Interaction of neuronal nitric oxide synthase with alpha(1)-adrenergic receptor subtypes in transfected HEK-293 cells

BACKGROUND: The C-terminal four amino acids (GEEV) of human α(1A)-adrenergic receptors (ARs) have been reported to interact with the PDZ domain of neuronal nitric oxide synthase (nNOS) in a yeast two-hybrid system. The other two α(1)-AR subtypes have no sequence homology in this region, raising the possibility of subtype-specific protein-protein interactions. RESULTS: We used co-immunoprecipitation and functional approaches with epitope-tagged α(1)-ARs to examine this interaction and the importance of the C-terminal tail. Following co-transfection of HEK-293 cells with hexahistidine/Flag (HF)-tagged α(1A)-ARs and nNOS, membranes were solubilized and immunoprecipitated with anti-FLAG affinity resin or anti-nNOS antibodies. Immunoprecipitation of HFα(1A)-ARs resulted in co-immunoprecipitation of nNOS and vice versa, confirming that these proteins interact. However, nNOS also co-immunoprecipitated with HFα(1B)- and HFα(1D)-ARs, suggesting that the interaction is not specific to the α(1A) subtype. In addition, nNOS co-immunoprecipitated with each of the three HFα(1)-AR subtypes which had been C-terminally truncated, suggesting that this interaction does not require the C-tails; and with Flag-tagged β(1)- and β(2)-ARs. Treatment of PC12 cells expressing HFα(1A)-ARs with an inhibitor of nitric oxide formation did not alter norepinephrine-mediated activation of mitogen activated protein kinases, suggesting nNOS is not involved in this response. CONCLUSIONS: These results show that nNOS does interact with full-length α(1A)-ARs, but that this interaction is not subtype-specific and does not require the C-terminal tail, raising questions about its functional significance

Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples

Funder: NCI U24CA211006Abstract: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts

alpha(1)-Adrenoceptors in proximal segments of tail arteries from control and reserpinised rats

It has been recently shown that the supersensitivity of distal segments of the rat tail artery to phenylephrine after chemical sympathectomy with reserpine results from the appearance of alpha(1D)-adrenoceptors. It is known that both alpha(1A)- and alpha(1D)-adrenoceptors are involved in the contractions of proximal portions of the rat tail artery. Therefore, this study investigated whether sympathectomy with reserpine would induce supersensitivity in proximal segments of the rat tail artery, a tissue in which alpha(1D)-adrenoceptors are already functional. Proximal segments of tail arteries from reserpinised rats were three- to sixfold more sensitive to phenylephrine and methoxamine than were arteries from control rats (n=6-2; p<0.05). The imidazolines N-[5-(4,5-Dihydro-1H-imidazol-2-yl)-2-hydroxy-5,6,7,8-tetrahydronaphthalen-1-yl]methanesulfonamide hydrobromide (A-61603) and oxymetazoline, which activate selectively alpha(1A)-adrenoceptors, were equipotent in tail arteries from control and reserpinised rats (n=4-2; p<0.05), whereas buspirone, which activates selectively alpha(1D)-adrenoceptor, was approximate to 4-fold more potent in tail arteries from reserpinised rats (n=4-6; p<0.05). Prazosin (nonselective) and 5- methylurapidil (alpha(1A)- selective), were competitive antagonists of contractions induced by phenylephrine and were equipotent in tail arteries from control and reserpinised rats (n=4-6). The selective alpha(1D)-adrenoceptor antagonist 8[ 2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5]decane-7,9-dione dihydrochloride (BMY-7378) presented similar complex antagonism in tail arteries from control and reserpinised rats, with Schild slopes much lower than 1.0 (p<0.05, n=4-6). Semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) revealed that mRNA encoding alpha(1A)- and alpha(1B)-adrenoceptors are similarly distributed in tail arteries from control and reserpinised rats, whereas mRNA for alpha(1D)-adrenoceptors is twice more abundant in the tail artery from reserpinised rats. In conclusion, the supersensitivity induced by reserpine is related only to alpha(1D)-adrenoceptors, even in tissues where this receptor subtype is already present and functional. Only the use of subtype-selective alpha(1)-adrenoceptor agonists detected the increased alpha(1D)-adrenoceptor component after reserpinisation, as the antagonists behaved similarly in tail arteries from control and reserpinised rats.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

The N Terminus of the Human ␣ 1D -Adrenergic Receptor Prevents Cell Surface Expression

