RESUMO
PURPOSE: To explore FGF1 and miR-143-3p expression in hepatocellular carcinoma (HCC) cells and its related mechanisms. METHODS: Eighty-two HCC patients treated at our hospital from January 2018 to January 2019 were enrolled as Group A, while further 80 healthy people undergoing physical examinations during the same time period were enrolled as Group B. HCC cells and normal human liver cells were purchased, with HepG2 and SMMC-7721 cells transfected with pcDNA3.1-FGF1, si-FGF1, NC, miR-143-3p-inhibitor and miR-143-3p-mimics. FGF1 and miR-143-3p expression was detected by qRT-PCR. The expression of N-cadherin, vimentin, Snail, Slug, E-cadherin and γ-catenin was detected by Western Blotting (WB). Cell proliferation was detected by MTT assay. Cell invasion was detected by Transwell. Cell apoptosis was detected by flow cytometry (FCM). RESULTS: FGF1 was highly expressed but miR-143-3p was poorly expressed in HCC cells. Areas under the curves (AUCs) of the two indicators were > 0.8. The indicators were correlated with the age, gender, tumor invasion, degree of differentiation, tumor location and TNM staging of the patients. Silencing FGF1 and overexpressing miR-143-3p could promote cell apoptosis, inhibit cell growth, cell epithelial-mesenchymal transition (EMT) and the expression of N-cadherin, vimentin, Snail and Slug, and increase the expression of E-cadherin and γ-catenin. Dual luciferase reporter gene assay (DLRGA) confirmed that FGF1 and miR-143-3p had a targeted relationship. The rescue experiment showed that the proliferation, invasion and apoptosis of HepG2 and SMMC-7721 cells in the miR-143-3p-mimics+pcDNA3.1-FGF1 and miR-143-3p-inhibitor+Si-FGF1 groups were not different from those in the miR-NC group. CONCLUSION: Inhibiting FGF1 can upregulate miR-143-3p-mediated Hedgehog signaling pathway, and affect cells' EMT, proliferation and invasion, so FGF1 is expected to become a potential therapeutic target for HCC.
Assuntos
Carcinoma Hepatocelular/metabolismo , Proliferação de Células , Fator 1 de Crescimento de Fibroblastos/metabolismo , Neoplasias Hepáticas/metabolismo , MicroRNAs/metabolismo , Fatores Etários , Apoptose , Área Sob a Curva , Caderinas/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal , Feminino , Fator 1 de Crescimento de Fibroblastos/genética , Citometria de Fluxo , Inativação Gênica , Humanos , Fígado/citologia , Neoplasias Hepáticas/patologia , Masculino , MicroRNAs/antagonistas & inibidores , Pessoa de Meia-Idade , Invasividade Neoplásica , Sondas RNA , Fatores Sexuais , Fatores de Transcrição da Família Snail/metabolismo , Vimentina/metabolismo , gama Catenina/metabolismoRESUMO
In situ hybridization (ISH) is a technique used for the spatial localization of nucleic acids within tissues and cells. It is based on the ability of labeled nucleic acids (probes) to hybridize under the right conditions with the nucleic acids present in fixed biological specimens. In this chapter, we describe protocols for detection of RNA by ISH using digoxigenin (DIG)-labeled probes for Fasciola hepatica adults (in cryosections, given their large size) and for newly excysted juveniles (NEJs, which are ideally suited given their small size for whole-mount ISH). We describe fluorogenic and chromogenic protocols, respectively, but the detection methods can be easily interchanged by using the appropriate enzyme-conjugated antibodies and detection solutions.
Assuntos
Fasciola hepatica/genética , Expressão Gênica/genética , Hibridização In Situ/métodos , Animais , Digoxigenina/química , Técnicas Genéticas , RNA/genética , Sondas RNA/genéticaRESUMO
Bovine viral diarrhea viruses (BVDV), segregated in BVDV-1 and BVDV-2 species, lead to substantial economic losses to the cattle industry worldwide. It has been hypothesized that there could be differences in level of replication, pathogenesis and tissue tropism between BVDV-1 and BVDV-2 strains. Thus, this study developed an in vitro method to evaluate virus competition between BVDV-1 and BVDV-2 strains. To this end the competitive dynamics of BVDV-1a, BVDV-1b, and BVDV-2a strains in cell cultures was evaluated by a PrimeFlow RNA assay. Similar results were observed in this study, as was observed in an earlier in vivo transmission study. Competitive exclusion was observed as the BVDV-2a strains dominated and excluded the BVDV-1a and BVDV-1b strains. The in vitro model developed can be used to identify viral variations that result in differences in frequency of subgenotypes detected in the field, vaccine failure, pathogenesis, and strain dependent variation in immune responses.
