A novel tilapia prolactin receptor is functionally distinct from its paralog

A novel tilapia prolactin (PRL) receptor (OmPRLR2) was identified based on its induction during hyperosmotic stress. OmPRLR2 protein shows 28% identity to tilapia OmPRLR1 and 26% identity to human PRLR. Comparison of OmPRLR1 and OmPRLR2 revealed conserved features of cytokine class I receptors (CKR1): a WS domain and transmembrane domain, two pairs of cysteines and N-glycosylation motifs in the extracellular region, CKR1 boxes I and II, and three tyrosines in the intracellular region. However, OmPRLR2 lacked the ubiquitin ligase and 14-3-3 binding motifs. OmPRLR2 mRNA was present in all tissues analyzed, with highest expression in gills, intestine, kidney and muscle, similar to OmPRLR1. Transfer of fish from fresh water to sea water transiently increased gill OmPRLR2 mRNA levels within 4h but decreased its protein abundance in the long term. OmPRLR2 is expressed in part as a truncated splice variant of 35kDa in addition to the 55kDa full-length protein. Cloning of the mRNA encoding the 35kDa variant revealed that it lacks the extracellular region. It is expressed at significantly higher levels in males than in females. In stably transfected HEK293 cells over-expressing tetracycline-inducible OmPRLR1 and OmPRLR2, activation of these receptors by tilapia PRL177 and PRL188 triggered different downstream signaling pathways. Moreover, OmPRLR2 significantly increased HEK293 salinity tolerance. Our data reveal that tilapia has two PRLR genes whose protein products respond uniquely to PRL and activate different downstream pathways. Expression of a short PRLR2 variant may serve to inhibit PRL binding during osmotic stress and in male tissues.Fil: Fiol, Diego Fernando. University of California at Davis; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Sanmarti, Enio. University of California at Davis; Estados UnidosFil: Sacchi, Romina. University of California at Davis; Estados UnidosFil: Kultz, Dietmar. University of California at Davis; Estados Unido

In Vitro Biologic Activities of the Antimicrobials Triclocarban, Its Analogs, and Triclosan in Bioassay Screens: Receptor-Based Bioassay Screens

BackgroundConcerns have been raised about the biological and toxicologic effects of the antimicrobials triclocarban (TCC) and triclosan (TCS) in personal care products. Few studies have evaluated their biological activities in mammalian cells to assess their potential for adverse effects.ObjectivesIn this study, we assessed the activity of TCC, its analogs, and TCS in in vitro nuclear-receptor-responsive and calcium signaling bioassays.Materials and methodsWe determined the biological activities of the compounds in in vitro, cell-based, and nuclear-receptor-responsive bioassays for receptors for aryl hydrocarbon (AhR), estrogen (ER), androgen (AR), and ryanodine (RyR1).ResultsSome carbanilide compounds, including TCC (1-10 muM), enhanced estradiol (E(2))-dependent or testosterone-dependent activation of ER- and AR-responsive gene expression up to 2.5-fold but exhibited little or no agonistic activity alone. Some carbanilides and TCS exhibited weak agonistic and/or antagonistic activity in the AhR-responsive bioassay. TCS exhibited antagonistic activity in both ER- and AR-responsive bioassays. TCS (0.1-10 muM) significantly enhanced the binding of [(3)H]ryanodine to RyR1 and caused elevation of resting cytosolic [Ca(2+)] in primary skeletal myotubes, but carbanilides had no effect.ConclusionsCarbanilides, including TCC, enhanced hormone-dependent induction of ER- and AR-dependent gene expression but had little agonist activity, suggesting a new mechanism of action of endocrine-disrupting compounds. TCS, structurally similar to noncoplanar ortho-substituted poly-chlorinated biphenyls, exhibited weak AhR activity but interacted with RyR1 and stimulated Ca(2+) mobilization. These observations have potential implications for human and animal health. Further investigations are needed into the biological and toxicologic effects of TCC, its analogs, and TCS

