RESUMO
The purpose of this study was to investigate the association between television (TV) viewing and all-cause mortality among Brazilian adults after 6 years of follow-up. This longitudinal study started in 2010 in the city of Bauru, SP, Brazil, and involved 970 adults aged ≥50 years. Mortality was reported by relatives and confirmed in medical records of the Brazilian National Health System. Physical activity (PA) and TV viewing were assessed by the Baecke questionnaire. Health status, sociodemographic and behavioral covariates were considered as potential confounders. After 6 years of follow-up, 89 deaths were registered (9.2% [95% CI=7.4%-11%]). Type 2 diabetes mellitus was associated with higher risk of mortality (P-value=.012). Deaths correlated significantly with age (ρ=.188; P-value=.001), overall PA score (ρ=-.128; P-value=.001) and TV viewing (ρ=.086; P-value=.007). Lower percentage of participants reported TV viewing time as often (16%) and very often (5.7%), but there was an association between higher TV viewing time ("often" and "very often" grouped together) and increased mortality after 6 years of follow-up (P-value=.006). The higher TV viewing time was associated with a 44.7% increase in all-cause mortality (HR=1.447 [1.019-2.055]), independently of other potential confounders. In conclusion, the findings from this cohort study identified increased risk of mortality among adults with higher TV viewing time, independently of PA and other variables.
Assuntos
Exercício Físico , Mortalidade , Comportamento Sedentário , Televisão , Idoso , Brasil/epidemiologia , Diabetes Mellitus Tipo 2/epidemiologia , Feminino , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Fatores SocioeconômicosRESUMO
S1P (sphingosine 1-phosphate) receptor modulator (SRM) drugs interfere with lymphocyte trafficking by downregulating lymphocyte S1P receptors. While the immunosuppressive activity of SRM drugs has proved useful in treating autoimmune diseases such as multiple sclerosis, that drug class is beset by on-target liabilities such as initial dose bradycardia. The S1P that binds to cell surface lymphocyte S1P receptors is provided by S1P transporters. Mice born deficient in one of these, spinster homolog 2 (Spns2), are lymphocytopenic and have low lymph S1P concentrations. Such observations suggest that inhibition of Spns2-mediated S1P transport might provide another therapeutically beneficial method to modulate immune cell positioning. We report here results using a novel S1P transport blocker (STB), SLF80821178, to investigate the consequences of S1P transport inhibition in rodents. We found that SLF80821178 is efficacious in a multiple sclerosis model but - unlike the SRM fingolimod - neither decreases heart rate nor compromises lung endothelial barrier function. Notably, although Spns2 null mice have a sensorineural hearing defect, mice treated chronically with SLF80821178 have normal hearing acuity. STBs such as SLF80821178 evoke a dose-dependent decrease in peripheral blood lymphocyte counts, which affords a reliable pharmacodynamic marker of target engagement. However, the maximal reduction in circulating lymphocyte counts in response to SLF80821178 is substantially less than the response to SRMs such as fingolimod (50% vs. 90%) due to a lesser effect on T lymphocyte sub-populations by SLF80821178. Finally, in contrast to results obtained with Spns2 deficient mice, lymph S1P concentrations were not significantly changed in response to administration of STBs at doses that evoke maximal lymphopenia, which indicates that current understanding of the mechanism of action of S1P transport inhibitors is incomplete.
RESUMO
The MuCap experiment at the Paul Scherrer Institute has measured the rate Λ(S) of muon capture from the singlet state of the muonic hydrogen atom to a precision of 1%. A muon beam was stopped in a time projection chamber filled with 10-bar, ultrapure hydrogen gas. Cylindrical wire chambers and a segmented scintillator barrel detected electrons from muon decay. Λ(S) is determined from the difference between the µ(-) disappearance rate in hydrogen and the free muon decay rate. The result is based on the analysis of 1.2 × 10(10) µ(-) decays, from which we extract the capture rate Λ(S) = (714.9 ± 5.4(stat) ± 5.1(syst)) s(-1) and derive the proton's pseudoscalar coupling g(P)(q(0)(2) = -0.88 m(µ)(2)) = 8.06 ± 0.55.
