominant localisation determinant.     

Distinct modulating effects of TipE-homologs 2–4 on Drosophila sodium channel splice variants

The Drosophila melanogaster TipE protein is thought to be an insect sodium channel auxiliary subunit functionally analogous to the β subunits of mammalian sodium channels. Besides TipE, four TipE-homologous proteins (TEH1–4) have been identified. It has been reported that TipE and TEH1 have both common and distinct effects on the gating properties of splice variants of the Drosophila sodium channel, DmNa_v. However, limited information is available on the effects of TEH2, TEH3 and TEH4 on the function of DmNa_v channel variants. In this study, we found that TEH2 increased the amplitude of peak current, but did not alter the gating properties of three examined DmNa_v splice variants expressed in Xenopus oocytes. In contrast, TEH4 had no effect on peak current, yet altered the gating properties of all three channel variants. Furthermore, TEH4 enhanced persistent current and slowed sodium current decay. The effects of TEH3 on DmNa_v variants are similar to those of TEH4, but the data were collected from a small portion of oocytes because co-expression of TEH3 with DmNa_v variants generated a large leak current in the majority of oocytes examined. In addition, TEH3 and TEH4 enhanced the expression of endogenous currents in oocytes. Taken together, our results reveal distinct roles of TEH proteins in modulating the function of sodium channels and suggest that TEH proteins might provide an important layer of regulation of membrane excitability in vivo. Our results also raise an intriguing possibility of TEH3/TEH4 as auxiliary subunits of other voltage-gated ion channels besides sodium channels.     

CFFM4: a new member of the CD20/FcεRIβ family

Proteins with transmembrane domains are classified in different families based on their structure, amino acid homology, and function. In this study, we report the identification, sequence, and expression profile of a new member of the CD20/FcepsilonRIbeta family, CD20/FcepsilonRIbeta family member 4 (CFFM4). The CFFM4 gene contains seven exons and six introns and is transcribed into an mRNA encoding a 240-amino acid protein with four hydrophobic regions. The CFFM4 protein shares a high degree of homology with the other members of the family, especially in the hydrophobic regions where several amino acids are conserved. However, the CFFM4 protein can be distinguished from the other members of the family based on the length of the second extracellular loop and the absence of an immunoreceptor tyrosine-based activation motif signal. Another distinct characteristic is that CFFM4 mRNA expression is not limited to the hematopoietic lineage. CFFM4 was detected by Northern dot blot in a variety of normal and cancerous tissues. CFFM4 expression was also compared in developmentally early hematopoietic human bone marrow CD34+ stem cells versus peripheral blood-derived CD14+ mature monocytes, in the undifferentiated versus differentiated myelomonocytic U937 cell line, and in acute myelogenous leukemia FAB1 versus FAB5. In each of these systems, cellular myelomonocytic differentiation correlated with an increase in CFFM4 mRNA expression. Such results indicate that CFFM4 is associated with mature cellular function in the monocytic lineage and like CD20 and FcepsilonRIbeta, it may be a component of a receptor complex involved in signal transduction.     

Identification of 2-(4-pyridyl)thienopyridinones as GSK-3β inhibitors

The discovery of a novel series of 2-(4-pyridyl)thienopyridinone GSK-3β inhibitors is reported. X-ray crystallography reveals its binding mode and enables rationalization of the SAR. The initial optimization of the template for improved cellular activity and predicted CNS penetration is also presented.     

Is Atlantic salmon production limited by number of territories?

Thirty Atlantic salmon Salmo salar parr (mean 135 mm total length, L _T) in the River Alta, Norway, were radio‐tagged and tracked during a 11–13 day period. The parr stayed within defined home ranges with a 95% probability of localization within an average area of 1286 m²(241–3484 m²). On average, each parr had overlapping home ranges with 7·7 other parr, and the overlap between pairs of fish covered on average 24% of their home range areas. Mean length of the river stretch used was 90 m (22–383 m). On average, during 38%(16–77%) of the tracking surveys, the fish had moved >10 m since the previous survey. Mean total distance moved during the whole study was 402 m (208–862 m). The Atlantic salmon were most often recorded in riffles, but 27% of the parr alternated between riffles and pools. The extensive movements, flexible habitat utilization and relatively large home ranges, together with the fact that all the parr had home ranges partly overlapping with other radio‐tagged parr, indicate a flexible and variable behaviour and, thereby, a more complex social structure than a rigid defence of a fixed territory. Thus, Atlantic salmon production seems not to be limited by the highest potential number of territories for large parr in a given area.     