Disseminated histiocytic sarcoma in a Silky Terrier

A 1-year-old male castrated Silky Terrier dog was presented to the Cornell University Emergency Service with a 2-week history of lameness and progressive stiffness. On physical examination, the patient was ambulatory but stiff with a stilted gait. He was hypertonic with multifocal, asymmetric soft tissue swellings and enlargement of the right antebrachium extensor muscles, right epaxial muscle, left oblique muscles, and left biceps femoris muscle. The cervical musculature was bilaterally swollen and hypertonic, and the patient had minimal range of motion of neck and was painful upon palpation. Pertinent results of a complete blood count included mild normocytic normochromic non-regenerative anemia, mild leukocytosis with a mature neutrophilia, and mild monocytosis. Results of a chemistry panel revealed low BUN and creatinine, mild hyperphosphotemia, moderate elevations in AST and GGT, moderately elevated creatine kinase, and mild hemolysis. Radiographs revealed moderate, multifocal, asymmetric soft tissue swellings with a subtle increase in opacity. MRI exam of the cervical spine revealed multifocal asymmetric myopathy, generalized lymphadenopathy, extradural compressive myelopathy of C5-7, and a questionable esophageal mass. EMG of the cervical muscles revealed a normal signal centrally within the muscle tissue and an abnormal signal in the periphery of the muscle. Cerebral spinal fluid (CSF) analysis showed a mild mononuclear pleocytosis. The patient’s CSF tested negative for Neospora (IFA), Canine Distemper Virus (PCR), and Toxoplasma (IgG/IgM). Serum was negative for Cryptococcus (Ag) and Rickettsia ristici (IFA) and 4DX Snap was negative for Borrelia burgdorferi, Erlichia canis, Anaplasma phagocytophilum, and Dirofilaria immitis. A skeletal muscle biopsy from the cervical region was composed of a poorly differentiated population of round pleomorphic cells which proved to be CD18 positive on immunohistological staining. The dog was ambulatory but his gait was stiff and began to regurgitate after feeding. The patient was discharged to the care of his owners on prednisone, clindamycin, doxycycline, tramadol, pregabalin, methocarbamol, and famotidine. The patient represented two weeks later for a scheduled euthanasia, as the clinical signs were worsening despite palliative therapy. Necropsy findings were consistent with disseminated histiocytic sarcoma with multifocal infiltration of the skeletal muscle, diaphragm, lymph nodes, esophagus and stomach

A novel GRAIL E3 ubiquitin ligase promotes environmental salinity tolerance in euryhaline tilapia

Background: Tilapia (Oreochromis mossambicus) are euryhaline fishes capable of tolerating large salinity changes. In a previous study aimed to identify genes involved in osmotolerance, we isolated an mRNA sequence with similarity to GRAIL (Gene Related to Anergy In Lymphocytes), which is a critical regulator of adaptive immunity and development. Tilapia GRAIL contains a PA (protease associated) domain and a C3H2C3 RING finger domain indicative of E3 ubiquitin ligase activity. Scope of review: Western blots analysis was used to assess GRAIL expression pattern and responses to hyperosmotic stress. Immunohistochemistry was used to reveal the cellular localization of GRAIL in gill epithelium. Overexpression in HEK293 T-Rex cells was used to functionally characterize tilapia GRAIL. Salinity stress causes strong up-regulation of both mRNA and protein levels of tilapia GRAIL in gill epithelium. Tissue distribution of GRAIL protein is mainly confined to gill epithelium, which is the primary tissue responsible for osmoregulation of teleost fishes. Overexpression of tilapia GRAIL in HEK293 cells increases cell survival (cell viability) while decreases apoptosis during salinity challenge. Major conclusions: Our data indicate that tilapia GRAIL is a novel E3 ubiquitin ligase involved in osmotic stress signaling, which promotes environmental salinity tolerance by supporting gill cell function during hyperosmotic stress. General significance: Involvement of tilapia GRAIL in the osmotic stress response suggests that GRAIL E3 ubiquitin ligases play a broader role in environmental stress responses, beyond their documented functions in adaptive immunity and development.Fil: Fiol, Diego Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata; Argentina. University Of California At Davis; Estados UnidosFil: Sanmarti, Enio. University Of California At Davis; Estados UnidosFil: Lim, Andreana H.. University Of California At Davis; Estados UnidosFil: Kültz, Dietmar. University Of California At Davis; Estados Unido

In vitro biologic activities of the antimicrobials triclocarban, its analogs, and triclosan in bioassay screens: receptor-based bioassay screens.

BackgroundConcerns have been raised about the biological and toxicologic effects of the antimicrobials triclocarban (TCC) and triclosan (TCS) in personal care products. Few studies have evaluated their biological activities in mammalian cells to assess their potential for adverse effects.ObjectivesIn this study, we assessed the activity of TCC, its analogs, and TCS in in vitro nuclear-receptor-responsive and calcium signaling bioassays.Materials and methodsWe determined the biological activities of the compounds in in vitro, cell-based, and nuclear-receptor-responsive bioassays for receptors for aryl hydrocarbon (AhR), estrogen (ER), androgen (AR), and ryanodine (RyR1).ResultsSome carbanilide compounds, including TCC (1-10 muM), enhanced estradiol (E(2))-dependent or testosterone-dependent activation of ER- and AR-responsive gene expression up to 2.5-fold but exhibited little or no agonistic activity alone. Some carbanilides and TCS exhibited weak agonistic and/or antagonistic activity in the AhR-responsive bioassay. TCS exhibited antagonistic activity in both ER- and AR-responsive bioassays. TCS (0.1-10 muM) significantly enhanced the binding of [(3)H]ryanodine to RyR1 and caused elevation of resting cytosolic [Ca(2+)] in primary skeletal myotubes, but carbanilides had no effect.ConclusionsCarbanilides, including TCC, enhanced hormone-dependent induction of ER- and AR-dependent gene expression but had little agonist activity, suggesting a new mechanism of action of endocrine-disrupting compounds. TCS, structurally similar to noncoplanar ortho-substituted poly-chlorinated biphenyls, exhibited weak AhR activity but interacted with RyR1 and stimulated Ca(2+) mobilization. These observations have potential implications for human and animal health. Further investigations are needed into the biological and toxicologic effects of TCC, its analogs, and TCS