RESUMO
OBJECTIVE: This study investigated the effects of chronic melatonin treatment on gene expression of α1-, α2-, ß1- and ß2-adrenoceptors in the hippocampus of rats subjected to chronic unpredictable mild stress (CUMS). BACKGROUND: Preclinical studies have also shown that melatonin prevented short- and long-term memory impairments and exhibited antidepressant-like actions. METHODS: For this study, we used 24 animals, which were divided into four groups, and the experiment lasted 4 weeks. We quantified the changes in mRNA and protein levels of α1-, α2-, ß1- and ß2-adrenoceptors in the hippocampus after melatonin treatment. RESULTS: Our results demonstrated a decreased gene expression of α1-, α2- and ß2-adrenoceptors in the hippocampus of rats subjected to unpredictable chronic mild stress, while there was no change in gene expression of ß1-adrenoceptors. Melatonin treatment in the CUMS rats prevented the stress-induced decrease in mRNA and protein levels of α1-and ß2-adrenoceptors, whereas did not affect either on mRNA or protein level of ß1-and α2-adrenoceptors. CONCLUSION: Our data suggest that melatonin, by increasing reduced levels of α1- and ß2-adrenoceptors mRNA and protein in the hippocampus of chronic stressed rats, may be beneficial in conditions such as chronic stress and provides an experimental opportunity to probe into further molecular mechanisms underlying the regulation of these receptor subtype (Fig. 2, Ref. 28).
Assuntos
Nível de Alerta/fisiologia , Expressão Gênica/fisiologia , Hipocampo/fisiopatologia , Melatonina/fisiologia , Receptores Adrenérgicos alfa 1/genética , Receptores Adrenérgicos beta 2/genética , Estresse Psicológico/fisiopatologia , Animais , Doença Crônica , Masculino , RNA Mensageiro/genética , Ratos , Receptores Adrenérgicos alfa 2/genética , Receptores Adrenérgicos beta 1/genética , Transdução de SinaisRESUMO
Preclinical studies have shown that melatonin exercised antidepressant-like and anxiolyticlike effects in animal models of anxiety. The aim of the present study was to correlate the changes in behaviour induced by melatonin treatment with the activity of the dopaminergic system in the hippocampus of Wistar rats exposed to chronic, unpredictable, mild stress (CUMS). Male Wistar rats, 11 weeks old, were subjected to chronic stress for 28 successive days. Separate groups of control and stressed rats were intraperitoneally injected daily either with melatonin (10 mg/kg/day, i.p.) or placebo (5% ethanol). The open-field and elevated plus-maze tests were used to assess locomotor activities and anxiety levels. The content of dopamine (DA) in the hippocampal tissues was determined using radioenzymatic assay, while changes in tyrosine hydroxylase (TH) mRNA and protein levels in the hippocampus were determined using real-time RT-PCR and Western immunoblotting. Chronic stress led to reduction in the hippocampal dopaminergic content without affecting the levels of TH protein. These changes were accompanied by increased locomotor activity and higher anxiety levels in the open-field test. Administration of melatonin for 28 days resulted in an increase in the hippocampal DA content as a result of elevated TH protein levels. Melatonin showed an improvement in anxiety-like behaviour along with significantly reduced exploration. We could conclude that melatonin may stimulate dopaminergic synthesis in the hippocampus in order to suppress stress-induced behaviour.
