RESUMO
BACKGROUND: Bacterial infections are responsible of high economic losses in aquaculture. Mexican golden trout (Oncorhynchus chrysogaster) is a threatened native trout species that has been introduced in aquaculture both for species conservation and breeding for production and for which no studies of bacterial infections have been reported. CASE PRESENTATION: Fish from juvenile stages of Mexican golden trout showed an infectious outbreak in a farm in co-culture with rainbow trout (Oncorhynchus mykiss), showing external puntiform red lesions around the mouth and caudal pedunculus resembling furuncles by Aeromonas spp. and causing an accumulated mortality of 91%. Isolation and molecular identification of bacteria from lesions and internal organs showed the presence of Aeromonas bestiarum, Aeromonas sobria, Plesiomonas shigelloides and Ichthyobodo necator isolated from a single individual. All bacterial isolates were resistant to amoxicillin-clavulanic acid and cefazoline. P. shigelloides was resistant to third generation ß-lactamics. CONCLUSIONS: This is the first report of coinfection by Aeromonas bestiarum, Aeromonas sobria, Plesiomonas shigelloides and Ichthyobodo necator in an individual of Mexican golden trout in co-culture with rainbow trout. Resistance to ß-lactams suggests the acquisition of genetic determinants from water contamination by human- or livestock-associated activities.
Assuntos
Aeromonas , Coinfecção , Doenças dos Peixes , Infecções por Bactérias Gram-Negativas , Oncorhynchus mykiss , Oncorhynchus , Parasitos , Plesiomonas , Aeromonas/genética , Animais , Coinfecção/veterinária , Doenças dos Peixes/microbiologia , Infecções por Bactérias Gram-Negativas/microbiologia , Infecções por Bactérias Gram-Negativas/veterinária , Necator , Plesiomonas/genéticaRESUMO
The dynamism of microbial populations in the rumen has been studied with molecular methods that analyze single nucleotide polymorphisms of ribosomal RNA gene fragments (rDNA). Therefore DNA of good quality is needed for this kind of analysis. In this work we report the evaluation of four DNA extraction protocols (mechanical lysis or chemical lysis with CTAB, ethylxanthogenate or DNAzol(®)) from ruminal fluid. The suitability of two of these protocols (mechanical lysis and DNAzol(®)) was tested on single-strand conformation polymorphism (SSCP) of rDNA of rumen microbial populations. DNAzol(®) was a simple method that rendered good integrity, yield and purity. With this method, subtle changes in protozoan populations were detected in young bulls fed with slightly different formulations of a supplement of multinutritional blocks of molasses and urea. Sequences related to Eudiplodinium maggi and a non-cultured Entodiniomorphid similar to Entodinium caudatum, were related to major fluctuating populations in an SSCP assay.
Assuntos
Métodos Analíticos de Preparação de Amostras/métodos , Bactérias/isolamento & purificação , DNA Bacteriano/química , DNA Bacteriano/isolamento & purificação , DNA Ribossômico/isolamento & purificação , Rúmen/microbiologia , Animais , Bactérias/classificação , Bactérias/genética , Bovinos , DNA Bacteriano/genética , DNA Ribossômico/genética , Masculino , Dados de Sequência Molecular , Filogenia , Polimorfismo Conformacional de Fita SimplesRESUMO
BACKGROUND: Inhibition of Candida albicans on denture resins could play a significant role in preventing the development of denture stomatitis. The safety of a new dental material with antifungal properties was analyzed in this work. METHODS: Poly(methyl methacrylate) [PMMA] discs and PMMA-silver nanoparticle discs were formulated, with the commercial acrylic resin, Nature-Cryl™, used as a control. Silver nanoparticles were synthesized and characterized by ultraviolet-visible spectroscopy, dispersive Raman spectroscopy, and transmission electron microscopy. The antifungal effect was assessed using a luminescent microbial cell viability assay. Biocompatibility tests were carried out using NIH-3T3 mouse embryonic fibroblasts and a Jurkat human lymphocyte cell line. Cells were cultured for 24 or 72 hours in the presence or absence of the polymer formulations and analyzed using three different tests, ie, cellular viability by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and cell proliferation by enzyme-linked immunosorbent assay BrdU, and genomic DNA damage (Comet assay). Finally, the samples were evaluated mechanically, and the polymer-bearing silver nanoparticles were analyzed microscopically to evaluate dispersion of the nanoparticles. RESULTS: The results show that PMMA-silver nanoparticle discs significantly reduce adherence of C. albicans and do not affect metabolism or proliferation. They also appear not to cause genotoxic damage to cells. CONCLUSION: The present work has developed a new biocompatible antifungal PMMA denture base material.
