RESUMO
The identification of tumour biomarkers that detect the presence of disease using noninvasive diagnostic procedures is a key part of cancer research. We determined in plasma the vesicle-related microRNA (miRNA) expression profile of nonsmall cell lung cancer (NSCLC) and evaluate whether plasma miRNAs can be both discriminating (between patients and healthy controls) and prognostic markers. 365 human miRNAs were analysed by Taqman® low-density arrays (Applied Biosystems, Foster City, CA, USA) in the plasma from 28 NSCLC patients and 20 controls. Five selected miRNAs (let-7f, miR-20b, miR-30e-3p, miR-223 and miR-301) were validated independently by real-time PCR in plasma from 78 NSCLC and 48 controls and correlated with pathologic parameters and survival. Levels of let-7f, miR-20b and miR-30e-3p were decreased in plasma vesicles of NSCLC patients. Moreover, levels of let-7f and miR-30e-3p distinguished between two groups of patients for stage of disease and therefore possibility of surgery. Plasma levels of miR-30e-3p and let-7f were associated with short disease-free survival and overall survival, respectively. NSCLC patients and healthy controls differ in vesicle-related miRNAs in plasma. Levels of let-7f and miR-30e-3p in NSCLC patients are associated with poor outcome. Thus, plasma vesicle-related miRNAs obtained by noninvasive methods could serve as circulating tumour biomarkers of discriminating and prognostic value.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Neoplasias Pulmonares/metabolismo , MicroRNAs/metabolismo , Biomarcadores Tumorais , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Estudos de Casos e Controles , Intervalo Livre de Doença , Reações Falso-Positivas , Feminino , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Marcadores Genéticos , Humanos , Neoplasias Pulmonares/mortalidade , Masculino , PrognósticoRESUMO
The chemokines are a homologous serum protein family characterized by their ability to induce activation of integrin adhesion molecules and leukocyte migration. Chemokines interact with their receptors, which are composed of a single-chain, seven-helix, membrane-spanning protein coupled to G proteins. Two CC chemokine receptors, CCR3 and CCR5, as well as the CXCR4 chemokine receptor, have been shown necessary for infection by several HIV-1 virus isolates. We studied the effect of the chemokine monocyte chemoattractant protein 1 (MCP-1) and of a panel of MCP-1 receptor (CCR2)-specific monoclonal antibodies (mAb) on the suppression of HIV-1 replication in peripheral blood mononuclear cells. We have compelling evidence that MCP-1 has potent HIV-1 suppressive activity when HIV-1-infected peripheral blood lymphocytes are used as target cells. Furthermore, mAb specific for the MCP-1R CCR2 which recognize the third extracellular CCR2 domain inhibit all MCP-1 activity and also block MCP-1 suppressive activity. Finally, a set of mAb specific for the CCR2 amino-terminal domain, one of which mimics MCP-1 activity, has a potent suppressive effect on HIV-1 replication in M- and T-tropic HIV-1 viral isolates. We conjecture a role for CCR2 as a coreceptor for HIV-1 infection and map the HIV-1 binding site to the amino-terminal part of this receptor. This concurs with results showing that the CCR5 amino terminus is relevant in HIV-1 infection, although chimeric fusion of various extracellular domains shows that other domains are also implicated. We discuss the importance of CCR2 structure relative to its coreceptor role and the role of anti-CCR2 receptor antibodies in the prevention of HIV-1 infection.
Assuntos
Infecções por HIV/virologia , HIV-1/fisiologia , Receptores de Quimiocinas , Receptores de Citocinas/fisiologia , Transdução de Sinais , Replicação Viral/fisiologia , Quimiocina CCL2/farmacologia , Infecções por HIV/metabolismo , Humanos , Células Jurkat , Receptores CCR2 , Receptores de Citocinas/agonistas , Replicação Viral/efeitos dos fármacosRESUMO
Here we present the identification and cloning of the NcBSR4 gene, the putative Neospora caninum orthologue to the Toxoplasma gondii TgBSR4 gene. To isolate NcBSR4, genome walking PCR was performed on N. caninum genomic DNA using the expressed sequence tag NcEST3c28h02.y1 sequence, which shares a 44% identity with the TgBSR4 gene, as a framework. Nucleotide sequencing of amplified DNA fragments revealed a single uninterrupted 1227 bp open reading frame that encodes a protein of 408 amino acids with 66% similarity to the TgBSR4 antigen. A putative 39-residue signal peptide was found at the NH2-terminus, followed by a hydrophilic region. At the COOH-terminus, a potential site for a glycosylphosphatidylinositol anchor was identified at amino acid 379. A polyclonal serum against recombinant NcBSR4 protein was raised in rabbits, and immunolabelling demonstrated stage-specific expression of the NcBSR4 antigen in N. caninum bradyzoites produced in vitro and in vivo. Furthermore, RT-PCR analysis showed a slight increase of NcBSR4 transcripts in bradyzoites generated during in vitro tachyzoite-to-bradyzoite stage-conversion, suggesting that this gene is specifically expressed at the bradyzoite stage and that its transcription relies on the switch to this stage.