Assuntos
Bioensaio , Vírus da Diarreia Viral Bovina Tipo 1/genética , Vírus da Diarreia Viral Bovina Tipo 2/genética , Células Epiteliais/virologia , RNA Viral/genética , Animais , Doença das Mucosas por Vírus da Diarreia Viral Bovina/diagnóstico , Doença das Mucosas por Vírus da Diarreia Viral Bovina/virologia , Bovinos , Linhagem Celular , Coinfecção , Vírus da Diarreia Viral Bovina Tipo 1/classificação , Vírus da Diarreia Viral Bovina Tipo 1/isolamento & purificação , Vírus da Diarreia Viral Bovina Tipo 1/metabolismo , Vírus da Diarreia Viral Bovina Tipo 2/classificação , Vírus da Diarreia Viral Bovina Tipo 2/isolamento & purificação , Vírus da Diarreia Viral Bovina Tipo 2/metabolismo , Cães , Células Epiteliais/patologia , Feminino , Células Madin Darby de Rim Canino , Gravidez , RNA/genética , RNA/metabolismo , Sondas RNA/genética , Sondas RNA/metabolismo , RNA Viral/metabolismo , Tropismo Viral , Replicação ViralAssuntos
DNA/genética , Código Genético , Genoma Humano/genética , Sondas RNA/química , RNA/química , Análise de Sequência de DNA/métodos , Animais , DNA/química , DNA/história , História Antiga , Humanos , Múmias , Peru , RNA/síntese química , RNA/genética , Sondas RNA/síntese química , Sondas RNA/genéticaRESUMO
Sugarcane is an important sugar and energy crop that can be used efficiently for biofuels production. The development of sugarcane cultivars tolerant to drought could allow for the expansion of plantations to sub-prime regions. Knowledge on the mechanisms underlying drought responses and its relationship with carbon partition would greatly help to define routes to increase yield. In this work we studied sugarcane responses to drought using a custom designed oligonucleotide array with 21,901 different probes. The oligoarrays were designed to contain probes that detect transcription in both sense and antisense orientation. We validated the results obtained using quantitative real-time PCR (qPCR). A total of 987 genes were differentially expressed in at least one sample of sugarcane plants submitted to drought for 24, 72 and 120 h. Among them, 928 were sense transcripts and 59 were antisense transcripts. Genes related to Carbohydrate Metabolism, RNA Metabolism and Signal Transduction were selected for gene expression validation by qPCR that indicated a validation percentage of 90%. From the probes presented on the array, 75% of the sense probes and 11.9% of the antisense probes have signal above background and can be classified as expressed sequences. Our custom sugarcane oligonucleotide array provides sensitivity and good coverage of sugarcane transcripts for the identification of a representative proportion of natural antisense transcripts (NATs) and sense-antisense transcript pairs (SATs). The antisense transcriptome showed, in most cases, co-expression with respective sense transcripts.
Assuntos
Secas , Saccharum/genética , Saccharum/metabolismo , Aclimatação/genética , Metabolismo dos Carboidratos/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Análise de Sequência com Séries de Oligonucleotídeos , Sondas RNA/genética , RNA Antissenso/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Plantas/genética , RNA de Plantas/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/genética , TranscriptomaRESUMO
Pre-mRNA splicing is an essential step in the post-transcriptional gene expression control involving protein-splicing factors like U2AF, which is exported to the cytoplasm and implicated in additional cellular functions. Identification of U2AF-associated mRNAs under native conditions was performed by immunoprecipitation and hybridization to Affymetrix GeneChip. Normalization and gene selection methods were performed, but the results were not reliable as they were different for different procedures, mainly because more than 20% of the mRNAs detected are differently enriched and the common normalization methods are based on small differences between them. We implemented a background correction method inspired in a non-specific hybridization method used for pre-processing data from ChIP-Chip technology. In this work, linear regression models are used to model in each array the non-specific hybridization, accounting for interactions between each three consecutive nucleotides into the probe sequence. Every probe intensity on the array was standardized using its predicted intensity and the probes' variance for similar predicted intensities. The standardized probe intensity values showed no need for further normalization and could be directly compared. We propose a probe set score, and a probe set enrichment value (ENRval) and its respective p-value for gene enrichment selection.