Assuntos
Ansiedade/tratamento farmacológico , Ansiedade/fisiopatologia , Locomoção , Melatonina/uso terapêutico , Estresse Psicológico/tratamento farmacológico , Estresse Psicológico/fisiopatologia , Animais , Ansiedade/complicações , Ansiedade/genética , Doença Crônica , Dopamina/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Asseio Animal/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Locomoção/efeitos dos fármacos , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Melatonina/farmacologia , Ratos Wistar , Estresse Psicológico/complicações , Estresse Psicológico/genética , Fatores de Tempo , Tirosina 3-Mono-Oxigenase/genética , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Breathing a mixture of 10% CO(2) with 90% O(2) (referred to here as carbogen-10) increases blood flow due to the vasodilatory effect of CO(2), and raises blood O(2) saturation due to the enriched oxygen level. These effects both tend to reduce the level of deoxygenated hemoglobin in brain tissues, thereby reducing the potential for further increases in BOLD contrast. In the present study, blocks of intense visual stimulation (60s) were presented amid longer blocks (180s) during which subjects breathed various fractional concentrations (0-100%) of carbogen-10 diluted with medical air. When breathing undiluted carbogen-10, the BOLD response to visual stimulation was reduced below the level of noise against the background of the carbogen-10 response. At these concentrations, the total (visual+carbogen) BOLD response amplitude (7.5±1.0%, n=6) converged toward that seen with carbogen alone (7.5±1.0%, n=6). In spite of the almost complete elimination of the visual BOLD response, pseudo-continuous arterial spin-labeling on a separate cohort indicated a largely preserved perfusion response (89±34%, n=5) to the visual stimulus during inhalation of carbogen-10. The previously discussed observations suggest that venous saturation can be driven to very high levels during carbogen inhalation, a finding which has significant implications for calibrated MRI techniques. The latter methods involve estimation of the relative change in venous O(2) saturation by expressing activation-induced BOLD signal increases as a fraction of the maximal BOLD signal M that would be observed as venous saturation approaches 100%. While the value of M has generally been extrapolated from much smaller BOLD responses induced using hypercapnia or hyperoxia, our results suggest that these effects could be combined through carbogen inhalation to obtain estimates of M based on larger BOLD increases. Using a hybrid BOLD calibration model taking into account changes in both blood flow and arterial oxygenation, we estimated that inhalation of carbogen-10 led to an average venous saturation of 91%, allowing us to compute an estimated M value of 9.5%.
Assuntos
Mapeamento Encefálico/métodos , Encéfalo/metabolismo , Dióxido de Carbono/farmacologia , Imageamento por Ressonância Magnética , Oxigênio/farmacologia , Adulto , Encéfalo/irrigação sanguínea , Dióxido de Carbono/química , Circulação Cerebrovascular/efeitos dos fármacos , Circulação Cerebrovascular/fisiologia , Potenciais Evocados Visuais/efeitos dos fármacos , Potenciais Evocados Visuais/fisiologia , Feminino , Humanos , Interpretação de Imagem Assistida por Computador , Inalação , Masculino , Oxigênio/sangue , Oxigênio/química , Consumo de Oxigênio/fisiologia , Estimulação Luminosa , Radiossensibilizantes/farmacologia , Adulto JovemRESUMO
The purpose of the present study was to compare the effects of cold water immersion (CWI) and active recovery (ACT) on resting limb blood flow, rectal temperature and repeated cycling performance in the heat. Ten subjects completed two testing sessions separated by 1 week; each trial consisted of an initial all-out 35-min exercise bout, one of two 15-min recovery interventions (randomised: CWI or ACT), followed by a 40-min passive recovery period before repeating the 35-min exercise bout. Performance was measured as the change in total work completed during the exercise bouts. Resting limb blood flow, heart rate, rectal temperature and blood lactate were recorded throughout the testing sessions. There was a significant decline in performance after ACT (mean (SD) -1.81% (1.05%)) compared with CWI where performance remained unchanged (0.10% (0.71%)). Rectal temperature was reduced after CWI (36.8°C (1.0°C)) compared with ACT (38.3°C (0.4°C)), as was blood flow to the arms (CWI 3.64 (1.47) ml/100 ml/min; ACT 16.85 (3.57) ml/100 ml/min) and legs (CW 4.83 (2.49) ml/100 ml/min; ACT 4.83 (2.49) ml/100 ml/min). Leg blood flow at the end of the second exercise bout was not different between the active (15.25 (4.33) ml/100 ml/min) and cold trials (14.99 (4.96) ml/100 ml/min), whereas rectal temperature (CWI 38.1°C (0.3°C); ACT 38.8°C (0.2°C)) and arm blood flow (CWI 20.55 (3.78) ml/100 ml/min; ACT 23.83 (5.32) ml/100 ml/min) remained depressed until the end of the cold trial. These findings indicate that CWI is an effective intervention for maintaining repeat cycling performance in the heat and this performance benefit is associated with alterations in core temperature and limb blood flow.