Assuntos
Resinas Acrílicas/síntese química , Candida albicans/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Materiais Dentários/síntese química , Dentaduras/microbiologia , Nanopartículas Metálicas/administração & dosagem , Prata/farmacologia , Animais , Antifúngicos/farmacologia , Materiais Biocompatíveis/síntese química , Materiais Biocompatíveis/farmacologia , Candida albicans/fisiologia , Adesão Celular/efeitos dos fármacos , Humanos , Células Jurkat , Teste de Materiais , Nanopartículas Metálicas/química , Camundongos , Células NIH 3T3 , Polimetil Metacrilato , Prata/químicaRESUMO
Internalization of Staphylococcus aureus in bovine endothelial cells (BEC) is increased by tumor necrosis factor alpha stimulation and NF-κB activation. Because the phosphoinositide-3-kinase (PI3K)-Akt signaling pathway also modulates NF-κB activity, we considered whether the internalization of S. aureus by BEC is associated with the activity of PI3K and Akt. We found a time- and multiplicity of infection-dependent phosphorylation of Akt on Ser473 in BEC infected with S. aureus. This phosphorylation was inhibited by LY294002 (LY), indicating the participation of PI3K. Inhibition of either PI3K with LY or wortmannin, or Akt with SH-5, strongly reduced the internalization of S. aureus. Transfection of BEC with a dominant-negative form of the Akt gene significantly decreased S. aureus internalization, whereas transfection with the constitutively active mutant increased the number of internalized bacterium. Inhibition of PDK1 activity with OSU-03012 did not affect the level of S. aureus internalization, demonstrating that phosphorylation of Akt on Thr308 is not important for this process. Compared to the untreated control, the adherence of S. aureus to the surface of BEC was unaltered when cells were transfected or incubated with the pharmacological inhibitors. Furthermore, Akt activation by internalized S. aureus triggered a time-dependent phosphorylation of glycogen synthase kinase-3α (GSK-3α) on Ser21 and GSK-3ß on Ser9 that was partially inhibited with SH-5. Finally, treatment of BEC with LY prior to S. aureus infection inhibited the NF-κB p65 subunit phosphorylation on Ser536, indicating the involvement of PI3K. These results suggest that PI3K-Akt activity is important for the internalization of S. aureus and phosphorylation of GSK-3α, GSK-3ß, and NF-κB.
Assuntos
Células Endoteliais/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/fisiologia , Staphylococcus aureus/fisiologia , Animais , Bovinos , Células Cultivadas , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Interleucina-1beta/metabolismo , Interleucina-1beta/farmacologia , NF-kappa B/metabolismo , NF-kappa B/farmacologia , Fosfatidilinositol 3-Quinases/genética , Fosforilação , Proteínas Proto-Oncogênicas c-akt/genética , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Electroporation has been the method of election for transfection of murine embryonic stem cells for over 15 years; however, it is a time consuming protocol because it requires large amounts of DNA and cells, as well as expensive and delicate equipment. Lipofection is a transfection method that requires lower amounts of cells and DNA than electroporation, and has proven to be efficient in a large number of cell lines. It has been shown that after lipofection, mouse embryonic stem cells remain pluripotent, capable of forming germ line chimeras and can be transfected with greater efficiency than with electroporation; however, gene targeting of mouse embryonic stem cells by lipofection has not been reported. The objective of this work was to find out if lipofection can be used as efficiently as electroporation for regular gene targeting protocols. This context compares gene targeting efficiency between these techniques in mouse embryonic stem cells E14TG2a, using a gene replacement type vector. No differences were found in gene targeting efficiency between groups; however, lipofection was three times more efficient than electroporation in transfection efficiency, which makes lipofection a less expensive alternative method to produce gene targeting in mouse embryonic stem cells.