Identification of the transgenic integration site in immunodeficient tgε26 human CD3ε transgenic mice

A strain of human CD3ε transgenic mice, tgε26, exhibits severe immunodeficiency associated with early arrest of T cell development. Complete loss of T cells is observed in homozygous tgε26 mice, but not in heterozygotes, suggesting that genomic disruption due to transgenic integration may contribute to the arrest of T cell development. Here we report the identification of the transgenic integration site in tgε26 mice. We found that multiple copies of the human CD3ε transgene are inserted between the Sstr5 and Metrn loci on chromosome 17, and that this is accompanied by duplication of the neighboring genomic region spanning 323 kb. However, none of the genes in this region were abrogated. These results suggest that the severe immunodeficiency seen in tgε26 mice is not due to gene disruption resulting from transgenic integration.     

Polyamines in liver and their influence on chromatin condensation after 17-β estradiol treatment of Atlantic salmon

Atlantic salmon (Salmo salar) were treated with 17- estradiol to induce vitellogenin synthesis in liver. This led to an increase in liver wet weight and total DNA. After incubation with micrococcal nuclease (EC 3.1.31.1) less soluble chromatin was obtained from nuclei of the estradiol treated than the control fish, but active gene regions were solubilized by the nuclease. Thus, in the estradiol treated fish soluble mononucleosomes contained hybridizable vitellogenin gene sequences. As a result of estradiol treatment the content in total liver of putrescine rose 3-fold, that of spermidine 2-fold, while spermine was unchanged. In muscle no significant changes were observed. The regulatory functions of polyamines during gene expression were investigated by binding (¹⁴C)spermine to isolated liver nuclei depleted of endogenous polyamines. The number of binding sites was higher in nuclei of estradiol treated than control fish. (^14C)spermine associated preferentially with micrococcal nuclease insensitive chromatin. Thus, the high content of putrescine and spermidine in liver supported the view of polyamine accumulation in proliferating tissues. The preferential binding to condensed chromatin indicated a stabilizing effect of polyamines on the organization of inactive chromatin structures.Abbreviations MNase micrococcal nuclease - PMSF phenylmethylsulfonylfluoride     

Interspecific hybridization, a matter of pioneering? Insights from Atlantic salmon and brown trout

Interspecific hybridization may occur in situations of recent contact between a colonizer and a resident species, being more intense in the colonization front. Atlantic salmon Salmo salar and brown trout S. trutta have been sympatric species since their origin and they share spatial and temporal spawning niches, exhibiting low levels of bidirectional interspecific hybridization and introgression throughout their distribution range. Different causes have been identified for increased hybridization, from escapes or deliberate releases of domesticated fish to sneaking male behavior. We have examined hybridization rates and direction in different situations of advance of one of these species into a territory formerly inhabited by the other (247 samples were analyzed in northern Spain and 487 in Kerguelen Islands). In all cases, hybrids found in the colonization front were offspring of colonizer females and resident males. We hypothesize that these findings are the result of adaptive relaxed mate choice of colonizing females, regardless of the relative abundance of each species.     

Contrasted TCRβ Diversity of CD8+ and CD8? T Cells in Rainbow Trout

Teleost fish express highly diverse naive TCRβ (TRB) repertoires and mount strong public and private clonal responses upon infection with pathogens. Fish T cells express typical markers such as CD8, CD4-1 and CD4-2, CD3, CD28 and CTLA4. Fish CD8⁺ T cells have been shown to be responsible for antigen-specific cell-mediated cytotoxicity in in vitro systems using histo-compatible effector and target cells. We compare here the complexity of TRB repertoires between FACS sorted CD8⁺ and CD8⁻ T cells from spleen and pronephros of rainbow trout. In contrast to human, while the TRB repertoire is highly diverse and polyclonal in CD8⁺ T cells of naïve fish, it appeared very different in CD8⁻ lymphocytes with irregular CDR3 length distributions suggesting a dominance of activated clones already in naïve fish or the presence of non conventional T cells. After infection with a systemic virus, CD8⁺ T cells mount a typical response with significant skewing of CDR3 length profiles. The infection also induces significant modifications of the TRB repertoire expressed by the CD8⁻ fraction, but for a different set of V/J combinations. In this fraction, the antiviral response results in an increase of the peak diversity of spectratypes. This unusual observation reflects the presence of a number of T cell expansions that rise the relative importance of minor peaks of the highly skewed distributions observed in unchallenged animals. These results suggest that the diversity of TRB expressed by CD8⁺ and CD8⁻ αβ T cells may be subjected to different regulatory patterns in fish and in mammals.     

Identification of an aminothiazole series of RORβ modulators

Crystallography has identified stearic acid, ALRT 1550 and ATRA as ligands that bind RORβ, however, none of these molecules represent good starting points to develop optimized small molecule modulators. Recently, Compound 1 was identified as a potent dual RORβ and RORγ inverse agonist with no activity towards RORα (Fig. 1). To our knowledge, this is one of only two small molecule RORβ inverse agonists identified in the primary literature from a tractable chemical series and represents an ideal starting point from which to design RORβ-selective modulators. Herein we describe our SAR optimization efforts that led to a series of potent neutral antagonists of RORβ.