RESUMO
We characterized Staphylococcus aureus small-colony variant (SCV) strains isolated from cystic fibrosis (CF) patients in southern Brazil. Smaller colonies of S. aureus were isolated from respiratory samples collected consecutively from 225 CF patients from July 2013 to November 2016. Two phenotypic methods-the auxotrophic classification and a modified method of antimicrobial susceptibility testing-were employed. PCR was conducted to detect the mecA, ermA, ermB, ermC, msrA, and msrB resistance genes. Furthermore, DNA sequencing was performed to determine the mutations in the thyA gene, and multilocus sequence typing was used to identify the genetic relatedness. S. aureus strains were isolated from 186 patients (82%); suggestive colonies of SCVs were obtained in 16 patients (8.6%). The clones CC1 (ST1, ST188, and ST2383), CC5 (ST5 and ST221), and ST398 were identified. Among SCVs, antimicrobial susceptibility testing showed that 77.7% of the isolates were resistant to multiple drugs, and all of them were susceptible to vancomycin. mecA (2), ermA (1), ermB (1), ermC (3), and msrB (18) were distributed among the isolates. Phenotypically thymidine-dependent isolates had different mutations in the thyA gene, and frameshift mutations were frequently observed. Of note, revertants showed nonconservative or conservative missense mutations. SCVs are rarely identified in routine laboratory tests. IMPORTANCE Similar findings have not yet been reported in Brazil, emphasizing the importance of monitoring small-colony variants (SCVs). Altogether, our results highlight the need to improve detection methods and review antimicrobial therapy protocols in cystic fibrosis (CF) patients.
Assuntos
Fibrose Cística/microbiologia , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus aureus/metabolismo , Timidina/metabolismo , Adolescente , Adulto , Idoso , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Brasil , Criança , Pré-Escolar , Farmacorresistência Bacteriana , Feminino , Humanos , Lactente , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Mutação , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , Adulto JovemRESUMO
Nucleotide biosynthesis proceeds through a de novo pathway and a salvage route. In the salvage route, free bases and/or nucleosides are recycled to generate the corresponding nucleotides. Thymidine kinase (TK) is the first enzyme in the salvage pathway to recycle thymidine nucleosides as it phosphorylates thymidine to yield thymidine monophosphate. The Arabidopsis genome contains two TK genes -TK1a and TK1b- that show similar expression patterns during development. In this work, we studied the respective roles of the two genes during early development and in response to genotoxic agents targeting the organellar or the nuclear genome. We found that the pyrimidine salvage pathway is crucial for chloroplast development and genome replication, as well as for the maintenance of its integrity, and is thus likely to play a crucial role during the transition from heterotrophy to autotrophy after germination. Interestingly, defects in TK activity could be partially compensated by supplementation of the medium with sugar, and this effect resulted from both the availability of a carbon source and the activation of the nucleotide de novo synthesis pathway, providing evidence for a compensation mechanism between two routes of nucleotide biosynthesis that depend on nutrient availability. Finally, we found differential roles of the TK1a and TK1b genes during the plant response to genotoxic stress, suggesting that different pools of nucleotides exist within the cells and are required to respond to different types of DNA damage. Altogether, our results highlight the importance of the pyrimidine salvage pathway, both during plant development and in response to genotoxic stress.
Assuntos
Arabidopsis/genética , Genoma de Planta/genética , Pirimidinas/metabolismo , Timidina Quinase/metabolismo , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Núcleo Celular/metabolismo , Cloroplastos/metabolismo , Dano ao DNA , Nucleotídeos/metabolismo , Timidina/metabolismo , Timidina Quinase/genéticaRESUMO
Benznidazole is the first-line drug used in treating Chagas disease, which is caused by the parasite Trypanosoma cruzi (T. cruzi). However, benznidazole has limited efficacy and several adverse reactions. Pentamidine is an antiprotozoal drug used in the treatment of leishmaniasis and African trypanosomiasis. In T. cruzi, pentamidine blocks the transport of putrescine, a precursor of trypanothione, which constitutes an essential molecule in the resistance of T. cruzi to benznidazole. In the present study, we describe the effect of the combination of benznidazole and pentamidine on isolated parasites, mammalian cells and in mice infected with T. cruzi. In isolated trypomastigotes, we performed a dose-matrix scheme of combinations, where pentamidine antagonized the effect of benznidazole, mainly at concentrations below the EC50 of pentamidine. In T. cruzi-infected mammalian cells, pentamidine reversed the effect of benznidazole (measured by qPCR). In comparison, in infected BALB/c mice, pentamidine failed to get synergy with benznidazole, measured on mice survival, parasitemia and amastigote nest quantification. To further explain the in vitro antagonism, we explored whether pentamidine affects intracellular trypanothione levels, however, pentamidine produced no change in trypanothione concentrations. Finally, the T. cruzi polyamine permease (TcPAT12) was overexpressed in epimastigotes, showing that pentamidine has the same trypanocidal effect, independently of transporter expression levels. These results suggest that, in spite of the high potency in the putrescine transport blockade, TcPAT12 permease is not the main target of pentamidine, and could explain the lack of synergism between pentamidine and benznidazole.
