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1.
Biol. Res ; 55: 22-22, 2022. ilus, graf
Artigo em Inglês | LILACS | ID: biblio-1383923

RESUMO

BACKGROUND: Retinal neurodegeneration is induced by a variety of environmental insults and stresses, but the exact mechanisms are unclear. In the present study, we explored the involvement of cytosolic mitochondrial DNA (mtDNA), resulting in the cGAS-STING dependent inflammatory response and apoptosis in retinal damage in vivo. METHODS: Retinal injury was induced with white light or intravitreal injection of lipopolysaccharide (LPS). After light-or LPS-induced injury, the amount of cytosolic mtDNA in the retina was detected by PCR. The mtDNA was isolated and used to transfect retinas in vivo. WB and real-time PCR were used to evaluate the activation of cGAS-STING path-way and the levels of apoptosis-associated protein at different times after mtDNA injection. Retinal cell apoptosis rate was detected by TUNEL staining. Full-field electroretinography (ERG) was used to assess the retinal function. RESULTS: Light injury and the intravitreal injection of LPS both caused the leakage of mtDNA into the cytoplasm in retinal tissue. After the transfection of mtDNA in vivo, the levels of cGAS, STING, and IFN-ß mRNAs and the protein levels of STING, phosph-TBK1, phospho-IRF3, and IFN-ß were upregulated. mtDNA injection also induced the activation of caspase 3 and caspase 9. BAX and BAK were increased at both the mRNA and protein levels. The release of cytochrome c from the mitochondria to the cytosol was increased after mtDNA injection. The wave amplitudes on ERG decreased and retinal cell apoptosis was detected after mtDNA injection. CONCLUSIONS: Cytosolic mtDNA triggers an inflammatory response. It also promotes apoptosis and the dysfunction of the retina.


Assuntos
Animais , Ratos , DNA Mitocondrial/genética , Lipopolissacarídeos , Injeções Intravítreas , Proteínas de Membrana/metabolismo , Mitocôndrias , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo
2.
Mem. Inst. Oswaldo Cruz ; 110(8): 1003-1009, Dec. 2015. tab, graf
Artigo em Inglês | LILACS | ID: lil-769825

RESUMO

An investigation was carried out into the genetic mechanisms responsible for multidrug resistance in nine carbapenem-resistant Pseudomonas aeruginosaisolates from different hospitals in Recife, Brazil. Susceptibility to antimicrobial agents was determined by broth microdilution. Polymerase chain reaction (PCR) was employed to detect the presence of genes encoding β-lactamases, aminoglycoside-modifying enzymes (AMEs), 16S rRNA methylases, integron-related genes and OprD. Expression of genes coding for efflux pumps and AmpC cephalosporinase were assessed by quantitative PCR. The outer membrane proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The blaSPM-1, blaKPC-2 and blaGES-1 genes were detected in P. aeruginosaisolates in addition to different AME genes. The loss of OprD in nine isolates was mainly due to frameshift mutations, premature stop codons and point mutations. An association of loss of OprD with the overexpression of MexAB-OprM and MexXY-OprM was observed in most isolates. Hyper-production of AmpC was also observed in three isolates. Clonal relationship of the isolates was determined by repetitive element palindromic-PCR and multilocus sequence typing. Our results show that the loss of OprD along with overexpression of efflux pumps and β-lactamase production were responsible for the multidrug resistance in the isolates analysed.


Assuntos
Humanos , Carbapenêmicos/metabolismo , Farmacorresistência Bacteriana Múltipla/genética , Mutação , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/genética , Resistência beta-Lactâmica/genética , beta-Lactamases/metabolismo , Aminoglicosídeos/metabolismo , Anfotericina B/análogos & derivados , Anfotericina B/metabolismo , Antifúngicos/metabolismo , Brasil , Cefalosporinase/classificação , Cefalosporinase/metabolismo , Códon sem Sentido/metabolismo , Ativação Enzimática/genética , Mutação da Fase de Leitura/genética , Regulação Bacteriana da Expressão Gênica/genética , Proteínas de Membrana Transportadoras/metabolismo , Metiltransferases/metabolismo , Nucleotidiltransferases/metabolismo , Mutação Puntual/genética , Porinas/metabolismo , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/isolamento & purificação , Sequências Repetitivas de Ácido Nucleico , beta-Lactamases/genética
3.
Clinics ; 66(9): 1519-1522, 2011. ilus, tab
Artigo em Inglês | LILACS | ID: lil-604286

