RESUMO
TAS1R taste receptors and their associated heterotrimeric G protein gustducin are involved in sugar and amino acid sensing in taste cells and in the gastrointestinal tract. They are also strongly expressed in testis and sperm, but their functions in these tissues were previously unknown. Using mouse models, we show that the genetic absence of both TAS1R3, a component of sweet and amino acid taste receptors, and the gustducin α-subunit GNAT3 leads to male-specific sterility. To gain further insight into this effect, we generated a mouse model that expressed a humanized form of TAS1R3 susceptible to inhibition by the antilipid medication clofibrate. Sperm formation in animals without functional TAS1R3 and GNAT3 is compromised, with malformed and immotile sperm. Furthermore, clofibrate inhibition of humanized TAS1R3 in the genetic background of Tas1r3(-/-), Gnat3(-/-) doubly null mice led to inducible male sterility. These results indicate a crucial role for these extraoral "taste" molecules in sperm development and maturation. We previously reported that blocking of human TAS1R3, but not mouse TAS1R3, can be achieved by common medications or chemicals in the environment. We hypothesize that even low levels of these compounds can lower sperm count and negatively affect human male fertility, which common mouse toxicology assays would not reveal. Conversely, we speculate that TAS1R3 and GNAT3 activators may help infertile men, particularly those that are affected by some of the mentioned inhibitors and/or are diagnosed with idiopathic infertility involving signaling pathway of these receptors.
Assuntos
Infertilidade Masculina/genética , Receptores Acoplados a Proteínas G/genética , Paladar/genética , Testículo/metabolismo , Transducina/genética , Animais , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Modelos Animais , Testículo/efeitos dos fármacosRESUMO
HIF-1alpha is overexpressed in many human cancers compared to normal tissues due to the interaction of a multiplicity of factors and pathways that reflect specific genetic alterations and extracellular stimuli. We developed two HIF-1alpha chimeric reporter systems, HIF-1alpha/FLuc and HIF-1alpha(DeltaODDD)/FLuc, to investigate the tightly controlled level of HIF-1alpha protein in normal (NIH3T3 and HEK293) and glioma (U87) cells. These reporter systems provided an opportunity to investigate the degradation of HIF-1alpha in different cell lines, both in culture and in xenografts. Using immunofluorescence microscopy, we observed different patterns of subcellular localization of HIF-1alpha/FLuc fusion protein between normal cells and cancer cells; similar differences were observed for HIF-1alpha in non-transduced, wild-type cells. A dynamic cytoplasmic-nuclear exchange of the fusion protein and HIF-1alpha was observed in NIH3T3 and HEK293 cells under different conditions (normoxia, CoCl2 treatment and hypoxia). In contrast, U87 cells showed a more persistent nuclear localization pattern that was less affected by different growing conditions. Employing a kinetic model for protein degradation, we were able to distinguish two components of HIF-1alpha/FLuc protein degradation and quantify the half-life of HIF-1alpha fusion proteins. The rapid clearance component (t(1/2) approximately 4-6 min) was abolished by the hypoxia-mimetic CoCl2 MG132 treatment and deletion of ODD domain, and reflects the oxygen/VHL-dependent degradation pathway. The slow clearance component (t(1/2) approximately 200 min) is consistent with other unidentified non-oxygen/VHL-dependent degradation pathways. Overall, the continuous bioluminescence readout of HIF-1alpha/FLuc stabilization in vitro and in vivo will facilitate the development and validation of therapeutics that affect the stability and accumulation of HIF-1alpha.
Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Primers do DNA , Camundongos , Microscopia de Fluorescência , Reação em Cadeia da PolimeraseRESUMO
BCL8 is a novel human gene family initially identified through cloning of BCL8A, located at the t(14;15)(q32;q11-q13) translocation breakpoint, in a case of diffuse large B-cell lymphoma. Multiple copies of BCL8A map to the 1-Mb proximal duplicated region at 15q. We identified additional copies on human chromosomes 13 (BCL8B), 22 (BCL8C), 2 (BCL8D), and 10 (BCL8E) by cDNA cloning and sequence analysis. BCL8A, BCL8C, BCL8D, and BCL8E are truncated at the genomic level and are presumably pseudogenes or sterile transcripts. BCL8B is expressed predominantly in human brain and encodes a 327-kDa protein with extensive homology to the Drosophila melanogaster protein kinase A anchoring protein RG. LRBA, a human gene with a ubiquitous expression pattern mapping to 4q32, encodes a protein closely related to BCL8. The phylogenetically conserved BCL8 gene family evolved by transchromosomal and intrachromosomal duplications within the human genome.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Família Multigênica , Proteínas de Neoplasias/genética , Proteínas Adaptadoras de Transdução de Sinal , Encéfalo/metabolismo , Proteínas de Transporte/genética , Sequência Conservada , Proteínas Quinases Dependentes de AMP Cíclico/genética , DNA Complementar , Evolução Molecular , Dosagem de Genes , Duplicação Gênica , Humanos , Hibridização in Situ Fluorescente , Análise de Sequência de DNARESUMO
Although it has been suggested that REL is the critical target gene of 2p12-16 amplification in diffuse large B-cell lymphoma (DLBCL), little experimental evidence supports this notion. In the present study, we sought to evaluate the relationship between REL amplification and REL function in a panel of 46 newly diagnosed DLBCLs and to correlate with DLBCL subgroups as identified by gene expression profiles and clinical features. The results indicate that amplification of the REL locus is not associated with accumulation of the active form of REL, as evaluated by immunofluorescence analysis. Upon subgrouping of the DLBCL cases based on gene expression signatures, REL amplification was detected in all subgroups, while high levels of nuclear-located REL were more frequently detected in activated B-cell-like DLBCL. Correlative analyses of REL copy number and REL nuclear accumulation with clinical parameters did not reveal any significant associations. Together these results indicate that 2p12-16 amplification does not lead to abnormal REL activation, suggesting that REL may not be the functional target of the amplification event. Nonetheless, these data indicate that DLBCLs are heterogeneous with respect to REL and thus nuclear factor-kappaB (NF-kappaB) activity.