Assuntos
Doença de Chagas/tratamento farmacológico , Nitroimidazóis/antagonistas & inibidores , Pentamidina/farmacologia , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Animais , Doença de Chagas/patologia , Chlorocebus aethiops , DNA de Protozoário/análise , DNA de Protozoário/isolamento & purificação , Relação Dose-Resposta a Droga , Glutationa/análogos & derivados , Glutationa/efeitos dos fármacos , Glutationa/metabolismo , Macrófagos/parasitologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Miocárdio/patologia , Parasitemia/tratamento farmacológico , Parasitemia/parasitologia , Putrescina/metabolismo , Distribuição Aleatória , Espermidina/análogos & derivados , Espermidina/metabolismo , Timidina/metabolismo , Tripanossomicidas/antagonistas & inibidores , Células VeroRESUMO
The aim of the present study was to characterize the mechanism underlying estrogen effects on the androgen-independent prostate cancer cell line PC-3. 17ß-estradiol and the ERß-selective agonist DPN, but not the ERα-selective agonist PPT, increased the incorporation of [methyl-(3)H]thymidine and the expression of Cyclin D2, suggesting that ERß mediates the proliferative effect of estrogen on PC-3 cells. In addition, upregulation of Cyclin D2 and incorporation of [methyl-(3)H]thymidine induced by 17ß-estradiol and DPN were blocked by the ERß-selective antagonist PHTPP in PC-3 cells. Upregulation of Cyclin D2 and incorporation of [methyl-(3)H]thymidine induced by DPN were also blocked by PKF118-310, a compound that disrupts ß-catenin-TCF (T-cell-specific transcription factor) complex, suggesting the involvement of ß-catenin in the estradiol effects in PC-3 cells. A diffuse immunostaining for non-phosphorylated ß-catenin was detected in the cytoplasm of PC-3 cells. Low levels of non-phosphorylated ß-catenin immunostaining were also detected near the plasma membrane and in nuclei. Treatment of PC-3 cells with 17ß-estradiol or DPN markedly increased non-phosphorylated ß-catenin expression. These effects were blocked by pretreatment with the ERß-selective antagonist PHTPP, PI3K inhibitor Wortmannin or AKT inhibitor MK-2206, indicating that ERß-PI3K/AKT mediates non-phosphorylated ß-catenin expression. Cycloheximide blocked the DPN-induced upregulation of non-phosphorylated ß-catenin, suggesting de novo synthesis of this protein. In conclusion, these results suggest that estrogen may play a role in androgen-independent prostate cancer cell proliferation through a novel pathway, involving ERß-mediated activation of ß-catenin.
Assuntos
Receptor beta de Estrogênio/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , beta Catenina/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ciclina D1/metabolismo , Ciclina D2/metabolismo , Cicloeximida/farmacologia , Estradiol/farmacologia , Receptor beta de Estrogênio/agonistas , Humanos , Masculino , Nitrilas/farmacologia , Fenóis/farmacologia , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , Timidina/metabolismoRESUMO
The aim of the present study was to investigate the role of each estrogen receptors on the regulation of proteins involved with proliferation and differentiation of Sertoli cells from 15-day-old rats. Activation of ESR1 by 17ß-estradiol (E2) and ESR1-selective agonist PPT increased CCND1 expression, and this effect was dependent on NF-kB activation. E2 and the ESR2-selective agonist DPN, but not PPT, increased, in a PI3K and CREB-dependent manner, the expression of CDKN1B and the transcription factors GATA-1 and DMRT1. Analyzing the expression of ESR1 and ESR2 in different stages of development of Sertoli cells, we observed that the ESR1/ESR2 ratio decreased with age, and this ratio seems to be important to determine the end of cell proliferation and the start of cell differentiation. In Sertoli cells from 15-day-old rats, the ESR1/ESR2 ratio favors the effect of ESR1 and the activation of this receptor increased [Methyl-(3)H]thymidine incorporation. We propose that in Sertoli cells from 15-day-old rats E2 modulates Sertoli cell proliferation through ESR1/NF-kB-mediated increase of CCND1, and cell cycle exit and differentiation through ESR2/CREB-mediated increase of CDKN1B, GATA-1 and DMRT1. The present study reinforces the important role of estrogen for normal testis development.