RESUMO
We report a measurement of the positive muon lifetime to a precision of 1.0 ppm; it is the most precise particle lifetime ever measured. The experiment used a time-structured, low-energy muon beam and a segmented plastic scintillator array to record more than 2×10(12) decays. Two different stopping target configurations were employed in independent data-taking periods. The combined results give τ(µ(+)) (MuLan)=2 196 980.3(2.2) ps, more than 15 times as precise as any previous experiment. The muon lifetime gives the most precise value for the Fermi constant: G(F) (MuLan)=1.166 378 8(7)×10(-5) GeV(-2) (0.6 ppm). It is also used to extract the µ(-)p singlet capture rate, which determines the proton's weak induced pseudoscalar coupling g(P).
RESUMO
We have analyzed requirements for IL-4-induced secretion of IgG1 from anti-Ig-activated B cells. Activated B cell blasts prepared by culture of high density B cells with anti-Ig failed to secrete IgG1 upon subsequent culture with LPS and IL-4. However, IL-4 markedly suppressed IgM secretion in the same cultures. Addition of a mixture of T cell-derived lymphokines or rIL-5 to LPS-stimulated anti-Ig blasts restored IL-4-stimulated IgG1 secretion; rIL-2 further enhanced the response to IL-4 + rIL-5. These results suggest that IL-4, IL-5, and IL-2 cooperate in the regulation of B lymphocyte Ig isotype expression.
Assuntos
Linfócitos B/imunologia , Imunoglobulina G/metabolismo , Isotipos de Imunoglobulinas/metabolismo , Interleucina-2/administração & dosagem , Interleucinas/administração & dosagem , Animais , Anticorpos Anti-Idiotípicos , Sinergismo Farmacológico , Regulação da Expressão Gênica , Imunoglobulina M/metabolismo , Interleucina-4 , Interleucina-5 , Ativação Linfocitária , CamundongosRESUMO
Globoid cell leukodystrophy (GLD) is characterized histopathologically by apoptosis of oligodendrocytes, progressive demyelination, and the existence of large, multinuclear (globoid) cells derived from perivascular microglia. The glycosphingolipid, psychosine (d-galactosyl-beta-1,1' sphingosine), accumulates to micromolar levels in GLD patients who lack the degradative enzyme galactosyl ceramidase. Here we document that an orphan G protein-coupled receptor, T cell death-associated gene 8, is a specific psychosine receptor. Treatment of cultured cells expressing this receptor with psychosine or structurally related glycosphingolipids results in the formation of globoid, multinuclear cells. Our discovery of a molecular target for psychosine suggests a mechanism for the globoid cell histology characteristic of GLD, provides a tool with which to explore the disjunction of mitosis and cytokinesis in cell cultures, and provides a platform for developing a medicinal chemistry for psychosine.
Assuntos
Divisão Celular/fisiologia , Metabolismo dos Lipídeos , Oligodendroglia/fisiologia , Psicosina/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores Acoplados a Proteínas G , Cálcio/metabolismo , Linhagem Celular , Separação Celular , AMP Cíclico/metabolismo , Citometria de Fluxo , Proteínas de Ligação ao GTP/metabolismo , Genes Reporter/genética , Humanos , Immunoblotting , Leucodistrofia de Células Globoides/patologia , Leucodistrofia de Células Globoides/fisiopatologia , Microscopia Confocal , Estrutura Molecular , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Cell types associated with angiotensinogen mRNA in rat brain were identified in individual brain sections by in situ hybridization with tritiated RNA probes or with a sulfur-35--labeled oligonucleotide combined with immunocytochemical detection of either glial fibrillary acidic protein (GFAP) for astrocytes or microtubule-associated protein (MAP-2) for neurons. Autoradiography revealed silver grains clustered primarily over GFAP-reactive soma and processes; most grain clusters were not associated with MAP-2--reactive cells. These results demonstrate that, in contrast to other known neuropeptide precursors, angiotensinogen is synthesized by glia.