RESUMO

INTRODUCTION: Pseudomonas aeruginosa (P. aeruginosa) is one of the primary opportunistic pathogens responsible for nosocomial infections. Aminoglycosides are an import ant component of antipseudomonal chemotherapy. The inactivation of drugs by modifying enzymes is the most common mechanism of aminoglycoside resistance. OBJECTIVES: The inactivation of aminoglycosides by modifying enzymes is the primary resistance mechanism employed by P. aeruginosa. The aim of the present study was to investigate the occurrence of aminoglycoside resistance and the prevalence of four import ant modifying enzyme genes (aac (6')-I, aac (6')-II, ant (2")-I, aph (3')-VI) in P. aeruginosa in Iran. METHODS: A total of 250 clinical isolates of P. aeruginosa were collected from several hospitals in seven cities in Iran. Antimicrobial susceptibility tests (using the disk diffusion method and E-tests) were performed for all 250 isolates. In addition, all isolates were screened for the presence of modifying enzyme genes by polymerase chain reaction. RESULTS: The resistance rates, as determined by the disk diffusion method, were as follows: gentamicin 43 percent, tobramycin 38 percent, and amikacin 24 percent. Of the genes examined, aac (6')-II (36 percent) was the most frequently identified gene in phenotypic resist ant isolates, followed by ant (2")-I, aph (3')-VI, and aac (6')-I. CONCLUSIONS: Aminoglycoside resistance in P. aeruginosa remains a signific ant problem in Iran. Therefore, there is considerable local surveillance of aminoglycoside resistance.


Assuntos
Feminino , Humanos , Masculino , Acetiltransferases/genética , Aminoglicosídeos/farmacologia , Antibacterianos/farmacologia , Farmacorresistência Bacteriana/genética , Canamicina Quinase/genética , Nucleotidiltransferases/genética , Pseudomonas aeruginosa/genética , Aminoglicosídeos/metabolismo , Antibacterianos/metabolismo , DNA Bacteriano/genética , Farmacorresistência Bacteriana/efeitos dos fármacos , Irã (Geográfico) , Pseudomonas aeruginosa/efeitos dos fármacos
4.
Braz. j. med. biol. res ; 41(4): 289-294, Apr. 2008. ilus
Artigo em Inglês | LILACS | ID: lil-479679

RESUMO

Azospirillum brasilense is a diazotroph found in association with important agricultural crops. In this organism, the regulation of nitrogen fixation by ammonium ions involves several proteins including the uridylyltransferase/uridylyl-removing enzyme, GlnD, which reversibly uridylylates the two PII proteins, GlnB and GlnZ, in response to the concentration of ammonium ions. In the present study, the uridylylation/deuridylylation cycle of A. brasilense GlnB and GlnZ proteins by GlnD was reconstituted in vitro using the purified proteins. The uridylylation assay was analyzed using non-denaturing polyacrylamide gel electrophoresis and fluorescent protein detection. Our results show that the purified A. brasilense GlnB and GlnZ proteins were uridylylated by the purified A. brasilense GlnD protein in a process dependent on ATP and 2-oxoglutarate. The dependence on ATP for uridylylation was similar for both proteins. On the other hand, at micromolar concentration of 2-oxoglutarate (up to 100 µM), GlnB uridylylation was almost twice that of GlnZ, an effect that was not observed at higher concentrations of 2-oxoglutarate (up to 10 mM). Glutamine inhibited uridylylation and stimulated deuridylylation of both GlnB and GlnZ. However, glutamine seemed to inhibit GlnZ uridylylation more efficiently. Our results suggest that the differences in the uridylylation pattern of GlnB and GlnZ might be important for fine-tuning of the signaling pathway of cellular nitrogen status in A. brasilense.


Assuntos
Humanos , Azospirillum brasilense/metabolismo , Proteínas de Bactérias/metabolismo , Azospirillum brasilense/genética , Proteínas de Bactérias/genética , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Escherichia coli/metabolismo , Nucleotidiltransferases , Proteínas PII Reguladoras de Nitrogênio/genética , Proteínas PII Reguladoras de Nitrogênio/metabolismo , Plasmídeos/genética , Transdução de Sinais
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