Assuntos
Biologia Computacional/métodos , Modelos Lineares , Hibridização de Ácido Nucleico/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Sondas de Oligonucleotídeos/genética , Sondas RNA , RNA/genética , Algoritmos , Regulação da Expressão Gênica , Imunoprecipitação/métodos , Sondas de Oligonucleotídeos/metabolismo , RNA/metabolismo , Sondas RNA/genética , Sondas RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
A polyclonal antibody was used to detect the expression of the homeodomain protein Lim1 (Lhx1) in embryos of Xenopus laevis, Engystomops randi, Colostethus machalilla and Gastrotheca riobambae. These frogs belong to four separate families, and have differences in their modes of reproduction and developmental rates. The expression of Lim1 in embryos of these frogs resembled the X. laevis expression pattern. Thus, the dorsal blastopore lip, axial mesoderm, pronephros and certain cells of the central nervous system were Lim1-positive in embryos of all frogs. There were, however, time differences; thus, in the mid-gastrula of the rapidly developing embryos of X. laevis and E. randi, the Lim1 protein was simultaneously detected in the prechordal plate (head organizer) and notochord (trunk organizer). In contrast, only the prechordal plate was Lim1-positive during gastrulation in the slow developing embryos of C. machalilla. The notochord elongated and became Lim1-positive after closure of the blastopore in C. machalilla and G. riobambae embryos. The prechordal plate of G. riobambae embryos could not be clearly detected, as the Lim1-signal remained around the blastopore during gastrulation. These observations indicate that the timing of gene expression at the dorsal blastopore lip in embryos of slow developing frogs differs from that of X. laevis. Moreover, the comparison shows that the developmental processes of the head and trunk organizers are basically separable and become dissociated in embryos of the slow developing frog, C. machalilla.
Assuntos
Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Proteínas de Homeodomínio/metabolismo , Reprodução/fisiologia , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologia , Xenopus laevis/metabolismo , Animais , Gástrula/metabolismo , Proteínas de Homeodomínio/genética , Hibridização In Situ , Proteínas com Homeodomínio LIM , Notocorda/metabolismo , Sondas RNA , Fatores de TranscriçãoRESUMO
A revisão sistemática teve como objetivo avaliar a efetividade dos testes de ácido nucleico no rastreio da C. trachomatis. A maioria dos estudos foi localizada via internet, entretanto, alguns deles foram encontrados em revistas que abordavam o tema e mediante contato com especialistas. Os artigos foram selecionados após criteriosa avaliação crítica da força de evidência científica, obedecendo às regras da Associação Médica Brasileira e do Conselho Federal de Medicina, além dos critérios de Irwig, para análise qualitativa dos artigos. A revisão incluiu todos os estudos publicados a partir de 1990 que avaliavam testes de ácido nucleico em mulheres sexualmente ativas, assintomáticas e que tivessem sido submetidas à avaliação clinica e a testes moleculares. Os testes de ácido nucleico que utilizavam sondas de RNA e amplificação de DNA (PCR) foram comparados à cultura (padrão-ouro) com o intuito de determinar se seriam método de diagnóstico adequado para o rastreio da infecção. Após análise qualitativa, foram selecionados 12 estudos, mas não foi possível realizar avaliação quantitativa dos mesmos devido à heterogeneidade dos dados. A efetividade e os benefícios dos testes de ácido nucleico justificam estudos de custo-efetividade, com o intuito de avaliar o impacto do rastreio universal na redução das complicações advindas da infecção clamidiana
This systematic review aims at evaluating the effectiveness of the nucleic acid test for detection of C. trachomatis. Most of the studies were searched electronically and key journals were hand-searched. Further studies were identified in the internet and by contacting experts in the field. The articles were selected after careful critical evaluation of the strength of scientific evidence, according to the rules of the Brazilian Medical Association and the Federal Council of Medicine, besides the Irwig's criteria for qualitative analysis of article The review included all studies published from 1990 onward that evaluated nucleic acid tests in asymptomatic, young and sexually active women that have been subjected to clinical evaluation and molecular testing. The nucleic acid tests taken with the use of probes of RNA and amplification (PCR) were compared to culture (gold standard) in order to determine if a method of diagnosis would be appropriate for screening of infection. After the qualitative analysis, we selected 12 studies; it has not been possible to perform their quantitative evaluation due to the heterogeneity of data. The effectiveness and benefits of DNA testing justify the cost-effectiveness studies in order to assess the impact of universal screening in reducing the complications that arise from chlamydial infection