Assuntos
Neospora/genética , Proteínas de Protozoários/genética , Sequência de Aminoácidos , Animais , Diferenciação Celular , Clonagem Molecular , Regulação da Expressão Gênica , Genes de Protozoários , Dados de Sequência Molecular , Neospora/citologia , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Proteínas RecombinantesRESUMO
We have analyzed the interleukin-4 (IL-4)-triggered mechanisms implicated in cell survival and show here that IL-4 deprivation induces apoptotic cell death but does not modulate Bcl-2 or Bcl-x expression. Since Bcl-x expression is insufficient to ensure cell survival in the absence of IL-4, we speculate that additional molecules replace the antiapoptotic role of Bcl-2 and Bcl-x in an alternative IL-4-triggered pathway. Cell death is associated with Bcl-3 downregulation and Bcl-3 expression blocks IL-4 deprivation-induced apoptosis, suggesting that Bcl-3 acts as a survival factor in the absence of growth factor. To characterize the IL-4-induced regulation of murine Bcl-3 expression, we cloned the promoter of this gene. Sequencing of the promoter showed no TATA box element but did reveal binding sites for AP1, AP1-like, and SP1 transcription factors. Retardation gels showed that IL-4 specifically induces AP1 and AP1-like binding activity and that mutation of these binding sites abolishes the IL-4-induced Bcl-3 promoter activity, suggesting that these transcription factors are important in Bcl-3 promoter transactivation. IL-4 deprivation induces downregulation of Jun expression and upregulation of Fos expression, both of which are proteins involved in the formation of AP1 and AP1-like transcription factors. Overexpression of Jun family proteins transactivates the promoter and restores Bcl-3 expression in the absence of IL-4 stimulation. Taken together, these data describe a new biological role for Bcl-3 and define the regulatory pathway implicated in Bcl-3 expression.
Assuntos
Interleucina-4/farmacologia , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/genética , Linfócitos T/citologia , Fator de Transcrição AP-1/metabolismo , Animais , Apoptose , Proteína 3 do Linfoma de Células B , Sequência de Bases , Divisão Celular , Linhagem Celular , Sobrevivência Celular , Clonagem Molecular , Regulação da Expressão Gênica , Camundongos , Dados de Sequência Molecular , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Receptores de Interleucina-2/genética , Análise de Sequência de DNA , Deleção de Sequência , Fatores de Transcrição , Proteína bcl-XRESUMO
When microcantilevers are used in the dynamic mode, the resonance shift uponmaterial adsorption depends on the position of the adsorbate along the microcantilever. Wehave previously described that the adsorbate stiffness needs to be considered in addition toits mass in order to correctly interpret the resonance shift. Here we describe a method thatallows obtaining the Young's modulus of the adsorbed bacteria derived from themeasurement of the frequency shift when adsorbates are placed close to the clampingregion. As a model system we have used E. Coli bacteria deposited on the cantileversurface by the ink-jet technique. We demonstrate that the correct information aboutadsorbed mass can be extracted by recording the cantilever profile and its resonanceresponse. Also, the position and extent of adsorbates is determined by recording themicrocantilever profile. We use a theoretical model based on the Euler - Bernouilliequation for a beam with both mass and flexural rigidity local increase due to the depositedmaterial.