Assuntos
Braço/irrigação sanguínea , Desempenho Atlético/fisiologia , Ciclismo/fisiologia , Temperatura Baixa , Imersão/fisiopatologia , Perna (Membro)/irrigação sanguínea , Adulto , Temperatura Corporal/fisiologia , Exercício Físico/fisiologia , Frequência Cardíaca/fisiologia , Humanos , Ácido Láctico/sangue , Masculino , Consumo de Oxigênio , Recuperação de Função Fisiológica/fisiologia , Reto/fisiologia , Fluxo Sanguíneo Regional , ÁguaRESUMO
The promoters of two U2 small nuclear RNA genes isolated from the sea urchin Lytechinus variegatus were mapped by microinjection of genes into sea urchin zygotes. One gene, LvU2E, is expressed only in oocytes and embryos and is found in a tandemly repeated gene set, while the other gene, LvU2L, is a single-copy gene and is expressed in embryos and somatic cells. The promoters each contain a TATA sequence at -25 which is required for expression, a proximal sequence element (PSE) centered at -55 required for expression, a sequence at -100 which couples the core promoter (PSE plus TATA box) to the upstream element, and an upstream sequence which stimulates expression fourfold. The PSE together with the TATA sequence is sufficient to determine the transcription start site. There is no sequence similarity between the -100 and PSE sequences of the two genes. The -100 sequences can be interchanged between the two genes. The LvU2E PSE functions in the context of the LvU2L gene, but the LvU2L PSE functions poorly in the context of the LvU2E gene.
Assuntos
Regulação da Expressão Gênica/genética , Regiões Promotoras Genéticas/genética , RNA Nuclear Pequeno/genética , Ouriços-do-Mar/genética , Animais , Sequência de Bases , Northern Blotting , Análise Mutacional de DNA , Microinjeções , Dados de Sequência Molecular , Sequências Reguladoras de Ácido Nucleico/genética , TATA Box/genéticaRESUMO
Three fibrillar collagen mRNAs, alpha1(I), alpha2(I), and alpha1(III), are coordinately upregulated in the activated hepatic stellate cell (hsc) in liver fibrosis. These three mRNAs contain sequences surrounding the start codon that can be folded into a stem-loop structure. We investigated the role of this stem-loop structure in expression of collagen alpha1(I) reporter mRNAs in hsc's and fibroblasts. The stem-loop dramatically decreases accumulation of mRNAs in quiescent hsc's and to a lesser extent in activated hsc's and fibroblasts. The stem-loop decreases mRNA stability in fibroblasts. In activated hsc's and fibroblasts, a protein complex binds to the stem-loop, and this binding requires the presence of a 7mG cap on the RNA. Placing the 3' untranslated region (UTR) of collagen alpha1(I) mRNA in a reporter mRNA containing this stem-loop further increases the steady-state level in activated hsc's. This 3' UTR binds alphaCP, a protein implicated in increasing stability of collagen alpha1(I) mRNA in activated hsc's (B. Stefanovic, C. Hellerbrand, M. Holcik, M. Briendl, S. A. Liebhaber, and D. A. Brenner, Mol. Cell. Biol. 17:5201-5209, 1997). A set of protein complexes assembles on the 7mG capped stem-loop RNA, and a 120-kDa protein is specifically cross-linked to this structure. Thus, collagen alpha1(I) mRNA is regulated by a complex interaction between the 5' stem-loop and the 3' UTR, which may optimize collagen production in activated hsc's.
Assuntos
Colágeno/genética , Fragmentos de Peptídeos/genética , Células 3T3 , Adenoviridae/genética , Animais , Linhagem Celular , Sequência Conservada , Genes Reporter , Fígado/metabolismo , Luciferases/metabolismo , Masculino , Camundongos , Modelos Genéticos , Plasmídeos , RNA Mensageiro/química , RNA Mensageiro/fisiologia , Proteínas de Ligação a RNA/fisiologia , Ratos , Ratos Sprague-Dawley , Análise de Sequência de RNARESUMO
The hepatic stellate cell (HSC) is the primary cell responsible for the dramatic increase in the synthesis of type I collagen in the cirrhotic liver. Quiescent HSCs contain a low level of collagen alpha1(I) mRNA, while activated HSCs contain about 60- to 70-fold more of this mRNA. The transcription rate of the collagen alpha1(I) gene is only two fold higher in activated HSCs than in quiescent HSCs. In assays using actinomycin D or 5,6-dichlorobenzimidazole riboside collagen alpha1(I) mRNA has estimated half-lives of 1.5 h in quiescent HSCs and 24 h in activated HSCs. Thus, this 16-fold change in mRNA stability is primarily responsible for the increase in collagen alpha1(I) mRNA steady-state level in activated HSCs. We have identified a novel RNA-protein interaction targeted to the C-rich sequence in the collagen alpha1(I) mRNA 3' untranslated region (UTR). This sequence is localized 24 nucleotides 3' to the stop codon. In transient transfection experiments, mutation of this sequence diminished accumulation of an mRNA transcribed from a collagen alpha1(I) minigene and in stable transfections decreased the half-life of collagen alpha1(I) minigene mRNA. Binding to the collagen alpha1(I) 3' UTR is present in cytoplasmic extracts of activated but not quiescent HSCs. It contains as a subunit alphaCP, which is also found in the complex involved in stabilization of alpha-globin mRNA. The auxiliary factors necessary to promote binding of alphaCP to the collagen 3' UTR are distinct from the factors necessary for binding to the alpha-globin sequence. Since alphaCP is expressed in both quiescent and activated HSCs, these auxiliary factors are responsible for the differentially expressed RNA-protein interaction at the collagen alpha1(I) mRNA 3' UTR.
