Both recombinant African catfish LH and FSH are able to activate the African catfish FSH receptor

LH and FSH are heterodimeric glycoprotein hormones, composed of a common alpha-subunit non-covalently associated with a hormone-specific beta-subunit. Repeated efforts to isolate catfish FSH (cfFSH) have not been successful and only catfish LH (cfLH) has been purified from catfish pituitaries. Recently, however, we succeeded in cloning the cDNA encoding the putative cfFSHbeta; the cDNAs for the alpha- and beta-subunit of cfLH have been cloned before. Here we report the expression of biologically active cfLH and cfFSH in the soil amoeba, Dictyostelium discoideum. The biological activity of the recombinant hormones was analyzed using cell lines transiently expressing either the cfLH receptor or the cfFSH receptor. Moreover, a primary testis tissue culture system served to study the steroidogenic potency of the recombinant hormones. Our results demonstrated that Dictyostelium produced biologically active, recombinant catfish gonadotropins, with recombinant cfLH being almost indistinguishable from its native counterpart, purified from pituitaries. Although recombinant cfFSH has significant effects in the bioassays used in this study, the specific function of native cfFSH in the control of reproduction and its expression patterns are not yet understood

The combined effect of two mutations that alter serially homologous color pattern elements on the fore and hindwings of a butterfly

<p>Abstract</p> <p>Background</p> <p>The ability for serially homologous structures to acquire a separate identity has been primarily investigated for structures dependent on Hox gene input but is still incompletely understood in other systems. The fore and hindwings of butterflies are serially homologous structures as are the serially homologous eyespots that can decorate each of these wings. Eyespots can vary in number between fore and hindwings of the same individual and mutations of large effect can control the total number of eyespots that each of the wings displays. Here we investigate the genetics of a new spontaneous color pattern mutation, <it>Missing</it>, that alters eyespot number in the nymphalid butterfly, <it>Bicyclus anynana</it>. We further test the interaction of <it>Missing </it>with a previously described mutation, <it>Spotty</it>, describe the developmental stage affected by <it>Missing</it>, and test whether <it>Missing </it>is a mutant variant of the gene <it>Distal-less </it>via a linkage association study.</p> <p>Results</p> <p><it>Missing </it>removes or greatly reduces the size of two of the hindwing eyespots from the row of seven eyespots, with no detectable effect on the rest of the wing pattern. Offspring carrying a single <it>Missing </it>allele display intermediate sized eyespots at these positions. <it>Spotty </it>has the opposite effect of <it>Missing</it>, i.e., it introduces two extra eyespots in homologous wing positions to those affected by <it>Missing</it>, but on the forewing. When <it>Missing </it>is combined with <it>Spotty </it>the size of the two forewing eyespots decreases but the size of the hindwing spots stays the same, suggesting that these two mutations have a combined effect on the forewing such that <it>Missing </it>reduces eyespot size when in the presence of a <it>Spotty </it>mutant allele, but that <it>Spotty </it>has no effect on the hindwing. <it>Missing </it>prevents the complete differentiation of two of the eyespot foci on the hindwing. We found no evidence for any linkage between the <it>Distal-less </it>and <it>Missing </it>genes.</p> <p>Conclusion</p> <p>The spontaneous mutation <it>Missing </it>controls the differentiation of the signaling centers of a subset of the serial homologous eyespots present on both the fore and the hindwing in a dose-dependent fashion. The effect of <it>Missing </it>on the forewing, however, is only observed when the mutation <it>Spotty </it>introduces additional eyespots on this wing. <it>Spotty</it>, on the other hand, controls the differentiation of eyespot centers only on the forewing. <it>Spotty</it>, unlike <it>Missing</it>, may be under Ubx gene regulation, since it affects a subset of eyespots on only one of the serially homologous wings.</p

Super-resolution imaging as a method to study GPCR dimers and higher-order oligomers

The study of G protein-coupled receptor (GPCR) dimers and higher-order oligomers has unveiled mechanisms for receptors to diversify signaling and potentially uncover novel therapeutic targets. The functional and clinical significance of these receptor–receptor associations has been facilitated by the development of techniques and protocols, enabling researchers to unpick their function from the molecular interfaces, to demonstrating functional significance in vivo, in both health and disease. Here we describe our methodology to study GPCR oligomerization at the single-molecule level via super-resolution imaging. Specifically, we have employed photoactivated localization microscopy, with photoactivatable dyes (PD-PALM) to visualize the spatial organization of these complexes to <10 nm resolution, and the quantitation of GPCR monomer, dimer, and oligomer in both homomeric and heteromeric forms. We provide guidelines on optimal sample preparation, imaging parameters, and necessary controls for resolving and quantifying single-molecule data. Finally, we discuss advantages and limitations of this imaging technique and its potential future applications to the study of GPCR function