Assuntos
Angiotensinogênio/biossíntese , Astrócitos/metabolismo , Encéfalo/metabolismo , Angiotensinogênio/genética , Animais , Proteína Glial Fibrilar Ácida/análise , Histocitoquímica , Proteínas Associadas aos Microtúbulos/análise , Hibridização de Ácido Nucleico , RNA Mensageiro/análise , RNA Mensageiro/genética , RatosRESUMO
The major sphingolipid metabolite, sphingosine-1-phosphate (S1P), has important biological functions. S1P is the ligand for a family of five G-protein-coupled receptors with distinct signaling pathways that regulate angiogenesis, vascular maturation, immunity, chemotaxis, and other important biological pathways. Recently, clinical trials have targeted S1P receptors (S1PRs) for autoimmune diseases and transplantation and have generated considerable interest in developing additional, more selective compounds. This review summarizes current knowledge on the biology of S1P and S1PRs that forms the basis for future drug development and the treatment of kidney disease.
Assuntos
Desenho de Fármacos , Nefropatias/tratamento farmacológico , Lisofosfolipídeos/metabolismo , Lisofosfolipídeos/uso terapêutico , Receptores de Lisoesfingolipídeo/metabolismo , Esfingosina/análogos & derivados , Animais , Sobrevivência Celular/efeitos dos fármacos , Humanos , Lisofosfolipídeos/farmacologia , Camundongos , Receptores de Lisoesfingolipídeo/agonistas , Receptores de Lisoesfingolipídeo/genética , Esfingosina/metabolismo , Esfingosina/farmacologia , Esfingosina/uso terapêuticoRESUMO
Mitogen-activated protein (MAP) kinases are serine/threonine protein kinases activated by dual phosphorylation on threonine and tyrosine residues. A MAP kinase kinase (MKK1 or MEK1) has been identified as a dual-specificity protein kinase that is sufficient to phosphorylate MAP kinases p42mapk and p44mapk on the regulatory threonine and tyrosine residues. Because of the multiplicity of MAP kinase isoforms and the diverse circumstances and agonists leading to their activation, we thought it unlikely that a single MKK could accommodate this complexity. Indeed, two protein bands with MKK activity have previously been identified after renaturation following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We now report the molecular cloning and characterization of a second rat MAP kinase kinase cDNA, MKK2. MKK2 cDNA contains an open reading frame encoding a protein of 400 amino acids, 7 residues longer than MKK1 (MEK1). The amino acid sequence of MKK2 is 81% identical to that of MKK1, but nucleotide sequence differences occur throughout the aligned MKK2 and MKK1 cDNAs, indicating that MKK2 is the product of a distinct gene. MKK1 and MKK2 mRNAs are expressed differently in rat tissues. Both cDNAs when expressed in COS cells displayed the ability to phosphorylate and activate p42mapk and p44mapk, both MKK1 and MKK2 were activated in vivo in response to serum, and both could be phosphorylated and activated by the v-Raf protein in vitro. However, differences between MKK1 and MKK2 in sites of phosphorylation by proline-directed protein kinases predict differences in feedback regulation.