Assuntos
Diferenciação Celular , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/metabolismo , Células de Sertoli/citologia , Células de Sertoli/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Ciclina D1/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Estradiol/farmacologia , Fator de Transcrição GATA1/metabolismo , Proteínas I-kappa B/metabolismo , Masculino , Modelos Biológicos , Inibidor de NF-kappaB alfa , NF-kappa B/metabolismo , Nitrilas/farmacologia , Fenóis/farmacologia , Fosforilação/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Pirazóis/farmacologia , Ratos , Ratos Wistar , Células de Sertoli/efeitos dos fármacos , Timidina/metabolismo , Fatores de Transcrição/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
Rutin is a flavonoid with several pharmacological properties and it has been demonstrated that rutin can modulate glucose homeostasis. In skeletal muscle, an increase in intracellular calcium concentration may induce glucose transporter-4 (GLUT-4) translocation with consequent glucose uptake. The aim of this study was to investigate the effect of rutin and intracellular pathways on calcium uptake as well as the involvement of calcium in glucose uptake in skeletal muscle. The results show that rutin significantly stimulated calcium uptake through voltage-dependent calcium channels as well as mitogen-activated kinase (MEK) and protein kinase A (PKA) signaling pathways. Also, rutin stimulated glucose uptake in the soleus muscle and this effect was mediated by extracellular calcium and calcium-calmodulin-dependent protein kinase II (CaMKII) activation. In conclusion, rutin significantly stimulates calcium uptake in rat soleus muscles. Furthermore, the increase in intracellular calcium concentration is involved in DNA activation by rutin. Also, rutin-induced glucose uptake via CaMKII may result in GLUT-4 translocation to the plasma membrane, characterizing an insulin-independent pathway. These findings indicate that rutin is a potential drug candidate for diabetes therapy.
Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Glucose/metabolismo , Músculo Esquelético/efeitos dos fármacos , Rutina/farmacologia , Animais , Antioxidantes/farmacologia , Transporte Biológico/efeitos dos fármacos , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , DNA/metabolismo , Masculino , Músculo Esquelético/metabolismo , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Timidina/metabolismoRESUMO
BACKGROUND: The secretory activity of Sertoli cells (SC) is dependent on ion channel functions and protein synthesis and is critical to ongoing spermatogenesis. The aim of this study was to investigate the mechanism of action associated with a non-metabolizable amino acid [14C]-MeAIB (alpha-(methyl-amino)isobutyric acid) accumulation stimulated by T4 and the role of the integrin receptor in this event, and also to clarify whether the T4 effect on MeAIB accumulation and on Ca2+ influx culminates in cell secretion. METHODS: We have studied the rapid and plasma membrane initiated effects of T4 by using 45Ca2+ uptake and [45C]-MeAIB accumulation assays, respectively. Thymidine incorporation into DNA was used to monitor nuclear activity and quinacrine to analyze the secretory activity on SC. RESULTS: The stimulation of MeAIB accumulation byT4 appears to be mediated by the integrin receptor in the plasma membrane since tetrac and RGD peptide were able to nullify the effect of this hormone. In addition, T4 increases extracellular Ca2+ uptake and Ca2+ from intracellular stocks to enhance nuclear activity, but this genomic action seems not to influence SC secretion mediated by T4. Also, the cytoskeleton and CIC-3 chloride channel contribute to the membrane-associated responses of SC. CONCLUSIONS: T4 integrin receptor activation ultimately determines the plasma membrane responses on amino acid transport in SC, but it is not involved in calcium influx, cell secretion or the nuclear effect of the hormone. GENERAL SIGNIFICANCE: The integrin receptor activation by T4 may take a role in plasma membrane processes involved in the male reproductive system.