Assuntos
Colágeno/genética , Fígado/citologia , Processamento de Proteína Pós-Traducional , RNA Mensageiro/metabolismo , Animais , Colágeno/metabolismo , Globinas/genética , Globinas/metabolismo , Fígado/metabolismo , Masculino , Proteínas de Ligação a RNA/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
The stability of collagen alpha1(I) mRNA is regulated by its 5' stem-loop, which binds a cytoplasmic protein in a cap-dependent manner, and its 3'-untranslated region (UTR), which binds alphaCP. When cultured in a three-dimensional gel composed of type I collagen, mouse fibroblasts had decreased collagen alpha1(I) mRNA steady-state levels, which resulted from a decreased mRNA half-life. In cells cultured in gel, hybrid mouse-human collagen alpha1(I) mRNA with a wild-type 5' stem-loop decayed faster than the same mRNA with a mutated stem-loop. When the 5' stem-loop was placed in a heterologous mRNA, the mRNA accumulated to a lower level in cells grown in gel than in cells grown on plastic. This suggests that the 5' stem-loop down-regulates collagen alpha1(I) mRNA. Protein binding to the 5' stem-loop was reduced in cells grown in gel, which was associated with destabilization of the collagen alpha1(I) mRNA. In addition to the binding of a cytoplasmic protein, there was also a nuclear binding activity directed to the collagen alpha1(I) 5' stem-loop. The nuclear binding was increased in cells grown in gel, suggesting that it may negatively regulate expression of collagen alpha1(I) mRNA. Binding of alphaCP, a protein involved in stabilization of collagen alpha1(I) mRNA, was unchanged by the culture conditions.
Assuntos
Colágeno/genética , Conformação de Ácido Nucleico , RNA Mensageiro/química , Células 3T3 , Animais , Colágeno/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Camundongos , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismoRESUMO
Activated hepatic stellate cells produce increased type I collagen in hepatic fibrosis. The increase in type I collagen protein results from an increase in mRNA levels that is mainly mediated by increased mRNA stability. Protein-RNA interactions in the 3'-UTR of the collagen alpha1(I) mRNA correlate with stabilization of the mRNA during hepatic stellate cell activation. A component of the binding complex is alphaCP(2). Recombinant alphaCP(2) is sufficient for binding to the 3'-UTR of collagen alpha1(I). To characterize the binding affinity of and specificity for alphaCP(2), we performed electrophoretic mobility shift assays using the poly(C)-rich sequence in the 3'-UTR of collagen alpha1(I) as probe. The binding affinity of alphaCP(2) for the 3'-UTR sequence is approximately 2 nM in vitro and the wild-type 3' sequence binds with high specificity. Furthermore, we demonstrate a system for detecting protein-nucleotide interactions that is suitable for high throughput assays using molecular beacons. Molecular beacons, developed for DNA-DNA hybridization, are oligonucleotides with a fluorophore and quencher brought together by a hairpin sequence. Fluorescence increases when the hairpin is disrupted by binding to an antisense sequence or interaction with a protein. Molecular beacons displayed a similar high affinity for binding to recombinant alphaCP(2) to the wild-type 3' sequence, although the kinetics of binding were slower.