Assuntos
Humanos , Feminino , Ácidos Nucleicos , Cervicite Uterina/diagnóstico , Chlamydia trachomatis/isolamento & purificação , Infecções por Chlamydia/complicações , Infecções por Chlamydia/diagnóstico , Infecções por Chlamydia/microbiologia , Programas de Rastreamento/métodos , Sondas RNA , Técnicas e Procedimentos DiagnósticosRESUMO
Cellular nucleic acid binding protein (CNBP) is a small single-stranded nucleic acid binding protein made of seven Zn knuckles and an Arg-Gly rich box. CNBP is strikingly conserved among vertebrates and was reported to play broad-spectrum functions in eukaryotic cells biology. Neither its biological function nor its mechanisms of action were elucidated yet. The main goal of this work was to gain further insights into the CNBP biochemical and molecular features. We studied Bufo arenarum CNBP (bCNBP) binding to single-stranded nucleic acid probes representing the main reported CNBP putative targets. We report that, although bCNBP is able to bind RNA and single-stranded DNA (ssDNA) probes in vitro, it binds RNA as a preformed dimer whereas both monomer and dimer are able to bind to ssDNA. A systematic analysis of variant probes shows that the preferred bCNBP targets contain unpaired guanosine-rich stretches. These data expand the knowledge about CNBP binding stoichiometry and begins to dissect the main features of CNBP nucleic acid targets. Besides, we show that bCNBP presents a highly disordered predicted structure and promotes the annealing and melting of nucleic acids in vitro. These features are typical of proteins that function as nucleic acid chaperones. Based on these data, we propose that CNBP may function as a nucleic acid chaperone through binding, remodeling, and stabilizing nucleic acids secondary structures. This novel CNBP biochemical activity broadens the field of study about its biological function and may be the basis to understand the diverse ways in which CNBP controls gene expression.
Assuntos
DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Quadruplex G , Chaperonas Moleculares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Regiões 5' não Traduzidas/química , Regiões 5' não Traduzidas/metabolismo , Animais , Sequência de Bases , Bufo arenarum , Sondas de DNA/química , Sondas de DNA/metabolismo , DNA de Cadeia Simples/química , Proteínas de Ligação a DNA/química , Embrião não Mamífero/química , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Guanosina/química , Guanosina/metabolismo , Humanos , Dados de Sequência Molecular , Ligação Proteica , Biossíntese de Proteínas , Sondas RNA/química , Sondas RNA/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Xenopus laevis/metabolismo , Dedos de ZincoRESUMO
Fluorescence in situ hybridization (FISH) using telomeric and ribosomal sequences was performed in four species of toad genus Chaunus: C. ictericus, C. jimi, C. rubescens and C. schneideri. Analyses based on conventional, C-banding and Ag-NOR staining were also carried out. The four species present a 2n = 22 karyotype, composed by metacentric and submetacentric chromosomes, which were indistinguishable either after conventional staining or banding techniques. Constitutive heterochromatin was predominantly located at pericentromeric regions, and telomeric sequences (TTAGGG)(n) were restricted to the end of all chromosomes. Silver staining revealed Ag-NORs located at the short arm of pair 7, and heteromorphism in size of NOR signals was also observed. By contrast, FISH with ribosomal probes clearly demonstrated absence of any heteromorphism in size of rDNA sequences, suggesting that the difference observed after Ag-staining should be attributed to differences in chromosomal condensation and/or gene activity rather than to the number of ribosomal cistrons.