RESUMO
Here, we identify and clone the NcSAG4 gene, orthologue to the Toxoplasma gondii TgSAG4 gene, and the first reported gene to be expressed specifically during the Neospora caninum bradyzoite stage. To isolate NcSAG4, we designed degenerate oligonucleotides based on the TgSAG4 protein amino acid sequence. A 312-bp DNA fragment was amplified by PCR from N. caninum genomic DNA, whose sequence showed 65% identity to TgSAG4 gene over 257 bp. NcSAG4 gene sequence was obtained by PCR genome walking. Nucleotide sequencing of amplified DNA fragments showed a single uninterrupted 522-bp ORF that encoded a 173-amino-acid protein with a predicted molecular mass of 18,394 Da, with 69% similarity to the TgSAG4 antigen. A 28-residue putative signal peptide was found at the NH2-terminus, followed by a strongly hydrophilic region. An amino acid motif for a phosphatidylinositol glycan anchor was identified at the COOH-terminus. The NcSAG4 protein lacking the putative signal peptide at the NH2-terminus was expressed in Escherichia coli and was recognized in western blot by sera from congenitally infected cattle. A mouse polyclonal anti-rNcSAG4 serum was produced for immunofluorescence studies, and revealed stage-specific NcSAG4 antigen expression in in vitro-cultured bradyzoites. Real-time reverse transcription-PCR analysis with samples from in vitro stage-conversion assay showed increasing levels of NcSAG4 transcript over time, suggesting a developmental upregulation of this gene.
Assuntos
Estágios do Ciclo de Vida/fisiologia , Glicoproteínas de Membrana/genética , Neospora/genética , Proteínas de Protozoários/genética , Sequência de Aminoácidos/genética , Animais , Western Blotting/métodos , Bovinos , Clonagem Molecular/métodos , Sequência Consenso , DNA de Protozoário/química , Eletroforese em Gel de Poliacrilamida/métodos , Técnica Indireta de Fluorescência para Anticorpo/métodos , Expressão Gênica , Glicoproteínas de Membrana/química , Microscopia de Contraste de Fase/métodos , Dados de Sequência Molecular , Neospora/citologia , Reação em Cadeia da Polimerase , Proteínas de Protozoários/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The replication of phi 29 DNA-protein p3 represents a simple model system to study the protein-priming mechanism of initiation of replication. The phi 29 DNA polymerase involved both in the initiation and elongation steps of phi 29 DNA-protein p3 replication, is a very processive enzyme and it is able to produce strand-displacement in the absence of other proteins. To correlate functional and structural domains in the phi 29 DNA polymerase point mutants in the most carboxyl region of amino-acid homology with other DNA polymerases have been constructed. Most of the mutations had a decreased initiation and elongation activity, but normal 3'----5' exonuclease activity, suggesting that this region contributes to the active domain for initiation and elongation. Point and deletion mutants in the terminal protein have allowed the mapping of one DNA-binding region and two DNA-polymerase-binding regions. The viral protein p6, which stimulates the initiation of replication, binds to a set of specific signals present at both phi 29 DNA ends. A good correlation of binding and stimulation of replication has been obtained by using fragments containing phi 29 DNA-terminal sequences and deletion mutants of protein p6. The viral protein p5 has been shown to bind to single-stranded DNA, to protect the latter against nuclease digetion, and to stimulate phi 29 DNA-protein p3 replication in vitro.
Assuntos
Bacteriófagos/genética , Replicação do DNA , Proteínas Virais/farmacologia , Replicação Viral , Bacteriófagos/fisiologia , Catálise , DNA Viral/metabolismo , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Exodesoxirribonuclease V , Exodesoxirribonucleases/metabolismo , Mutação , Homologia de Sequência do Ácido Nucleico , Proteínas Virais/metabolismoRESUMO
Transcription initiation from the Bacillus subtilis phage phi 29 late A3 promoter requires the viral protein p4, a transcriptional activator. Protein p4 binds to a region of the A3 promoter, located between nucleotides -50 and -100 relative to the transcription start site, that presents a sequence-directed curvature. This curvature is enhanced when protein p4 binds to the promoter. A number of deletion mutants at the carboxyl end of protein p4 have been constructed and their behavior as transcriptional activators of the late A3 promoter has been investigated. The binding of these deletion mutants to the late A3 promoter has been analyzed by gel retardation, DNase I footprinting, methylation interference and circular permutation assays. The results suggest that the last 12 amino acid residues of protein p4, six of which are positively charged, although not involved in the specific recognition of the promoter are responsible for part of the bend induced by protein p4 in its binding site. Evidence is presented which suggests that full induction of this curvature is needed for the transcription activation process. A model is proposed for protein p4 interaction with the A3 promoter, in which the bend is induced in two steps: first, two monomers of protein p4 bind to the inverted recognition sequences, subsequent interaction between them generating a bend between these sequences; second, the highly basic carboxyl terminus of protein p4 establishes non-specific electrostatic interactions with the DNA backbone inducing a bend at both ends of the protein p4 binding region.