Assuntos
Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas Quinases Dependentes de Cálcio-Calmodulina , Clonagem Molecular , DNA/genética , Ativação Enzimática , Expressão Gênica , MAP Quinase Quinase 2 , Quinases de Proteína Quinase Ativadas por Mitógeno , Dados de Sequência Molecular , Proteínas Oncogênicas v-raf , Fosforilação , RNA Mensageiro/genética , Ratos , Proteínas Recombinantes , Proteínas Oncogênicas de Retroviridae/metabolismo , Alinhamento de SequênciaRESUMO
A search of the expressed sequence tag (EST) database retrieved a human cDNA sequence which partially encoded a novel G protein-coupled receptor (GPCR) GPR26. A human genomic DNA fragment encoding a partial open reading frame (ORF) and a rat cDNA encoding the full length ORF of GPR26 were obtained by library screening. The rat GPR26 cDNA encoded a protein of 317 amino acids, most similar (albeit distantly related) to the serotonin 5-HT(5A) and gastrin releasing hormone BB2 receptors. GPR26 mRNA expression analysis revealed signals in the striatum, pons, cerebellum and cortex. HEK293 and Rh7777 cells transfected with GPR26 cDNA displayed high basal cAMP levels, slow growth rate of clonal populations and derangements of normal cell shape. We also used a sequence reported only in the patent literature encoding GPR57 (a.k.a. HNHCI32) to PCR amplify a DNA fragment which was used to screen a human genomic library. This resulted in the cloning of a genomic fragment containing a pseudogene, psiGPR57, with a 99.6% nucleotide identity to GPR57. Based on shared sequence identities, the receptor encoded by GPR57 was predicted to belong to a novel subfamily of GPCRs together with GPR58 (a.k.a. phBL5, reported only in the patent literature), putative neurotransmitter receptor (PNR) and a 5-HT(4) pseudogene. Analysis of this subfamily revealed greatest identities (approximately 56%) between the receptors encoded by GPR57 and GPR58, each with shared identities of approximately 40% with PNR. Furthermore, psiGPR57, GPR58, PNR and the 5-HT(4) pseudogene were mapped in a cluster localized to chromosome 6q22-24. PNR and GPR58 were expressed in COS cells, however no specific binding was observed for various serotonin receptor-specific ligands.
Assuntos
Encéfalo/metabolismo , Receptores de Superfície Celular/genética , Receptores Acoplados a Proteínas G , 1-Metil-3-Isobutilxantina , Sequência de Aminoácidos , Animais , Sequência de Bases , Células COS , Linhagem Celular , Clonagem Molecular , DNA Complementar/isolamento & purificação , Humanos , Hibridização in Situ Fluorescente , Ligantes , Dados de Sequência Molecular , Fases de Leitura Aberta , Pseudogenes , RNA Mensageiro/metabolismo , Ratos , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , TransfecçãoRESUMO
Lysophosphatidic acid (LPA) is the simplest natural phospholipid, primarily known as a membrane component and metabolic intermediate. However, a remarkable variety of biological effects of this compound have come to light, seemingly pointing to an additional role for LPA as a signalling molecule. In this review, Marcel Durieux and Kevin Lynch integrate the recent information that indicates that LPA could be an intercellular messenger, possibly acting through a G protein-coupled receptor, and with a role in cell growth and motility.
Assuntos
Lisofosfolipídeos/fisiologia , Transdução de Sinais/fisiologia , Animais , HumanosRESUMO
Three 'alpha 1-adrenoceptors' and three 'alpha 2-adrenoceptors' have now been cloned. How closely do these receptors match the native receptors that have been identified pharmacologically? What are the properties of these receptors, and how do they relate to other members of the cationic amine receptor family? Kevin Lynch and his colleagues discuss these questions in this review.
Assuntos
Receptores Adrenérgicos , Sequência de Aminoácidos , Animais , Clonagem Molecular , Humanos , Dados de Sequência Molecular , Receptores Adrenérgicos/análise , Receptores Adrenérgicos/classificação , Receptores Adrenérgicos/metabolismoRESUMO
Nearly all molecules known to signal cells via G proteins have been assigned a cloned G-protein-coupled-receptor (GPCR) gene. This has been the result of a decade-long genetic search that has also identified some receptors for which ligands are unknown; these receptors are described as orphans (oGPCRs). More than 80 of these novel receptor systems have been identified and the emphasis has shifted to searching for novel signalling molecules. Thus, multiple neurotransmitter systems have eluded pharmacological detection by conventional means and the tremendous physiological implications and potential for these novel systems as targets for drug discovery remains unexploited. The discovery of all the GPCR genes in the genome and the identification of the unsolved receptor-transmitter systems, by determining the endogenous ligands, represents one of the most important tasks in modern pharmacology.
