RESUMO
We investigated whether bacterial DNA (CpG-DNA)-induced IL-1beta expression in the mouse hypothalamus is mediated via afferent vagus nerve. Subdiaphragmatic vagotomy did not modify the CpG-DNA (i.p.)-induced IL-1beta expression in the hypothalamus, indicating that CpG-DNA-induced IL-1beta expression is independent of the afferent vagus nerve originating from the subdiaphragmatic organs. On the other hand, we observed the Toll-like receptor 9 mRNA expression in the hypothalamus, suggesting that circulating CpG-DNA acts directly in the brain.
Assuntos
DNA Bacteriano/farmacologia , Expressão Gênica/efeitos dos fármacos , Hipotálamo/efeitos dos fármacos , Interleucina-1/metabolismo , Nervo Vago/fisiologia , Animais , Comportamento Animal/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Ingestão de Alimentos/efeitos dos fármacos , Expressão Gênica/fisiologia , Hipotálamo/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Tamanho do Órgão/efeitos dos fármacos , RNA Mensageiro/biossíntese , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Sincalida/farmacologia , Estômago , Receptor Toll-Like 9 , Vagotomia/métodos , Nervo Vago/cirurgiaRESUMO
Mammalian interleukin-1beta (IL-1beta) is a secretory cytokine lacking a signal peptide, which does not follow the classical endoplasmic reticulum to Golgi pathway of secretion. Its post-translational processing by IL-1beta-converting enzyme (ICE) and subsequent release from activated macrophages requires ATP acting on P2X7 receptors. Little information is available on the production and release of fish IL-1beta, but the IL-1beta gene sequences reported to date lack a conserved ICE recognition site. We show for the first time that lipopolysaccharide (LPS)/macrophage-activating factor/bacterial DNA (VaDNA)-primed immune cells of the marine fish gilthead seabream (Sparus aurata) accumulate intracellular IL-1beta as a approximately 30 kDa polypeptide (proIL-1beta). The combination of LPS and VaDNA was found to be synergistic, suggesting that each ligand is recognized by a different pattern recognition receptor. More importantly, addition of extracellular ATP does not promote IL-1beta secretion by immune cells and fails to induce phosphatidylserine flip. In contrast, gilthead seabream SAF-1 fibroblasts shed microvesicles containing a 22 kDa IL-1beta form within 30 min of activation with ATP. Notably, the post-translational processing of IL-1beta by SAF-1 cells is abrogated by a specific ICE inhibitor.
Assuntos
Interleucina-1/metabolismo , Leucócitos/metabolismo , Dourada/fisiologia , Trifosfato de Adenosina/farmacologia , Animais , Anexina A5/análise , Anexina A5/farmacologia , Anticorpos/imunologia , Western Blotting , Inibidores de Caspase , Linhagem Celular , DNA Bacteriano/farmacologia , DNA Complementar/genética , Escherichia coli/genética , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Citometria de Fluxo , Vetores Genéticos/genética , Interleucina-1/genética , Cinética , Leucócitos/química , Leucócitos/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Fatores Ativadores de Macrófagos/farmacologia , Macrófagos/química , Macrófagos/efeitos dos fármacos , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Oligopeptídeos/farmacologia , Reação em Cadeia da Polimerase , Dourada/imunologia , Transformação Genética , Vibrio/genéticaAssuntos
Adjuvantes Imunológicos , Ilhas de CpG/imunologia , Citocinas/biossíntese , DNA Bacteriano/imunologia , Imunidade Inata/imunologia , Imunoglobulina M/biossíntese , Adjuvantes Imunológicos/toxicidade , Animais , Formação de Anticorpos/efeitos dos fármacos , Vacinas Bacterianas/imunologia , Vacinas Bacterianas/toxicidade , Ensaios Clínicos como Assunto , DNA Bacteriano/farmacologia , DNA Bacteriano/toxicidade , Avaliação Pré-Clínica de Medicamentos , Francisella tularensis/imunologia , Galactosamina/toxicidade , Humanos , Controle de Infecções , Lipopolissacarídeos/toxicidade , Listeria monocytogenes/imunologia , Listeriose/prevenção & controle , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Oligodesoxirribonucleotídeos/síntese química , Oligodesoxirribonucleotídeos/imunologia , Oligodesoxirribonucleotídeos/farmacologia , Segurança , Tularemia/prevenção & controle , Vacinas de DNA/imunologia , Vacinas de DNA/toxicidade , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/toxicidadeRESUMO
A method of transformation on solid medium especially adapted for pneumococcus has been developed. Under specific conditions, all colonies that are allowed to grow in the presence of transforming DNA for six hours give rise to transformed bacteria. Combined with replica plating this technique has been used to isolate mutants modified with regard to recombination. Most of the mutants found are transformation-defective and show a large diversity in their response to ultraviolet light. Some of these mutants have lost their ability to take up transforming DNA. One shows a reduced yield of transformants for a given quantity of DNA taken up. Mutants that manifest altered behavior with regard to marker efficiencies have also been isolated. One of these exhibits a decrease in the transformation efficiency of only the high efficiency markers and two mutants show a decrease in the transformation efficiency of the low efficiency markers.
Assuntos
DNA Bacteriano/farmacologia , Streptococcus pneumoniae/metabolismo , Transformação Genética/efeitos dos fármacos , Aminopterina , Técnicas Bacteriológicas , Transporte Biológico , Cinchona , DNA Bacteriano/metabolismo , Resistência a Medicamentos , Eritromicina , Métodos , Mutação , Nitrosoguanidinas , Plantas Medicinais , Efeitos da Radiação , Recombinação Genética , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/efeitos da radiação , Estreptomicina , Timidina/metabolismo , Fatores de Tempo , Trítio , Raios UltravioletaRESUMO
Transformation of the pneumococcus mutant 401 by DNA's bearing the standard reference marker and several other markers belonging to two unlinked loci has shown that differences in the integration efficiencies of these markers were considerably reduced in this strain compared to the wild-type strain Cl(3). The sensitivities of mutant 401 to ultraviolet light and to X-ray irradiation are the same as those of Cl(3). However, in 401 all the markers tested are more resistant to inactivation as shown by transformation of 401 and Cl(3) by ultraviolet-irradiated DNA. The increase in resistance is greater for low efficiency (LE) markers than for high efficiency (HE) markers.-The decreased discrimination between LE and HE markers in strain 401 is not due to a mechanism related to modification of markers in the transforming DNA by the recipient cells, nor are the proteins inducing competence of the cells responsible for the differences in the integration efficiencies of various markers.-Genetic studies of the fate of recombinants as well as the measure of the amount of DNA taken up have shown that all the markers are integrated in strain 401 by the same recombination process, that specific to high efficiency markers.