Assuntos
Bufonidae/genética , DNA Ribossômico/genética , Região Organizadora do Nucléolo/genética , Telômero/genética , Animais , Bufonidae/sangue , Bufonidae/classificação , Bandeamento Cromossômico , Cromossomos , Análise Citogenética , Sondas de DNA , Hibridização in Situ Fluorescente , Cariotipagem , Região Organizadora do Nucléolo/ultraestrutura , Sondas RNA , Coloração pela Prata , Telômero/ultraestruturaRESUMO
The chromosomal localization of ribosomal DNA (rDNA) was studied in polytene and diploid tissues of four sciarid species, Trichosia pubescens, Rhynchosciara americana, R. milleri and Schwenkfeldina sp. While hybridization to mitotic chromosomes showed the existence of a single rDNA locus, ribosomal probes hybridized to more than one polytene chromosome region in all the species analyzed as a result of micronucleolar attachment to specific chromosome sites. Micronucleoli are small, round bodies containing transcriptionally active, probably extrachromosomal rDNA. In T. pubescens the rDNA is predominantly localized in chromosome sections X-10 and X-8. In R. americana the rDNA is frequently found associated with centromeric heterochromatin of the chromosomes X, C, B and A, and also with sections X-1 and B-13. Ribosomal probes in R. milleri hybridized with high frequency to pericentric and telomeric regions of its polytene complement. Schwfenkfeldina sp. displays a remarkably unusual distribution of rDNA in polytene nuclei, characterized by the attachment of micronucleoli to many chromosome regions. The results showed that micronucleoli preferentially associate with intercalary or terminal heterochromatin of all sciarid flies analyzed and, depending on the species, are attached to a few (Trichosia), moderate (Rhynchosciara) or a large (Schwenkfeldina sp.) number of polytene chromosome sites.
Assuntos
DNA Ribossômico/genética , DNA Ribossômico/metabolismo , Dípteros/genética , Dípteros/metabolismo , Animais , Nucléolo Celular/genética , Nucléolo Celular/metabolismo , Cromossomos/genética , Cromossomos/metabolismo , Feminino , Hibridização in Situ Fluorescente , Masculino , Sondas RNARESUMO
A técnica de hibridização in situ (ISH) tem sido usada para identificar mRNA (ou DNA) em amostras de tecido de material humano e animal. Embora uma série de protocolos para essa técnica seja utilizada, as descrições não são bem detalhadas. O objetivo deste trabalho é descrever a reação de hibridização in situ em tecido fresco e sua aplicação em patologia, tornando mais compreensível essa técnica tão importante, que possibilita observar a localização tecidual e a expressão temporal e espacial dos transcritos de um determinado gene (mRNA). Resultados de reações com as ribossondas PITX1, SHH e WNT-5A, realizadas em amostras de tecido congelado, são apresentados.
In situ hybridization (ISH) has been used to identify mRNA (or DNA) in fresh tissue samples of humans and animals. Several protocols describing this technique are available, although its description is not usually detailed enough. The present work describes in situ hybridization reaction on fresh tissue in a way to make understandable this important technique, which allows verifying the cellular localization, and the spatial and temporal expression, of gene transcripts (mRNA). Results with PITX1, TGIF, SHH and WNT-5A riboprobes, in fresh tissue samples, are presented.
Assuntos
Hibridização In Situ/métodos , Sondas RNA , Fixação de Tecidos , Técnicas HistológicasRESUMO
Here we analyse the structural organisation and expression of the zebrafish cellular nucleic acid-binding protein (zCNBP) gene and protein. The gene is organised in five exons and four introns. A noteworthy feature of the gene is the absence of a predicted promoter region. The coding region encodes a 163-amino acid polypeptide with the highly conserved general structural organisation of seven CCHC Zn knuckle domains and an RGG box between the first and the second Zn knuckles. Although theoretical alternative splicing is possible, only one form of zCNBP is actually detected. This form is able to bind to single-stranded DNA and RNA probes in vitro. The analysis of zCNBP developmental expression shows a high amount of CNBP-mRNA in ovary and during the first developmental stages. CNBP-mRNA levels decrease while early development progresses until the midblastula transition (MBT) stage and increases again thereafter. The protein is localised in the cytoplasm of blastomeres whereas it is mainly nuclear in developmental stages after the MBT. These findings suggest that CNBP is a strikingly conserved single-stranded nucleic acid-binding protein which might interact with maternal mRNA during its storage in the embryo cell cytoplasm. It becomes nuclear once MBT takes place possibly in order to modulate zygotic transcription and/or to associate with newly synthesised transcripts.