ABSTRACT We previously reported that truncation of the N-terminal 79 amino acids of ␣ 1D -adrenoceptors (⌬ 1-79 ␣ 1D -ARs) greatly increases binding site density. In this study, we determined whether this effect was associated with changes in ␣ 1D -AR subcellular localization. Confocal imaging of green fluorescent protein (GFP)-tagged receptors and sucrose density gradient fractionation suggested that full-length ␣ 1D -ARs were found primarily in intracellular compartments, whereas ⌬ 1-79 ␣ 1D -ARs were translocated to the plasma membrane. This resulted in a 3-to 4-fold increase in intrinsic activity for stimulation of inositol phosphate formation by norepinephrine. We determined whether this effect was transplantable by creating N-terminal chimeras of ␣ 1 -ARs containing the body of one subtype and the N terminus of another (␣ 1A NT-D, ␣ 1B NT-D, ␣ 1D NT-A, and ␣ 1D NT-B). When expressed in human embryonic kidney 293 cells, radioligand binding revealed that binding densities of ␣ 1A -or ␣ 1B -ARs containing the ␣ 1D -N terminus decreased by 86 to 93%, whereas substitution of ␣ 1A -or ␣ 1B -N termini increased ␣ 1D -AR binding site density by 2-to 3-fold. Confocal microscopy showed that GFP-tagged ␣ 1D NT-B-ARs were found only on the cell surface, whereas GFP-tagged ␣ 1B NT-D-ARs were completely intracellular. Radioligand binding and confocal imaging of GFP-tagged ␣ 1D -and ⌬ 1-79 ␣ 1D -ARs expressed in rat aortic smooth muscle cells produced similar results, suggesting these effects are generalizable to cell types that endogenously express ␣ 1D -ARs. These findings demonstrate that the N-terminal region of ␣ 1D -ARs contain a transplantable signal that is critical for regulating formation of functional bindings, through regulating cellular localization

Heuristics to reduce linear combinations of activation functions to improve image classification

International audienceImage classification is one of the classical problems in computer vision, and CNNs (Convolutional Neural Networks) are widely used for this task. However, the choice of a CNN can vary depending on the chosen dataset. In this context, we have trainable activation functions that are crucial in CNNs and adapt to the data. One technique for constructing these functions is to write them as a linear combination of other activation functions, where the coefficients of this combination are learned during training. However, if we have a large number of activation functions to combine, the computational cost can be very high, and manually testing and choosing these functions may be impractical, depending on the number of available activation functions. To alleviate the difficulty of choosing which activation functions should be part of the linear combination, we propose two heuristics: Linear Combination Approximator by Coefficients (LCAC) and Major and Uniform Coefficient Extractor (MUCE). Our heuristics provide an efficient selection of a subset of activation functions so that their results are better or equivalent to the linear combination that uses all 34 available activation functions in our experiments (C34), considering the image classification problem. Compared to the C34 function, the LCAC function was better or equivalent in 62.5%, and the MUCE function in 87.5% of the conducted experiments

Multiple effects of sibutramine on ejaculation and on vas deferens and seminal vesicle contractility

Sibutramine is an inhibitor of norepinephrine and 5-HT reuptake largely used in the management of obesity. Although a fairly safe drug, postmarketing adverse effects of sibutramine were reported including abnormal ejaculation in men. This study investigates the effects of sibutramine on ejaculation and vas deferens and seminal vesicle contractility. Adult male rats received sibutramine (5; 20; or 50 mg kg(-1), ip) and after 60 min were exposed to receptive females for determination of ejaculation parameters. The vasa deferentia and seminal vesicles of untreated rats were mounted in isolated organ baths for recording of isometric contractions and HEK293 cells loaded with fluorescent calcium indicator were used to measure intracellular Ca(2+) transients. Sibutramine 5 and 20 mg kg-1 reduced ejaculation latency whereas 50 mg kg(-1) increased ejaculation latency. Sibutramine 3 to 30 mu M greatly increased the sensitivity of the seminal vesicle and vas deferens to norepinephrine, but at concentrations higher than 10 mu M there were striking depressions of maximal contractions induced by norepinephrine, carbachol and CaCl(2). In HEK293 cells, sibutramine 10 to 100 mu M inhibited intracellular Ca(2+) transients induced by carbachol. Depending on the doses, sibutramine either facilitates or inhibits ejaculation. Apart from its actions in the central nervous system, facilitation of ejaculation may result from augmented sensitivity of smooth muscles to norepinephrine while reductions of intracellular Ca(2+) may be involved in the delayed ejaculation observed with high doses of sibutramine. (C) 2009 Elsevier B.V. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Implication of Rho-kinase and soluble guanylyl cyclase enzymes in prostate smooth muscle dysfunction in middle-aged rats

FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ – CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOAging is highly associated with benign prostate hyperplasia (BPH). We investigated here the alterations of the contractile and relaxant machinery in prostates of middle-aged rats, focusing on the Rho-kinase, nitric oxide (NO)-soluble guanylyl cyclase (sGC), 1- and -adrenoceptor pathways.Methods: Male Wistar young (3.5-month old) and middle-aged rats (10-month old) were used. Quantitative image analysis of prostates and functional assays evaluating the prostate contractions and relaxations were employed. Measurement of [H-3]-noradrenaline efflux, western blotting for 1 and 1 sGC subunits, and cyclic nucleotide levels were carried out.Results: Prostates of middle-aged rats showed significant increases in lumen and smooth muscle cells, but no alterations in the relative prostate weight were observed. In vivo, noradrenaline (10(-7)-10(-4)g/kg) produced greater prostatic contractions in middle-aged compared with control rats. Likewise, the in vitro contractions to phenylephrine (1nM-100M) and ,-methylene ATP (1-10M) were greater in middle-aged rats. Electrical-field stimulation (EFS, 1-32Hz) promoted higher [H-3]-noradrenaline efflux and prostate contractions in middle-aged rats. Reduced expressions of 1 and 1 sGC subunits and diminished NO-mediated prostate relaxations in middle-age were observed. Isoproterenol-induced relaxations and cAMP levels were reduced in prostates of middle-aged rats. The Rho-kinase inhibitor fasudil (50mg/kg, 2 weeks) normalized the prostate hypercontractility in middle-age rats.Conclusions: Prostate hypercontractility in middle-aging is associated with increased release of noradrenaline and Rho-kinase pathway, as well as with impairments of NO-sGC and -adrenoceptor pathways. Middle-aged rats are suitable to explore the enhanced prostatic tone in the absence of prostate overgrowth. Neurourol. Urodynam. (C) 2016 Wiley Periodicals, Inc.Aging is highly associated with benign prostate hyperplasia (BPH). We investigated here the alterations of the contractile and relaxant machinery in prostates of middle-aged rats, focusing on the Rho-kinase, nitric oxide (NO)-soluble guanylyl cyclase (sGC), 1- and -adrenoceptor pathways.Methods: Male Wistar young (3.5-month old) and middle-aged rats (10-month old) were used. Quantitative image analysis of prostates and functional assays evaluating the prostate contractions and relaxations were employed. Measurement of [H-3]-noradrenaline efflux, western blotting for 1 and 1 sGC subunits, and cyclic nucleotide levels were carried out.Results: Prostates of middle-aged rats showed significant increases in lumen and smooth muscle cells, but no alterations in the relative prostate weight were observed. In vivo, noradrenaline (10(-7)-10(-4)g/kg) produced greater prostatic contractions in middle-aged compared with control rats. Likewise, the in vitro contractions to phenylephrine (1nM-100M) and ,-methylene ATP (1-10M) were greater in middle-aged rats. Electrical-field stimulation (EFS, 1-32Hz) promoted higher [H-3]-noradrenaline efflux and prostate contractions in middle-aged rats. Reduced expressions of 1 and 1 sGC subunits and diminished NO-mediated prostate relaxations in middle-age were observed. Isoproterenol-induced relaxations and cAMP levels were reduced in prostates of middle-aged rats. The Rho-kinase inhibitor fasudil (50mg/kg, 2 weeks) normalized the prostate hypercontractility in middle-age rats.Conclusions: Prostate hypercontractility in middle-aging is associated with increased release of noradrenaline and Rho-kinase pathway, as well as with impairments of NO-sGC and -adrenoceptor pathways. Middle-aged rats are suitable to explore the enhanced prostatic tone in the absence of prostate overgrowth363589596FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ – CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ – CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOsem informaçãosem informaçã

Sex differences in oncogenic mutational processes

Funder: Canadian Network for Research and Innovation in Machining Technology, Natural Sciences and Engineering Research Council of Canada (NSERC Canadian Network for Research and Innovation in Machining Technology); doi: https://doi.org/10.13039/501100002790Funder: Genome Canada (Génome Canada); doi: https://doi.org/10.13039/100008762Funder: Canada Foundation for Innovation (Fondation canadienne pour l'innovation); doi: https://doi.org/10.13039/501100000196Funder: Terry Fox Research Institute (Institut de Recherche Terry Fox); doi: https://doi.org/10.13039/501100004376Abstract: Sex differences have been observed in multiple facets of cancer epidemiology, treatment and biology, and in most cancers outside the sex organs. Efforts to link these clinical differences to specific molecular features have focused on somatic mutations within the coding regions of the genome. Here we report a pan-cancer analysis of sex differences in whole genomes of 1983 tumours of 28 subtypes as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium. We both confirm the results of exome studies, and also uncover previously undescribed sex differences. These include sex-biases in coding and non-coding cancer drivers, mutation prevalence and strikingly, in mutational signatures related to underlying mutational processes. These results underline the pervasiveness of molecular sex differences and strengthen the call for increased consideration of sex in molecular cancer research

Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples

The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts.The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that -80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAFPeer reviewe