Assuntos
Regiões 3' não Traduzidas/metabolismo , Colágeno/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/metabolismo , Regiões 3' não Traduzidas/química , Regiões 3' não Traduzidas/genética , Células 3T3 , Animais , Pareamento de Bases , Técnicas Biossensoriais , Sondas de DNA/química , Sondas de DNA/genética , Sondas de DNA/metabolismo , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Corantes Fluorescentes , Cinética , Cirrose Hepática/genética , Camundongos , Desnaturação de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/genética , Oligodesoxirribonucleotídeos/metabolismo , Poli C/química , Poli C/genética , Poli C/metabolismo , Ligação Proteica , Estabilidade de RNA , Proteínas Recombinantes de Fusão/metabolismo , Especificidade por Substrato , TermodinâmicaRESUMO
BACKGROUND/AIMS: The aim of the study is detection and evaluation of the abdominal and gastrointestinal infective foci using 99mTc-ciprofloxacin (Laboratory for radioactive isotopes, Vinca). METHODOLOGY: In total 21 patients with clinical suspicion on abdominal or gastrointestinal infection were investigated. In all the patients, planar liver/spleen scintigraphy was performed. Ciprofloxacin chloride (3.5 mg) was mixed with 555 MBq of 99mTc in 3 mL of physiological solution and incubated for 20 min. After slow i.v. injection in a cubital vein, dynamic acquisition (1 f/min) was performed during the first 60 min in the position of interest, followed by static acquisition (500,000 imp) anterior and posterior view, abdomen and pelvis after 1 h and 4 h in all patients. When necessary, additional scintigrams were acquired after 24 h. In all the patients with negative or equivocal findings of planar scintigraphy, emission computerized tomography (SPECT) was performed (60 positions, 6 degrees). Interpretation was made by three independent observers. Additional data were provided using clinical findings, ultrasonography, computed tomography and magnetic resonance imaging, laboratory analyses, and surgical or microbiological confirmation of infection. RESULTS: There were eleven true-positive findings, seven true negative, two were false negative while one was false positive due to intestinal obstruction. Sensitivity was 79%, specificity 91%, positive predictive value 92%, negative predictive value 77%, accuracy 84%. CONCLUSIONS: According to our results, scintigraphy with radiolabeled ciprofloxacin is a useful method for detection and assessment of exact localization of abdominal and gastrointestinal infections.
Assuntos
Abdome/diagnóstico por imagem , Infecções Bacterianas/diagnóstico por imagem , Ciprofloxacina/análogos & derivados , Gastroenteropatias/diagnóstico por imagem , Compostos de Organotecnécio , Infecções Bacterianas/metabolismo , Ciprofloxacina/farmacocinética , Reações Falso-Negativas , Reações Falso-Positivas , Gastroenteropatias/metabolismo , Humanos , Fígado/diagnóstico por imagem , Fígado/metabolismo , Compostos de Organotecnécio/farmacocinética , Valor Preditivo dos Testes , Cintilografia , Sensibilidade e Especificidade , Baço/diagnóstico por imagem , Baço/metabolismo , Distribuição TecidualRESUMO
BACKGROUND AND PURPOSE: Arterial transit time is the time needed for blood to travel from large arteries to capillaries, as estimated from arterial spin-labeling MR imaging. The purpose of this study was to determine whether vascular risk factors and cognitive performance are related to regional differences in cerebral arterial transit time in patients with coronary artery disease who are at risk for cognitive decline. MATERIALS AND METHODS: Arterial transit time was estimated from multiple postlabel delay pseudocontinuous arterial spin-labeling images obtained from 29 men with coronary artery disease. Tests of memory, attention, processing speed, and executive function were administered. Principal component analysis was used to create separate models of cognition and vascular risk, which were related to brain regions through voxelwise analyses of arterial transit time maps. RESULTS: Principal component analysis identified 2 components of vascular risk: 1) "pressor" (age, systolic blood pressure, and pulse pressure) and 2) "obesity" (body fat percentage and body mass index). Obesity was inversely related to arterial transit time in the posterior cingulate, precuneus, lateral occipital cortices, middle temporal gyrus, and frontal pole (P corrected < .05), whereas pressor was not significant. Cognitive scores were factored into a single component. Poor performance was inversely related to precuneus arterial transit time (P corrected < .05). The average arterial transit time in regions identified by obesity was associated with poorer cognitive function (r(2) = 0.21, t = -2.65, P = .01). CONCLUSIONS: Altered cerebral hemodynamics, notably in nodal structures of the default mode network, may be one way that vascular risk factors impact cognition in patients with coronary artery disease.