Assuntos
Membrana Celular/efeitos dos fármacos , Integrina alfaVbeta3/genética , Receptores de Superfície Celular/genética , Células de Sertoli/efeitos dos fármacos , Espermatogênese/fisiologia , Tiroxina/farmacologia , Aminoácidos/metabolismo , Ácidos Aminoisobutíricos/metabolismo , Animais , Animais Recém-Nascidos , Transporte Biológico/efeitos dos fármacos , Cálcio/metabolismo , Radioisótopos de Carbono , Canais de Cloreto/metabolismo , Citoesqueleto/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Integrina alfaVbeta3/metabolismo , Masculino , Oligopeptídeos/farmacologia , Ratos , Ratos Wistar , Receptores de Superfície Celular/metabolismo , Células de Sertoli/citologia , Células de Sertoli/fisiologia , Transdução de Sinais/efeitos dos fármacos , Espermatogênese/efeitos dos fármacos , Timidina/metabolismo , Tiroxina/análogos & derivadosRESUMO
This work describes an efficient, simple, and green bioprocess for obtaining 5-halogenated pyrimidine nucleosides from thymidine by transglycosylation using whole cells. Biosynthesis of 5-fluoro-2'-deoxyuridine (floxuridine) was achieved by free and immobilized Aeromonas salmonicida ATCC 27013 with an 80% and 65% conversion occurring in 1 h, respectively. The immobilized biocatalyst was stable for more than 4 months in storage conditions (4 °C) and could be reused at least 30 times without loss of its activity. This microorganism was able to biosynthesize 2.0 mg L(-1) min(-1) (60%) of 5-chloro-2'-deoxyuridine in 3 h. These halogenated pyrimidine 2'-deoxynucleosides are used as antitumoral agents.
Assuntos
Aeromonas salmonicida/metabolismo , Floxuridina/metabolismo , Biotecnologia/métodos , Biotransformação , Células Imobilizadas/metabolismo , Glicosilação , Timidina/metabolismo , Fatores de TempoRESUMO
Numerous natural compounds have been used against Trypanosoma cruzi, the causative agent of Chagas' disease. Here, we studied the effect of the diterpene 5-epi-icetexone on growth and morphology of parasites synchronized with hydroxyurea, at different periods of time after removal of the nucleotide. We observed that the diterpene does not affect the growth of the parasites when added within 10 h after removal of hydroxyurea, but the compound was effective on growth when added to the cultures after 12 h. Thymidine incorporation was somewhat inhibited when the diterpene was added at 12 h after removal of hydroxyurea, possibly on the transition S/G2. Using transmission electron microscopy we observed that the diterpene induced a delay in the progression of cell division. We conclude that the compound, at cytostatic dose, affects the cell cycle of T. cruzi, possibly in the transition S/G2 phase and cell division. Further studies will focus to identify the molecular targets for the action of 5-epi-icetexone.
Assuntos
Ciclo Celular/efeitos dos fármacos , Doença de Chagas/parasitologia , Diterpenos/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Humanos , Hidroxiureia/farmacologia , Timidina/metabolismo , Fatores de Tempo , Trypanosoma cruzi/citologia , Trypanosoma cruzi/crescimento & desenvolvimentoRESUMO
Microglial activation is a key event in the progression and infiltration of tumors. We have previously demonstrated that the co-chaperone stress inducible protein 1 (STI1), a cellular prion protein (PrP(C)) ligand, promotes glioblastoma (GBM) proliferation. In the present study, we examined the influence of microglial STI1 in the growth and invasion of the human glioblastoma cell line GBM95. We demonstrated that soluble factors secreted by microglia into the culture medium (microglia conditioned medium; MG CM) caused a two-fold increase in the proliferation of GBM95 cells. This effect was reversed when STI1 was removed from the MG CM. In this context, we have shown that microglial cells synthesize and secrete STI1. Interestingly, no difference was observed in proliferation rates when GBM cells were maintained in MG CM or MG CM containing an anti-PrP(C) neutralizing antibody. Moreover, rec STI1 and rec STI1(Δ230-245), which lack the PrP(C) binding site, both promoted similar levels of GBM95 proliferation. In the migration assays, MG CM favored the migration of GBM95 cells, but migration failed when STI1 was removed from the MG CM. We detected metalloproteinase 9 (MMP-9) activity in the MG CM, and when cultured microglia were treated with an anti-STI1 antibody, MMP-9 activity decreased. Our results suggest that STI1 is secreted by microglia and favors tumor growth and invasion through the participation of MMP-9 in a PrP(C)-independent manner.