Assuntos
Proteínas de Ligação a RNA/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Processamento Alternativo , Sequência de Aminoácidos , Animais , Clonagem Molecular , DNA Complementar/química , DNA Complementar/genética , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genes/genética , Imuno-Histoquímica , Hibridização In Situ , Masculino , Dados de Sequência Molecular , Ovário/embriologia , Ovário/crescimento & desenvolvimento , Ovário/metabolismo , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Sondas RNA/genética , Sondas RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Peixe-Zebra/embriologia , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/metabolismoRESUMO
The KAL1 gene has a closely related nonfunctional pseudogene on the Y chromosome; a high degree of X-Y sequence similarity is observed. Some individuals present a T to C substitution at position 1833 (exon 12). Because this nucleotide differs in the X (thymine) and in the Y (cytosine) chromosome, we investigated if this was truly a polymorphism, or if in some cases the Y sequence had been amplified. The complete sequence of exon 12 of KAL1 was analyzed in 11 Kallmann Syndrome (KS) males, in 50 normal males, in 50 normal females, and in 16 patients with Ullrich-Turner Syndrome (UTS). Nucleotide 1833 was found in a heterozygous or a homozygous state in KS, normal males and normal females; UTS patients were always homozygous. Of the 61 males, 17 were heterozygous, while 11 were TT and 33 were CC. With these observations we can not assure whether these patients present a "real" polymorphism. Besides, all males were heterozygous in nucleotides 1678, 1694, 1699, 1708 and 1825, whilst females were homozygous; and in these positions, KAL1 also differs from its pseudogene. These results indicate that we are identifying the X and the Y nucleotide and these variants are not polymorphisms. Sequence variations may be pseudogene products rather than true polymorphisms, so we should always determine if the position where the variation is located differs between KAL1 and its pseudogene, because it has been suggested that the presence of various polymorphisms in affected individuals could be the cause of KS.
Assuntos
Proteínas da Matriz Extracelular/genética , Proteínas do Tecido Nervoso/genética , Adulto , Cromossomos Humanos X , Cromossomos Humanos Y , Éxons/genética , Feminino , Hormônios/sangue , Humanos , Síndrome de Kallmann/genética , Masculino , Dados de Sequência Molecular , Polimorfismo Genético/genética , Sondas RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Síndrome de Turner/genéticaRESUMO
The present study was undertaken to determine tonin expression and activity in rat heart presenting isoproterenol-induced hypertrophy. Renin, angiotensin-converting enzyme (ACE), and angiotensinogen (AG) expression were also determined. Wistar rats were treated with isoproterenol for 7 days (5 mg x kg(-1) x day(-1) sc). For untreated animals, the levels of tonin-specific activity in the atrium were 2.6- and 5.5-fold higher than those of the left and right ventricle, respectively. After treatment, the levels of tonin-specific activity increased twofold in the atrium but did not change in the ventricles. Renin expression was not detectable in these structures, and ACE expression levels did not change with treatment. AG expression was detected in the left ventricle at very low levels compared with the atrium and increased significantly only in the hypertrophied atrium (1.8-fold). Tonin mRNA was not detected in the ventricle but was found at low levels in the atrium, which increased after isoproterenol treatment. Our results permit us to conclude that tonin may play a role in the process of heart hypertrophy in the rat.