Assuntos
Bacillus subtilis/genética , Bacteriófagos/genética , DNA Viral/genética , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , Transativadores/metabolismo , Transcrição Gênica , Sequência de Aminoácidos , Sequência de Bases , Deleção Cromossômica , Desoxirribonuclease I , Genes Virais , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Mapeamento de Nucleotídeos , Plasmídeos , Ligação Proteica , Transativadores/genéticaRESUMO
Chemokines are involved in the control of dendritic cell (DC) trafficking, which is critical for the immune response. We have generated DC from human umbilical cord blood CD34+ progenitors cultured with granulocyte-macrophage colony-stimulating factor, tumor necrosis factor alpha (TNF-alpha), and stem cell factor. Using an anti-CCR6 monoclonal antibody, we observed that these cells showed maximum expression of this beta-chemokine receptor when they were immature, as determined by their relatively low expression of several DC maturation markers such as CD1a, CD11c, CD14, CD40, CD80, and CD83. Immature DC responded strongly to macrophage inflammatory protein-3alpha (MIP-3alpha), the CCR6 ligand, in migration and calcium mobilization assays. CCR6 expression decreased in parallel with the DC maturation induced by prolonged TNF-alphaq treatments. Interleukin-4 was also able to decrease CCR6 protein levels. Our findings suggest that the MIP-3alpha/CCR6 interaction plays an important role in the trafficking of immature DC to chemokine production sites such as injured or inflamed peripheral tissues, where DC undergo maturation on contact with antigens.
Assuntos
Células Dendríticas/metabolismo , Regulação para Baixo , Interleucina-4/metabolismo , Proteínas Inflamatórias de Macrófagos , Receptores de Quimiocinas/biossíntese , Fator de Necrose Tumoral alfa/metabolismo , Animais , Anticorpos Monoclonais/biossíntese , Linhagem Celular , Quimiocina CCL20 , Quimiocinas CC/metabolismo , Quimiocinas CC/farmacologia , Células Dendríticas/efeitos dos fármacos , Humanos , Interleucina-4/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Receptores CCR6 , Receptores de Quimiocinas/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Spodoptera/citologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Series of deletions at the amino end of protein p3, the phage phi 29 DNA terminal protein (TP), have been constructed and characterized. Measurements of the activity of the deletion mutants in the formation of the protein p3-dAMP initiation complex in vitro indicate the dispensability of the first 13 amino acids (aa) of the protein. The activity of protein p3 decreased considerably when 17 or more aa were deleted. The results on the in vitro phi 29 DNA replication primed by the p3 deletion mutants correlated very well with those obtained in the formation of the TP-dAMP initiation complex.
Assuntos
Bacteriófagos/fisiologia , Replicação do DNA , Proteínas Virais/fisiologia , Replicação Viral , Adamantano/análogos & derivados , Adamantano/metabolismo , Sequência de Aminoácidos , Bacillus subtilis , Bacteriófagos/genética , DNA Viral/biossíntese , DNA Viral/genética , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Virais/genéticaRESUMO
A set of plasmid expression vectors for cloning of DNA fragments containing open reading frames has been obtained. The plasmids carry the strong leftward promoter of bacteriophage lambda and the translation initiation signals from either the gene ner of bacteriophage Mu or the gene 4 of bacteriophage phi 29. The vectors could overexpress the cloned sequences as fusion peptides at the N terminus with the N-terminal segment of the phi 29 protein p4 or at the C terminus with the Escherichia coli beta-galactosidase from its 8th residue, or both. Alternatively, the cloned sequences could be directed to overproduce proteins in an unfused form. DNA fragments of the hemagglutinin gene from human influenza A virus, have been cloned in one of the plasmid vectors and some potential antigenic determinants have been characterized using monoclonal antibodies.