Assuntos
Proteínas de Ligação ao GTP/fisiologia , Receptores de Superfície Celular/fisiologia , Animais , Humanos , Ligantes , Receptores de Superfície Celular/efeitos dos fármacos , Receptores de Superfície Celular/genéticaRESUMO
The majority of genes encoding G protein-coupled receptors were isolated by methods based on sequence similarities found throughout this family. Experimental techniques have exploited these similarities (including low-stringency hybridization, polymerase chain reaction and electronic database searching) to identify genes encoding many pharmacologically recognized receptors and their subtypes. Homology-based searches have revealed receptors for which the endogenous ligands were unknown and these were named orphan receptors. Many orphan receptors are expressed in the brain, suggesting the existence of unidentified neurotransmitters. Methods used to identify ligands for these orphan receptors resulted in the identification of novel ligands and succeeded in pairing previously identified ligands with their receptors. Similar successful strategies are required to characterize the physiological and pathological importance of the remaining orphan receptors to facilitate the discovery of novel drugs for these systems.
Assuntos
Sistema Nervoso Central/efeitos dos fármacos , Receptores de Superfície Celular/efeitos dos fármacos , Animais , Sistema Nervoso Central/metabolismo , Humanos , Receptores de Angiotensina/efeitos dos fármacos , Receptores de Angiotensina/metabolismo , Receptores de Canabinoides , Receptores de Superfície Celular/metabolismo , Receptores de Droga/efeitos dos fármacos , Receptores de Droga/metabolismo , Receptores de Galanina , Receptores de Neuropeptídeos/efeitos dos fármacos , Receptores de Neuropeptídeos/metabolismo , Receptores de Neuropeptídeo Y/efeitos dos fármacos , Receptores de Neuropeptídeo Y/metabolismo , Receptores de Somatostatina/efeitos dos fármacos , Receptores de Somatostatina/metabolismoRESUMO
GH-releasing hormone (GHRH), acting through the GHRH receptor (GHRH-R), plays a pivotal role in the regulation of GH synthesis and secretion in the pituitary. It is possible that GHRH may serve other roles in other tissues. Here we report the cloning of a cDNA encoding a human GHRH-R from an acromegalic pituitary cDNA library. The isolated cDNA encodes a 423-amino acid protein that has seven putative transmembrane domains characteristic of G-protein-coupled receptors. It is a member of the secretin family of G-protein-coupled receptors and has 47%, 42%, 35%, and 28% identity with receptors for vasoactive intestinal peptide, secretin, calcitonin, and PTH, respectively. Transient expression of this cDNA in COS cells induced saturable, high affinity, GHRH-specific binding and also stimulated intracellular cAMP accumulation in response to physiological concentrations of GHRH. A specific GHRH antagonist blocked both binding and second messenger response. Northern analysis indicated that GHRH-R mRNA was most abundant in extracts of pituitary and was not detected in other tissues.
Assuntos
Adeno-Hipófise/metabolismo , Receptores de Neuropeptídeos , Receptores de Neurotransmissores/genética , Receptores de Hormônios Reguladores de Hormônio Hipofisário , Acromegalia/genética , Acromegalia/metabolismo , Adenoma/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Células Cultivadas , Chlorocebus aethiops , Clonagem Molecular , AMP Cíclico/metabolismo , DNA/genética , DNA de Neoplasias/genética , Humanos , Dados de Sequência Molecular , Neoplasias Hipofisárias/genética , RNA/genética , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-Dawley , Receptores de Superfície Celular/genética , Receptores de Neurotransmissores/biossíntese , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Ovinos , Transdução de SinaisRESUMO
To determine whether the angiotensinogen (Ao) gene is expressed in multiple organs of the fetal rat and the changes associated with maturation, fetal (15-20 days of gestation), newborn (1-10 days old), and adult (90 days old) rat tissues were subjected to Northern analysis and hybridization with a full length Ao complementary DNA (cDNA). Whereas Ao messenger RNA (mRNA) was undetectable in fetal livers, Ao sequences were readily detectable 1 h after birth and reached a peak at 24 h of birth. Levels remained elevated at 5 and 10 days after birth to decrease slightly at 90 days of postnatal life. Poly A+ enriched liver RNA was subjected to a similar analysis demonstrating that fetal liver Ao mRNA levels were 50-fold less than the corresponding adult levels. In contrast to the finding in the fetal liver, Ao mRNA was found in fetal brown fat, brains, and kidneys. We conclude that 1) Expression of the Ao gene is developmentally regulated in a tissue-specific manner; 2) Unlike the adult animal, the liver may not be the primary source of Ao in the fetus; 3) Alternate sources of Ao synthesis include fetal brown fat, brain, and kidneys.