Assuntos
Cardiomegalia/metabolismo , Miocárdio/metabolismo , Calicreínas Teciduais/metabolismo , Agonistas Adrenérgicos beta , Angiotensina II/biossíntese , Animais , Fator Natriurético Atrial/biossíntese , Cardiomegalia/induzido quimicamente , Progressão da Doença , Regulação da Expressão Gênica , Isoproterenol , Masculino , Tamanho do Órgão/efeitos dos fármacos , Sondas RNA , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Calicreínas Teciduais/biossínteseRESUMO
O objetivo deste artigo é revisar e comentar as vantagens e desvantagens dos diferentes tipos de testes de detecçäo de Chlamydia trachomatis na rotina de laboratórios clínicos, com ênfase nas técnicas de amplificaçäo. A Chlamydia trachomatis é considerada a bactéria sexualmente transmissível mais freqüente em países desenvolvidos e de grande impacto no sistema reprodutivo das mulheres. É o agente causador de doenças do trato urogenital, linfogranuloma venéreo (LGV), tracoma, conjutivite de inclusäo e pneumonia no recémðnascido. Um dos fatores de risco para a infecçäo é a prática sexual entre adolescentes. A recorrência das infecções é comum. Episódios sucessivos de infecçäo aumentam o risco de desenvolver seqüelas e a chance de contrair a infecçäo pelo vírus da imunodeficiência humana. O dignóstico da infecçäo pela Chlamydia trachomatis ainda é crítico, devido à freqüência de infecções assintomáticas. As técnicas de amplificaçäo de ácidos nucléicos permitem utilizar urina para a detecçäo da clamídia, simplificando a coleta. Apresentam maior sensibilidade do que a cultura e do que os testes mais utilizados, como a imunofluorescência direta e o enzimaimunoensaio. A cultura celular, utilizada como padräoðouro, tem especificidade de 100 por cento e sensibilidade de 70 por cento a 85 por cento. De acordo com o Centers for Disease Control (CDC), um diagnóstico é considerado definitivo quando positivo em cultura ou em pelo menos dois testes näoðculturais distintos. Os testes de amplificaçäo säo mais dispendiosos do que os demais testes näoðculturais, mas de menor custo que a cultura
Assuntos
Humanos , Técnicas Bacteriológicas , Chlamydia trachomatis , Técnicas de Laboratório Clínico , Técnica Direta de Fluorescência para Anticorpo , Técnica Indireta de Fluorescência para Anticorpo , Técnicas Imunoenzimáticas , Infecções por Chlamydia/diagnóstico , Infecções por Chlamydia/microbiologia , Técnica de Amplificação ao Acaso de DNA Polimórfico , Sondas RNA , Sensibilidade e EspecificidadeRESUMO
Background: Restenosis post stenting is due to the deposit of extracellular matrix, mainly collagen in the neointima. Controversy exists regarding if collagen is generated locally or by immigration from the adventitia. Aim: To study the fibrocellular response after stent implantation in rabbit iliac arteries. To observe, by immunohistochemistry and in situ hybridization, if collagen type I mRNA is expressed in the neointima, in the media or in the adventitia. Material and methods: Thirty eight white rabbits (New Zealand) of 4 kg received an hypercholesterolemic diet during 1 month. After this period, in all but 6 of them, an angioplasty with stent implantation was performed via right carotid artery in both iliac arteries, using a 1:1.3 relationship regarding the reference vessel. Angiograms were performed at day 0, 4, 21, and 40, followed by paraffin fixation of the injured segments, immunohistochemistry for a-actin and in situ hybridization to detect procollagen type I (a1R1) mRNA. Results: No hybridization was observed in non injured arteries or at day 0 (n= 6). Expression of a1R1 mRNA was observed in the neointima starting at day 4 after stenting (n= 8). At day 21 (n= 8) hybridization of procollagen type I was not only observed in the neointima, but also in the media, which became equally intense in both areas. At day 40 (n= 6) hybridization was observed similarly in the media and adventitia. Conclusions: In this model, hybridization of procollagen type I started in the neointima, then involved the media and finally the adventitia. This finding might be useful for designing therapies to be delivered locally at the end of an angioplasty to prevent collagen deposition in the neointima
Assuntos
Animais , Coelhos , Angioplastia , Colágeno/biossíntese , Oclusão de Enxerto Vascular/fisiopatologia , Sondas RNA , Modelos Animais de Doenças , Imuno-Histoquímica/métodosRESUMO
The hepatitis B virus protein HBx has been implicated in the development of liver cancer. It has been shown that the HBx protein is able to bind to single-stranded DNA in a specific manner. This DNA binding activity might be relevant for HBx oncogene character. To study the HBx interaction with nucleic acids in more detail we expressed full-length HBx as well as several N- and C-terminally truncated HBx proteins as 6xHis and GST-fusions in E. coli. Using a gel shift assay, we were able to demonstrate that all of the truncated HBx proteins have the ability to bind to an AU-rich RNA. The affinity of GST-HBx #3 (residues 80-142) was an order of magnitude higher than that of GST-HBx #2 (residues 5-79), indicating that a high affinity RNA binding site is located in HBx C-terminal half. AUF1 is the protein ligand that binds to AU-rich RNA regions present in certain proto-oncogene mRNAs and causes their rapid degradation. By a competitive binding experiment of AUF1 and HBx to the AU-rich RNA oligonucleotide, we show that HBx is able to displace AUF1 from its binding site on the RNA oligonucleotide. This new aspect of HBx function is discussed in the context of cellular transformation.