Assuntos
Encéfalo/irrigação sanguínea , Circulação Cerebrovascular/fisiologia , Transtornos Cognitivos/fisiopatologia , Doença da Artéria Coronariana/fisiopatologia , Hemodinâmica/fisiologia , Idoso , Encéfalo/fisiopatologia , Transtornos Cognitivos/etiologia , Doença da Artéria Coronariana/complicações , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Componente Principal , Fatores de RiscoRESUMO
Six members of a family--the mother, three daughters, and two sons--have a unique syndrome consisting of congenital external ophthalmoplegia, bilateral facial weakness, lingua scrotalis, progressive chorioretinal sclerosis, and an intellectual deficit. Bilateral ptosis and almost complete ophthalmoplegia were found in three of the family members, bilateral facial weakness in two, and Parinaud's syndrome and convergence paresis in one. Electromyographically, a lesion of the lower motor neurons--"nuclear ophthalmoplegia"--was found. Three members of the family had different stages of progressive chorioretinal sclerosis and two had myopia. All the family members had lingua scrotalis, and all of those who had ophthalmoplegia had low IQs. Electroretinographic reactions were subnormal or absent in patients with chorioretinal degeneration. It was concluded that an extensive abiotrophic process, genetically conditioned, was a possibility.
Assuntos
Corioide , Paralisia Facial/genética , Deficiência Intelectual/genética , Oftalmoplegia/genética , Doenças Retinianas/genética , Língua Fissurada/genética , Adolescente , Adulto , Criança , Eletromiografia , Eletrorretinografia , Oftalmopatias/genética , Feminino , Fundo de Olho , Humanos , Masculino , Oftalmoplegia/diagnóstico , Reflexo Pupilar , Doenças Retinianas/diagnóstico , Esclerose/genética , Síndrome , Acuidade VisualRESUMO
Fibrosis results from an increase in the synthesis and deposition of type I collagen. Fibrosis is frequently associated with inflammation, which is accompanied by increased levels of tumor necrosis factor-alpha (TNFalpha) and activation of the transcription factor NF-kappaB. However, several agents known to activate NF-kappaB, such as phorbol 12-myristate 13-acetate (PMA) and TNFalpha, result in decreased expression of type I collagen. Therefore, we directly examined the effects of NF-kappaB on alpha1(I) collagen gene expression in two collagen-producing cells, NIH 3T3 fibroblasts and hepatic stellate cells (HSCs). Transient transfections of NIH 3T3 cells or HSCs using NF-kappaB p50, p65, and c-Rel expression plasmids with collagen reporter gene plasmids demonstrated a strong inhibitory effect on transcription of the collagen gene promoter. Dose-response curves showed that p65 was a stronger inhibitor of collagen gene expression than was NF-kappaB p50 or c-Rel (maximum inhibition 90%). Transient transfections with reporter gene plasmids containing one or two Spl binding sites demonstrated similar inhibitory effects of NF-kappaB p65 on the activity of these reporter genes, suggesting that the inhibitory effects of NF-kappaB p65 are mediated through the critical Spl binding sites in the alpha1(I) collagen gene promoter. Cotransfection experiments using either a super-repressor I[ke]B or Spl partially blocked the inhibitory effects of p65 on collagen reporter gene activity. Coimmunoprecipitation experiments demonstrated that NF-kappaB and Spl do interact in vivo. Nuclear run-on assays showed that NF-kappaB p65 inhibited transcription of the endogenous alpha1(I) collagen gene. Together, these results demonstrate that NF-kappaB decreases transcription of the alpha1(I) collagen gene.
Assuntos
Colágeno/genética , Regulação da Expressão Gênica , NF-kappa B/fisiologia , Isoformas de Proteínas/genética , Proteínas Proto-Oncogênicas c-rel/fisiologia , Células 3T3 , Animais , Sítios de Ligação , Colágeno/biossíntese , Sequência Consenso , Fibrose , Genes Reporter , Vetores Genéticos/genética , Humanos , Proteínas I-kappa B/fisiologia , Fígado/citologia , Cirrose Hepática/genética , Substâncias Macromoleculares , Masculino , Camundongos , NF-kappa B/genética , Subunidade p50 de NF-kappa B , Regiões Promotoras Genéticas/genética , Ligação Proteica , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/fisiologia , Proteínas Proto-Oncogênicas c-rel/genética , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-Dawley , Fator de Transcrição Sp1/metabolismo , Fator de Transcrição RelA , TransfecçãoRESUMO
The regulation of collagen alpha1(I) expression in hepatic stellate cells (HSCs) occurs in a complex fashion that is just beginning to be determined. The presence of regulatory sequences in both the 5' and 3' regions of the mRNA appear to be critical to its regulation in HSCs and are involved in the increased expression of collagen in activated HSCs. The 3' UTR contains a C-rich site that binds alphaCP, a known RNA-binding protein that is responsible for the increased stability of the mRNA in activated HSCs. Given that alphaCP is present in both activated and quiescent HSCs, there must be a mechanism for modifying alpha(CP to bind to the RNA in activated but not quiescent HSCs. The 5' region contains an evolutionary conserved stem-loop region that encompasses the translation initiation codon. This stem-loop can bind protein(s) in activated HSCs in an RNA cap-dependent manner. Such binding, together with the binding of alphaCP to the 3' UTR, can facilitate translation of collagen alpha1(I) mRNA, resulting in increased mRNA steady-state levels and collagen synthesis. A role of alphaCP in activating translation initiation has also been demonstrated. These two mechanisms work together to upregulate collagen alpha1(I) production in activated but not quiescent HSCs.