Assuntos
Escherichia coli/genética , Peptídeos/genética , Plasmídeos , Sequência de Bases , Clonagem Molecular , Escherichia coli/metabolismo , Vetores Genéticos , Hemaglutininas Virais/genética , Dados de Sequência Molecular , Biossíntese Peptídica , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genéticaRESUMO
Series of deletions at the C end of p3, the phage phi 29 DNA terminal protein (TP), have been constructed and characterized. Measurements of the activity of those deletion mutants in the formation of the p3-dAMP initiation complex in vitro indicate the need of an intact C-end for the normal TP primer function in DNA replication. It appears that the region at the C-end between aa 240 and 262 of p3, or part of it, might be essential for the normal TP function.
Assuntos
Deleção Cromossômica , Colífagos/genética , Replicação do DNA , Escherichia coli/genética , Mutação , Proteínas Virais/genética , Sequência de Aminoácidos , Enzimas de Restrição do DNA , Genes , Genes Virais , PlasmídeosRESUMO
A series of internal deletions of gene 3, coding for the phage phi 29 DNA terminal protein, have been constructed and characterized. In addition, a substitution mutant in the sequence corresponding to amino acids (aa) 49-51 was obtained. The priming activity of the substitution mutant protein, in the formation of the protein p3-dAMP initiation complex, was drastically reduced suggesting that some of the aa present at position 49-51 are essential for p3 function. Deletions of 8 to 33 aa, from aa residue 48 towards the N terminus of the substitution mutant, further decreased the priming activity of the protein. The activity of deletion mutants lacking 15 or 21 aa from residue 57 towards the C terminus, and also containing a point mutation at position 56, was greatly reduced, and no activity was seen when 24 aa were lacking.
Assuntos
Bacillus subtilis/genética , Bacteriófagos/genética , Deleção Cromossômica , Genes Virais , Proteínas Virais/genética , Proteínas Estruturais Virais/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Escherichia coli/genética , Dados de Sequência Molecular , Mutação , Sondas de Oligonucleotídeos , Plasmídeos , Regiões Promotoras Genéticas , Proteínas Virais/biossínteseRESUMO
We report the molecular cloning of a novel gene family. The first member of this family was cloned from a mouse lambda gt11 cDNA library using the B92 monoclonal antibody (mAb) raised against stromal cell extracts. This was followed by RACE-PCR using mRNA from the stromal cell line. A 4 kb cDNA was obtained encoding a unique protein sequence of 1258 aa, that we designate stromal antigen (SA)-1. The human SA-1 gene was cloned by homology from a thymus cDNA library and the sequence of the predicted protein was found to be highly homologous to the murine SA-1 (>98.9%). Another cDNA was cloned and the deduced protein (SA-2) was 71% homologous to SA-1. Northern blot and PCR analysis indicated that on the mRNA level the SA-1 gene is expressed in all tissues analyzed and probably encodes a single transcript. The identification of SA-1 protein in tissues and cells required combined immunoprecipitation and Western blotting using a polyclonal antiserum raised against a predicted peptide of SA-1 and the B92 mAb. Using this assay we identified a protein of about 120 kDa in hemopoietic organs. Subcellular fractionation indicated that SA-1 is a nuclear protein. Thus, despite the ubiquitous expression on the mRNA level, the protein was predominantly detected in hemopoietic organs and may therefore be controlled on a post-transcriptional level. The SA-1 gene described in this study is highly conserved between mouse and man. This implies a crucial function for this protein.
Assuntos
Expressão Gênica , Proteínas Nucleares/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Células Cultivadas , Clonagem Molecular , Feminino , Biblioteca Gênica , Humanos , Linfócitos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Proteínas Nucleares/síntese química , Proteínas Nucleares/metabolismo , Peptídeos/síntese química , Peptídeos/imunologia , Reação em Cadeia da Polimerase , Testes de Precipitina , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Recombinação Genética , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Células Estromais , Timo/metabolismo , Transcrição GênicaRESUMO
We have cloned the murine CCR6 receptor and its ligand, the beta-chemokine mMIP-3alpha. Calcium mobilization assays performed with mCCR6 transfectants showed significant responses upon addition of mMIP-3alpha. Murine MIP-3alpha RNA is expressed in thymus, small intestine and colon, whereas mCCR6 RNA is expressed in spleen and lymph nodes. RT-PCR analysis of FACS-sorted lymphoid and antigen presenting cell subsets showed mCCR6 expression mainly in B cells, CD8- splenic dendritic cells and CD4+ T cells. The cloning and functional characterization of the mCCR6 and mMIP-3alpha will allow the study of the role of these proteins in mouse models of inflammation and immunity.