Assuntos
Antígenos da Hepatite B/metabolismo , Vírus da Hepatite B/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo D , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Transativadores/metabolismo , Sítios de Ligação , Ligação Competitiva , Cromatografia de Afinidade , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Enteropeptidase/metabolismo , Escherichia coli , Expressão Gênica , Vetores Genéticos , Antígenos da Hepatite B/genética , Antígenos da Hepatite B/isolamento & purificação , Vírus da Hepatite B/genética , Hepatite B Crônica/epidemiologia , Ribonucleoproteína Nuclear Heterogênea D0 , Reação em Cadeia da Polimerase , Ligação Proteica , Sondas RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/isolamento & purificação , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Deleção de Sequência , Transativadores/genética , Transativadores/isolamento & purificação , Proteínas Virais Reguladoras e AcessóriasRESUMO
The targeting of polypeptides to restricted cytoplasmic domains by means of mRNA sorting is a widespread phenomena utilized by many cell types. In the central nervous system, in situ hybridization analysis has shown previously that the mRNAs encoding several myelin-specific proteins are specifically located within the myelinating processes of oligodendrocytes. Here, by means of biochemical and subcellular fractionation methods, we show that a myelin fraction is selectively enriched in those mRNAs. The four major myelin basic protein (MBP) mRNAs that arise by alternative splicing of exons II and VI of the MBP gene are concentrated in this subcellular fraction. Furthermore, an interaction of MBP and MOBP 81A mRNAs with the cytoskeleton was observed. This interaction might serve to mediate the anchoring of these messengers after translocation to the subcellular site of translation.
Assuntos
Citoesqueleto/metabolismo , Proteínas da Mielina/biossíntese , RNA Mensageiro/biossíntese , Frações Subcelulares/metabolismo , Processamento Alternativo , Animais , Northern Blotting , Western Blotting , Membrana Celular/metabolismo , Citoesqueleto/química , Éxons/fisiologia , Immunoblotting , Camundongos , Camundongos Endogâmicos C57BL , Proteínas da Mielina/análise , Bainha de Mielina/metabolismo , Oligodendroglia/metabolismo , Pressão Osmótica , Plasmídeos , Sondas RNA , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Frações Subcelulares/químicaRESUMO
We have developed RNA probes for the direct identification of wild poliovirus isolates by blot hybridization. The probes are complementary to sequences of the first 30 to 32 codons of VP1, which evolve more extensively (approximately 1.5-fold) than the rest of VP1. To illustrate our general approach, we describe the design of probes specific to each of four major genotypes recently endemic (1981 to 1991) to the Americas: Andean type 1, Brazil type 1, Brazil type 3, and Central America-Mexico type 3. A wild isolate of each genotype was selected according to molecular and epidemiologic criteria to be representative of the principal lineages in circulation. Variable VP1 sequences of the representative isolates were amplified by the reverse transcriptase PCR and were inserted into a plasmid vector containing a T7 promoter. The in vitro transcripts, labeled with digoxigenin, served as probes. These formed stable hybrids only with RNAs of isolates of the corresponding genotypes. Hybrids were detected by a sensitive chemiluminescence assay, capable under normal diagnostic conditions of detecting specific wild poliovirus sequences in samples containing up to a 100-fold excess of Sabin vaccine strain-related sequences of the same serotype.