Assuntos
Colágeno/genética , Cirrose Hepática/genética , Fígado/metabolismo , Regiões 5' não Traduzidas/genética , Animais , Regulação da Expressão Gênica/genética , Humanos , Camundongos , Processamento Pós-Transcricional do RNA/genética , RNA Mensageiro/genéticaRESUMO
There are two gene sets for U2 snRNA in the sea urchin L. variegatus, the U2E gene, which is expressed in oogenesis and early embryogenesis and then silenced, and the U2L gene, which is expressed constitutively. There are four major promoter elements found in both U2 snRNA genes; an essential TATA box at -25 to -30, a proximal element (PSE) at -55 required for expression, an element at -100 necessary for maximal expression, and an upstream activating sequence (UAS) necessary for maximal expression. The elements of the U2E and U2L promoters were interchanged and the effect on the temporal pattern of expression determined after microinjection of the genes into sea urchin zygotes. When the U2E PSE element was introduced into the U2L gene, the temporal pattern of expression of the gene was changed to that of the U2E gene. Converting the U2L gene proximal element into the early U2 gene proximal element by altering 14 nucleotides in the promoter also changed the temporal pattern of expression of the U2L gene. Factors that interact with the U2E PSE, detected by a gel mobility shift assay and DNaseI footprinting, were present in blastula but not late gastrula embryos. In contrast, interchanging the -100 element did not greatly affect the temporal pattern of expression, and factors that interact with the U2E gene -100 box were present in both late gastrula and blastula embryos.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , RNA Nuclear Pequeno/genética , Animais , Sequência de Bases , Núcleo Celular/metabolismo , Gástrula/metabolismo , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Ouriços-do-Mar/embriologia , Fatores de TempoRESUMO
Thirty-eight children with signs of hydrocephalus have been tested for toxoplasmosis. On the basis of clinical data, including roentgenographical and ophthalmological findings, and serological data (Sabin-Feldman and IgM-IFA tests), congenital toxoplasmosis was confirmed in 15 (39%) infants. The diagnosis was established by demonstration of the persistence of Toxoplasma antibodies. In 13 (34%) infants aged 1 to 3 months there was not enough serological data and these were placed in a group of cases with "suspected congenital toxoplasmosis." Ten children with negative serology for toxoplasmosis and with hydrocephalus were considered not to be infected.
Assuntos
Hidrocefalia/congênito , Toxoplasmose Congênita/complicações , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Toxoplasmose Congênita/diagnósticoRESUMO
The mechanisms of Golgi impregnation of neurons has remained enigmatic for decades. Recently, it was suggested that divalent (di)chromate anions play a role in the Golgi impregnation process. Therefore, we incubated slices of (para)formaldehyde-fixed rat brain tissue in solutions of potassium (di)chromate, phosphate, chloride or nitrate at pH 6 or 7. Slices were then immersed in solutions of silver nitrate and processed for light microscopical analysis. At pH 6, dichromate probes resulted in dense and homogeneous impregnation of neuronal cytoplasm (typical impregnation). At pH 7, chromate probes showed solely partial cytoplasmic and heavy nuclear-region neuron impregnation (atypical impregnation). Phosphate probes at pH 6 resulted in typical impregnation, whereas at pH 7 phosphate probes gave atypical impregnation. Both at pH 6 and 7, chloride and nitrate probes did not yield any Golgi impregnation. These findings confirmed the pH-dependence of silver-chromate Golgi impregnation as well as the correctness of corresponding acidic silver-phosphate impregnation. Our study revealed a previously unknown, strong anion-dependence of Golgi impregnation, suggesting that hydrogenated monovalent anions are carriers of the neuron impregnation.