Assuntos
Angiotensinogênio/genética , Feto/metabolismo , Regulação da Expressão Gênica , Tecido Adiposo Marrom/embriologia , Tecido Adiposo Marrom/metabolismo , Animais , Animais Recém-Nascidos/metabolismo , Encéfalo/embriologia , Encéfalo/metabolismo , DNA/genética , Idade Gestacional , Rim/embriologia , Rim/metabolismo , Fígado/embriologia , Fígado/crescimento & desenvolvimento , Fígado/metabolismo , Hibridização de Ácido Nucleico , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos WKY , Distribuição TecidualRESUMO
Angiotensinogen messenger RNA (mRNA) has been identified in both brown and white adipose tissue. Recently we have shown that when 3T3-L1 cells were treated with isobutylmethylxanthine (IBMX) to accelerate differentiation, angiotensinogen mRNA increased markedly in adipocytes as compared with preadipocytes. To determine if a correlation existed between the regulatory events associated with the differentiation process, we compared the change in angiotensinogen mRNA in spontaneously differentiating 3T3-F442A cells with two established parameters of differentiation in adipocyte cell lines. Differentiation was assessed by visual examination of cells for lipid droplets, fluorescent staining of the F-actin fibers, and increases in glycerol phosphate dehydrogenase mRNA. F-actin fibers were highly structured in preadipocytes, becoming disassembled and very disorganized as cells differentiated into adipocytes. The quantity of angiotensinogen mRNA increased as the number of lipid-containing cells increased within a culture. Glycerol phosphate dehydrogenase mRNA accumulated in differentiated adipocytes to about the same extent as angiotensinogen mRNA. Thus, increases in angiotensinogen mRNA were associated with the morphological and biochemical changes that occur during the phenotypic modulation of 3T3-F442A cells.
Assuntos
Tecido Adiposo/metabolismo , Angiotensinogênio/genética , RNA Mensageiro/metabolismo , Actinas/metabolismo , Tecido Adiposo/citologia , Diferenciação Celular , Linhagem Celular , Glicerolfosfato Desidrogenase/genética , Metabolismo dos LipídeosRESUMO
The presence of angiotensinogen messenger RNA (mRNA) was assessed in total RNA extracted from hepatoma, glioma, neuroblastoma, and glioma-neuroblastoma hybrid cell lines. Total RNA from 1 X 10(7) cells was extracted, transferred to a membrane, and hybridized with a 32P-labeled, full-length (1650-base pair) rat angiotensinogen complementary DNA (cDNA). Angiotensinogen RNA sequences could be definitively detected only in hepatoma cells. Steroids were used in an attempt to increase the angiotensinogen mRNA level. Dexamethasone (2 X 10(-6) M) or 17 beta-estradiol (1 X 10(-7) M) was added to the cultures 18 to 24 hours prior to harvest. Dexamethasone treatment of the hepatoma cells resulted in a large increase in angiotensinogen mRNA, whereas estradiol had no effect. Steroids failed to induce detectable levels of angiotensinogen mRNA in total RNA from the other cell lines. That the RNA was intact was ensured by hybridizing duplicate Northern blots to a 32P-labeled actin cDNA. Actin mRNA sequences were detected in all cell lines. Blot hybridization of poly(A)+RNA resulted in the visualization of a weak angiotensinogen mRNA signal for a glioma cell line and a glioma-neuroblastoma hybrid line. However, the ability to detect angiotensinogen mRNA in a cell may depend on the phenotype expressed, which can be governed by culture conditions.