Neoplastic Transformation of T Lymphocytes through Transgenic Expression of a Virus Host Modification Protein

Virus host evasion genes are ready-made tools for gene manipulation and therapy. In this work we have assessed the impact in vivo of the evasion gene A238L of the African Swine Fever Virus, a gene which inhibits transcription mediated by both NF-κB and NFAT. The A238L gene has been selectively expressed in mouse T lymphocytes using tissue specific promoter, enhancer and locus control region sequences for CD2. The resulting two independently derived transgenic mice expressed the transgene and developed a metastasic, angiogenic and transplantable CD4+CD8+CD69– lymphoma. The CD4+CD8+CD69– cells also grew vigorously in vitro. The absence of CD69 from the tumour cells suggests that they were derived from T cells at a stage prior to positive selection. In contrast, transgenic mice similarly expressing a mutant A238L, solely inhibiting transcription mediated by NF-κB, were indistinguishable from wild type mice. Expression of Rag1, Rag2, TCRβ-V8.2, CD25, FoxP3, Bcl3, Bcl2 l14, Myc, IL-2, NFAT1 and Itk, by purified CD4+CD8+CD69– thymocytes from A238L transgenic mice was consistent with the phenotype. Similarly evaluated expression profiles of CD4+CD8+ CD69– thymocytes from the mutant A238L transgenic mice were comparable to those of wild type mice. These features, together with the demonstration of (mono-)oligoclonality, suggest a transgene-NFAT-dependent transformation yielding a lymphoma with a phenotype reminiscent of some acute lymphoblastic lymphomas

Ubiquitination of CXCR7 Controls Receptor Trafficking

The chemokine receptor CXCR7 binds CXCL11 and CXCL12 with high affinity, chemokines that were previously thought to bind exclusively to CXCR4 and CXCR3, respectively. Expression of CXCR7 has been associated with cardiac development as well as with tumor growth and progression. Despite having all the canonical features of G protein-coupled receptors (GPCRs), the signalling pathways following CXCR7 activation remain controversial, since unlike typical chemokine receptors, CXCR7 fails to activate Gαi-proteins. CXCR7 has recently been shown to interact with β-arrestins and such interaction has been suggested to be responsible for G protein-independent signals through ERK-1/2 phosphorylation. Signal transduction by CXCR7 is controlled at the membrane by the process of GPCR trafficking. In the present study we investigated the regulatory processes triggered by CXCR7 activation as well as the molecular interactions that participate in such processes. We show that, CXCR7 internalizes and recycles back to the cell surface after agonist exposure, and that internalization is not only β-arrestin-mediated but also dependent on the Serine/Threonine residues at the C-terminus of the receptor. Furthermore we describe, for the first time, the constitutive ubiquitination of CXCR7. Such ubiquitination is a key modification responsible for the correct trafficking of CXCR7 from and to the plasma membrane. Moreover, we found that CXCR7 is reversibly de-ubiquitinated upon treatment with CXCL12. Finally, we have also identified the Lysine residues at the C-terminus of CXCR7 to be essential for receptor cell surface delivery. Together these data demonstrate the differential regulation of CXCR7 compared to the related CXCR3 and CXCR4 receptors, and highlight the importance of understanding the molecular determinants responsible for this process

Cholinergic receptor pathways involved in apoptosis, cell proliferation and neuronal differentiation

Acetylcholine (ACh) has been shown to modulate neuronal differentiation during early development. Both muscarinic and nicotinic acetylcholine receptors (AChRs) regulate a wide variety of physiological responses, including apoptosis, cellular proliferation and neuronal differentiation. However, the intracellular mechanisms underlying these effects of AChR signaling are not fully understood. It is known that activation of AChRs increase cellular proliferation and neurogenesis and that regulation of intracellular calcium through AChRs may underlie the many functions of ACh. Intriguingly, activation of diverse signaling molecules such as Ras-mitogen-activated protein kinase, phosphatidylinositol 3-kinase-Akt, protein kinase C and c-Src is modulated by AChRs. Here we discuss the roles of ACh in neuronal differentiation, cell proliferation and apoptosis. We also discuss the pathways involved in these processes, as well as the effects of novel endogenous AChRs agonists and strategies to enhance neuronal-differentiation of stem and neural progenitor cells. Further understanding of the intracellular mechanisms underlying AChR signaling may provide insights for novel therapeutic strategies, as abnormal AChR activity is present in many diseases