Assuntos
Quimiocinas CC/genética , Quimiocinas CC/farmacologia , Clonagem Molecular , Expressão Gênica , Proteínas Inflamatórias de Macrófagos , Receptores de Quimiocinas/genética , Sequência de Aminoácidos , Animais , Células Apresentadoras de Antígenos/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Linhagem Celular , Quimiocina CCL20 , Quimiocinas CC/síntese química , Quimiocinas CC/química , Biblioteca Gênica , Humanos , Ligantes , Tecido Linfoide/metabolismo , Camundongos , Dados de Sequência Molecular , Especificidade de Órgãos , Sinais Direcionadores de Proteínas , RNA Mensageiro/análise , RNA Mensageiro/genética , Receptores CCR6 , Receptores de Quimiocinas/química , Receptores de Quimiocinas/metabolismo , Alinhamento de Sequência , TransfecçãoRESUMO
Thyroid hormone (triiodothyronine; T3) has been shown to control the expression of beta 1-adrenergic receptors (beta 1-AR) in cardiac myocytes, but not in C6 glioma cells. This cell specificity has been attributed to low expression of T3 receptors and high expression of the c-erbA alpha 2 splice variant that interferes with the action of T3. To check this hypothesis we have expressed the c-erbA/thyroid hormone receptor (TR) alpha 1 gene in C6 glioma cells and investigated their response to thyroid hormone. Cells expressing TR alpha 1, but not wild-type cells, were responsive to T3 as shown by increased expression of mitochrondrial hydroxymethylglutaryl CoA synthase after T3 exposure. However, T3 had no effect on beta 1-AR gene expression in either set of cells. The beta 1-AR mRNA concentrations were, however, altered by retinoic acid (RA) treatment. Retinoic acid caused a rapid up-regulation of beta 1-AR mRNA levels that was blocked by cycloheximide. Retinoic acid did not increase the beta 1-AR gene transcription rate in run-on experiments. These results indicate an indirect post-transcriptional effect of RA. Control of beta 1-AR expression in C6 cells is also exerted at the translational level, because there was no correlation between mRNA and protein induction, as determined by radioligand binding studies. We conclude that lack of responsiveness of the beta 1-AR gene in C6 cells to T3 is not due to high expression of c-erbA alpha 2 but to undefined cell-specific factors.
Assuntos
Regulação Neoplásica da Expressão Gênica/genética , Genes erbA/genética , Glioma/genética , Receptores Adrenérgicos beta 1/genética , Receptores dos Hormônios Tireóideos/genética , Ativação Transcricional/genética , Tretinoína/farmacologia , Animais , Autorradiografia , Northern Blotting , Densitometria , Di-Hidroalprenolol/análise , Di-Hidroalprenolol/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioma/metabolismo , RNA Mensageiro/análise , RNA Mensageiro/genética , Ensaio Radioligante , Ratos , Receptores Adrenérgicos beta 1/metabolismo , Receptores dos Hormônios Tireóideos/metabolismo , Fatores de Tempo , Ativação Transcricional/efeitos dos fármacos , Tri-Iodotironina/farmacologia , Trítio , Células Tumorais CultivadasRESUMO
OBJECTIVE: To study the effect of the trauma of anesthesia and surgery, and their duration, on immune status. PATIENTS AND METHODS: Fifty patients undergoing cholecystectomy were studied in 2 groups. In group A surgery was of short duration ( < 60 min) and in group B surgery was longer ( > 60 min). Immunological analysis were performed at 5 times: t0 (before surgery), t1 (1 h), t2 (24 h), t3 (4 days) and t4 (7 days). RESULTS: Group A patients experienced a non significant decrease in T lymphocytes, activated T lymphocytes and B lymphocytes, with levels returning to normal after 7 days. T-suppression, on the other hand, decreased significantly in the first 24 h, but gradually returned to normal after 7 days. T lymphocytes, activated T lymphocytes and B lymphocytes decreased in group B and regressed after 7 days. The population of B lymphocytes decreased significantly and had not fully recovered 7 days after surgery. CONCLUSIONS: The act of anesthesia/surgery depresses immune response in function of duration, with the effect being greater when surgery lasts longer.