Assuntos
Angiotensinogênio/genética , RNA Mensageiro/metabolismo , Animais , Linhagem Celular , Meios de Cultura , Densitometria , Dexametasona/farmacologia , Estradiol/farmacologiaRESUMO
Angiotensinogen (renin substrate) and its messenger RNA are known to accumulate in the rat brain. We have cloned rat preangiotensinogen cDNAs and used them as probes to measure the accumulation of preangiotensinogen messenger RNA sequences in eight regions of rat brain, as well as in liver and kidney. The brain regions examined were the cerebral cortex, hippocampus, striatum, cerebellum, diencephalon (including basal forebrain structures), midbrain, brainstem, and pituitary. On a tissue weight basis, the accumulation of preangiotensinogen RNA sequences was greatest in the liver, midbrain, and brainstem. The relative concentrations of messenger RNA were ranked as follows: liver, brainstem, midbrain greater than cerebellum, diencephalon greater than hippocampus greater than cortex, striatum, kidney greater than pituitary. Relative RNA concentrations from liver to kidney varied over a 16-fold range. Liver and brain preangiotensinogen RNA sequences were indistinguishable in size as measured by gel electrophoresis; however, the kidney sequences appeared some 100 nucleotides larger. Our data agree with previous measurements of angiotensinogen in the rat brain as assayed by renin-catalyzed angiotensin I release.
Assuntos
Angiotensinogênio/análise , Angiotensinas/análise , Química Encefálica , RNA Mensageiro/análise , Angiotensinogênio/metabolismo , Animais , Autorradiografia , Encéfalo/metabolismo , Eletroforese em Gel de Ágar , Rim/análise , Rim/metabolismo , Fígado/análise , Fígado/metabolismo , Masculino , Hibridização de Ácido Nucleico , RNA Mensageiro/metabolismo , Ratos , Ratos EndogâmicosRESUMO
Constitutively active nitric oxide synthases (NOS) are a unique class of NADPH-dependent, calcium/calmodulin-dependent enzymes that catalyze the conversion of L-arginine to nitric oxide and L-citrulline. However, little is known about the molecular similarities or differences between the two prototypical constitutive NOS enzymes, endothelial NOS (ECNOS) and brain NOS (bNOS). The aims of this study were to begin characterizing the gene structure and tissue distribution of messenger RNAs (mRNAs) for ECNOS and bNOS and to examine the immunological resemblance of the proteins by Western blotting. Full-length complementary DNAs (cDNAs) encoding bovine ECNOS and rat bNOS hybridized, under high stringency, to different-sized fragments of endonuclease-digested bovine, rat, and human genomic DNA. In addition, more than one fragment was detected with both cDNAs, suggesting that ECNOS and bNOS genes contained multiple introns. Tissue distribution of ECNOS mRNA (4.4 kb) and bNOS mRNA (9.5 kb) in the rat was detected by Northern blotting. Patterns among tissue extracts were strikingly different, with ECNOS mRNA being most abundant in aorta, heart, lung, kidney, adrenal gland, spinal cord, and urogenital tissues and bNOS mRNA most prominent in brain regions, intestine, stomach, spinal cord, adrenal gland, and aorta. Interestingly, ECNOS cDNA detected two equally abundant RNA transcripts (4.4 and 4.0 kb) in most brain regions tested, suggesting an alternative splicing of the ECNOS pre-mRNA. Western blotting, using an ECNOS monoclonal antibody, recognized ECNOS protein from native bovine endothelial cells, cultured bovine endothelial cells, and COS cells transfected with ECNOS cDNA but did not recognize purified bNOS.(ABSTRACT TRUNCATED AT 250 WORDS)