Durante los últimos 15 años se ha demostrado que la electroporación representa el método ideal para la transfección de células troncoembrionarias de ratón; sin embargo, demanda grandes cantidades de ADN y células, así como equipo caro y delicado, ello hace que este proceso sea costoso y laborioso. La lipofección es un método de transfección que requiere menos de células y ADN que la electroporación; asimismo, ha probado ser eficiente en gran número de líneas celulares. Se ha demostrado que después de lipofectar células troncoembrionarias de ratón, éstas mantienen su pluripotencia y son capaces de formar quimeras de línea germinal y se transfectan con mayor eficiencia que con electroporación, pero no se ha notificado la mutagénesis dirigida mediante la lipofección de células troncoembrionarias de ratón. El objetivo del presente trabajo fue saber si la lipofección puede ser utilizada con la misma o mayor eficiencia que la electroporación para los protocolos regulares de mutagénesis dirigida; en este contexto, se compara la eficiencia en mutagénesis dirigida entre estas técnicas en células troncoembrionarias de ratón E14TG2a, utilizando un vector de reemplazo. Entre las células transfectadas no se hallan diferencias en la eficiencia en mutagénesis dirigida entre grupos; sin embargo, los resultados que aquí se ofrecen muestran que la lipofección es tres veces más eficiente en la transfección, lo cual indica que la lipofección es un método alternativo menos costoso para obtener mutagénesis dirigida en células troncoembrionarias de ratón.
RESUMO
Staphylococcus epidermidis is an environmental opportunistic pathogen associated with bovine intramammary infections. In bacterial infections, the endothelial tissue plays an important role during inflammation and it is the target of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-alpha). Therefore, this work was designed to explore the effect of TNF-alpha on the interaction of S. epidermidis with bovine endothelial cells (BEC). We show that cell signaling activated by TNF-alpha caused a marked reduction in the number of intracellular S. epidermidis, suggesting that molecules participating in this pathway were involved in the internalization of this bacterium. We also found that S. epidermidis internalization was not associated with basal levels of nuclear factor kappa B (NF-kappaB) activity because the intracellular number of bacteria recovered after treating BEC with the NF-kappaB inhibitors, SN50 or BAY 11-7083, was similar to that of the untreated control. Interestingly, inhibition of the basal activity of JNK with SP600125 and p38 with SB203580 caused a decrease in the number of intracellular S. epidermidis. These results suggest that activation of the signaling pathway initiated by TNF-alpha could play an important role in the phagocytosis of this bacterium. However, the basal activity of NF-kappaB was shown not to be important for the internalization process of S. epidermidis.
Assuntos
Células Endoteliais/microbiologia , Staphylococcus epidermidis/imunologia , Fator de Necrose Tumoral alfa/imunologia , Animais , Antracenos/farmacologia , Bovinos , Contagem de Colônia Microbiana , Citoplasma/microbiologia , Imidazóis/farmacologia , Fatores Imunológicos/farmacologia , MAP Quinase Quinase 4/antagonistas & inibidores , NF-kappa B/antagonistas & inibidores , NF-kappa B/imunologia , Nitrilas/farmacologia , Peptídeos/farmacologia , Piridinas/farmacologia , Sulfonas/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
The Notch signaling pathway (NSP) is an important intercellular communication mechanism that regulates embryo development and adult physiological functions. The Hairless (H) protein is a powerful antagonist of the NSP by its interaction with the Suppressor of Hairless (Su[H]) protein, recruiting the corepressors Gro and CtBP. In the present work, we examined the role of several important amino acids in different H protein domains analyzing four mutant lines of Drosophila melanogaster. The mutant alleles were evaluated by single-strand conformational polymorphism (SSCP) analysis and we located mutated regions at different positions along the sequence of the Hairless gene.
Assuntos
Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Mutação Puntual/genética , Fatores de Transcrição/química , Fatores de Transcrição/genética , Sequência de Aminoácidos , Animais , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/química , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Dados de Sequência Molecular , Mutação Puntual/fisiologia , Polimorfismo Conformacional de Fita Simples/genética , Receptores Notch/genética , Transdução de Sinais/genética , Fatores de Transcrição/metabolismoRESUMO
The previous findings that both a long and a short type 1 deiodinase (D1) mRNA are present in different tissues and that the D1 gene contains two potential polyA signals suggest that the two mRNAs result from differential polyA signal usage. In this study, we examined the properties of the two D1 mRNAs generated in HEK 293 cells by the alternative use of each of the poly A signals in order to ascertain the potential regulatory role of the 3'UTR of this gene. Our results showed that the long mRNA is less stable, but that it is translated more efficiently than the short mRNA. The net result of these differences is a higher D1 activity with the long message. These data suggest that the D1 3'UTR may play an important role in regulating the stability and translational efficiency of the D1 mRNA, both of which could be physiologically relevant when the demand for D1 activity is high.