Assuntos
Anestesia , Sistema Imunitário/imunologia , Procedimentos Cirúrgicos Operatórios , Adulto , Feminino , Humanos , Imunidade Celular , Masculino , Pessoa de Meia-IdadeRESUMO
OBJECTIVE: To study changes in the immune system appearing after general and regional (intradural anesthesia). PATIENTS AND METHODS: Fifty patients scheduled for elective surgery to repair inguinal hernias were studied. The patients were randomly assigned to 2 groups: group A patients received general anesthesia and group B patients received intradural anesthesia. Blood was extracted 5 times to determine levels of lymphocytes, activated T cells and B lymphocytes, and T-suppressant activity: t0 (before surgery), t1 (1 h), t2 (4 h), t3 (24 h) y t4 (7 days). RESULTS: The patients in group A showed slight increases in T lymphocytes, activated T lymphocytes and B lymphocytes, whereas T-suppressant activity decreased significantly. All lymphoid population decreased in group B. Baseline levels had recovered in both groups by 7 days after surgery. CONCLUSION: Anesthetic procedures have an effect on immune responses but the impact is only temporary.
Assuntos
Anestesia Geral , Raquianestesia , Sistema Imunitário , Adulto , Humanos , Pessoa de Meia-IdadeRESUMO
We have measured the effect of bacteria adsorption on the resonant frequency of microcantilevers as a function of the adsorption position and vibration mode. The resonant frequencies were measured from the Brownian fluctuations of the cantilever tip. We found that the sign and amount of the resonant frequency change is determined by the position and extent of the adsorption on the cantilever with regard to the shape of the vibration mode. To explain these results, a theoretical one-dimensional model is proposed. We obtain analytical expressions for the resonant frequency that accurately fit the data obtained by the finite element method. More importantly, the theory data shows a good agreement with the experiments. Our results indicate that there exist two opposite mechanisms that can produce a significant resonant frequency shift: the stiffness and the mass of the bacterial cells. Based on the thermomechanical noise, we analyse the regions of the cantilever of lowest and highest sensitivity to the attachment of bacteria. The combination of high vibration modes and the confinement of the adsorption to defined regions of the cantilever allows the detection of single bacterial cells by only measuring the Brownian fluctuations. This study can be extended to smaller cantilevers and other biological systems such as proteins and nucleic acids.
RESUMO
A Neospora caninum 17-19 kDa antigenic protein fraction (p17) in one-dimensional polyacrylamide gel electrophoresis (SDS-PAGE) is the immunodominant antigen recognized by sera from bovines naturally infected by N. caninum. To identify the proteins making up the p17 fraction, we screened a new N. caninum tachyzoite cDNA library with an affinity-purified antibody against p17 (APA17). We isolated several cDNA clones with 100% sequence identity to the NcGRA7 gene. This previously described gene encodes a dense granule protein with an apparent molecular mass of 33 kDa. A second line of evidence emerged through a combined proteomic approach associating two-dimensional PAGE (2D-PAGE) to Western blotting and to mass spectrometry to characterize the p17 fraction. Two acidic immunodominant but minority protein spots were recognized by APA17 and by bovine sera. These antigens of 17 and 33 kDa are respectively composed of 4 and 2 isoforms. Furthermore, p17 isolation by 2D-PAGE and peptide sequencing by tandem mass spectrometry yielded a partial sequence of 17 amino acids, which allowed the putative amino terminal region of the NcGRA7 protein to be identified unambiguously. The NcGRA7 protein, without the putative signal peptide at the NH2-terminus, was cloned and expressed in Escherichia coli and when the purified recombinant protein (rNcGRA7) was analysed by SDS-PAGE and mass spectrometry, 2 bands of 24 and 33 kDa were resolved and identified as NcGRA7. These results demonstrate that the immunodominant 17 kDa antigen of N. caninum is encoded by the NcGRA7 gene.