Assuntos
Neurônios/metabolismo , Coloração e Rotulagem/métodos , Animais , Encéfalo/citologia , Cromatos , Formaldeído , Concentração de Íons de Hidrogênio , Masculino , Sondas Moleculares , Neurônios/citologia , Nitratos , Fosfatos , Polímeros , Cloreto de Potássio , Compostos de Potássio , Ratos , Ratos Wistar , Nitrato de Prata , Fixação de TecidosRESUMO
Chronic isolation stress of adult rat males acted increasing gene expression of tyrosine hydroxylase (TH) and neuronal norepinephrine transporter (NET) in the right stellate ganglia, while vesicular monoamine transporter 2 (VMAT2) level remained unchanged. The stress decreased protein level of TH, as well as mRNA levels for NET and VMAT2 in the left stellate ganglia, but expressed no effect on protein levels of these two transporters. These results demonstrate asymmetry in noradrenergic genes in the right and left stellate ganglia during stress and provide molecular evidence to help explain the difference in response to the stress.
Assuntos
Proteínas da Membrana Plasmática de Transporte de Norepinefrina/metabolismo , Isolamento Social , Gânglio Estrelado/metabolismo , Estresse Psicológico/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo , Proteínas Vesiculares de Transporte de Monoamina/metabolismo , Análise de Variância , Animais , Doença Crônica , Lateralidade Funcional/fisiologia , Regulação da Expressão Gênica , Masculino , RNA Mensageiro/metabolismo , Ratos , Ratos WistarRESUMO
The neuropeptide oxytocin has been shown to influence on neuroendocrine function. The aim of the present study was to investigate the effect of peripheral oxytocin treatment on the synthesis, uptake and content of adreno-medullary catecholamine. For this purpose oxytocin (3.6µg/100g body weight, s.c) was administrated to male rats once a day over 14 days. In order to assess the effect of peripheral oxytocin treatment on adreno-medullary catecholamine we measured epinephrine and norepinephrine content and gene expression of tyrosine hydroxylase (TH), norepinephrine transporter (NET) and vesicular monoamine transporter 2 (VMAT2) in the adrenal medulla. Our results show a significant increase of epinephrine (1.7-fold, p<0.05) and norepinephrine (1.5-fold, p<0.05) content in oxytocin treated animals compared to saline treated ones. Oxytocin treatment had no effect either on mRNA or protein level of TH and NET. Under oxytocin treatment the increase in VMAT2 mRNA level was not statistically significant, but it caused a significant increase in protein level of VMAT2 (3.7-fold, p<0.001). These findings indicate that oxytocin treatment increases catecholamine content in the rat adrenal medulla modulating VMAT2 expression.
Assuntos
Medula Suprarrenal/metabolismo , Ocitocina/farmacologia , Proteínas Vesiculares de Transporte de Monoamina/metabolismo , Medula Suprarrenal/efeitos dos fármacos , Animais , Epinefrina/metabolismo , Expressão Gênica , Regulação da Expressão Gênica , Masculino , Norepinefrina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Norepinefrina/genética , Proteínas da Membrana Plasmática de Transporte de Norepinefrina/metabolismo , Ratos , Ratos Wistar , Tirosina 3-Mono-Oxigenase/genética , Tirosina 3-Mono-Oxigenase/metabolismo , Proteínas Vesiculares de Transporte de Monoamina/genéticaRESUMO
Type I collagen is the most abundant protein in human body. Fibrosis is characterized by excessive synthesis of type I collagen in parenchymal organs. It is a leading cause of morbidity and mortality worldwide, about 45% of all natural deaths are attributable to some fibroproliferative disease. There is no cure for fibrosis. To find specific antifibrotic therapy targeting type I collagen, critical molecular interactions regulating its synthesis must be elucidated. Type I and type III collagen mRNAs have a unique sequence element at the 5' end, the 5' stem-loop. This stem-loop is not found in any other mRNA. We cloned LARP6 as the protein which binds collagen 5' stem-loop with high affinity and specificity. Mutation of the 5' stem-loop or knock down of LARP6 greatly diminishes collagen expression. Mice with mutation of the 5' stem-loop are resistant to development of liver fibrosis. LARP6 associates collagen mRNAs with filaments composed of nonmuscle myosin; disruption of these filaments abolishes synthesis of type I collagen. Thus, LARP6 dependent collagen synthesis is the specific mechanism of high collagen expression seen in fibrosis. We developed fluorescence polarization (FP) method to screen for drugs that can inhibit binding of LARP6 to 5' stem-loop RNA. FP is high when LARP6 is bound, but decreases to low levels when the binding is competed out. Thus, by measuring decrease in FP it is possible to identify chemical compounds that can dissociate LARP6 from the 5' stem-loop. The method is simple, fast and suitable for high throughput screening.