RESUMO
Candida species are amongst the commensals of the mucosal surfaces of the human body which include the oral cavity, vagina, and intestinal mucosa. Fungal infections are on the rise worldwide. The overall burden of infections due to fungi is difficult to estimate because the majority of them remain undiagnosed. The present study aims to determine the burden of antifungal resistance in low socioeconomic country, Pakistan and the frequency of ERG11 and MDR1 genes involved. A total of 636 Candida isolates were obtained from various tertiary care institutions in Lahore in the form of culture on various culture plates. Sabouraud agar culture plates were used to culture the Candida spp. Antifungal resistance was determined against Fluconazole, Itraconazole, Ketoconazole, and Nystatin via disk diffusion technique. Most resistance was observed against Fluconazole followed by Itraconazole, Ketoconazole, and Nystatin. The Candida isolates recovering from CVP tip and tissue have a high resistance profile. Candida species resistant to at least two antifungals were chosen for further ERG11 and MDR1 detection through real-time PCR. Among 255 Candida isolates, 240 contained ERG11 gene while MDR1 gene is present in 149 Candida isolates. The isolates carrying both genes were tested by the broth microdilution technique for the susceptibility against cycloheximide, all of them were able to grow in cycloheximide. The genetic determinants of antifungal resistance such as ERG11 and MDR1 are as important in the multidrug resistance against a variety of compounds and antifungal drugs.
Assuntos
Antifúngicos , Candida , Cicloeximida , Testes de Sensibilidade Microbiana , Antifúngicos/farmacologia , Humanos , Candida/efeitos dos fármacos , Candida/genética , Candida/classificação , Candida/isolamento & purificação , Cicloeximida/farmacologia , Paquistão , Candidíase/microbiologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Farmacorresistência Fúngica Múltipla/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismoRESUMO
Oocyte activation via dual inhibition of protein synthesis and phosphorylation has improved in vitro embryo production in different mammalian species. In this study, we evaluated the effects of the combination of cycloheximide (CHX), dimethyl amino purine (DMAP), and anisomycin (ANY) on the activation of bovine oocytes, particularly on dynamics of MPF and MAPKs, embryonic developmental potential, and quality. The results showed that the cleavage and blastocyst rates, as well as levels of CCNB1, CDK1, p-CDK1Thr161, and p-CDK1Thr14-Tyr15, were similar among groups; ANY and ANY + CHX reduced the expression of ERK1/2 compared to DMAP-combinations (p < 0.05), whereas ANY + DMAP, CHX + DMAP, and ANY + CHX + DMAP reduced p-ERK1/2 compared to ANY and ANY + CHX treatments (p < 0.05). The quality of blastocysts in terms of cell counts, their allocation, and the numbers of TUNEL-positive cells did not differ among groups. However, transcript levels of POU5F1 were higher in embryos derived from ANY + CHX + DMAP treatment compared to other groups, while expression levels of CDX2 did not show differences. In addition, the BCL2A1/BAX ratio of the ANY + CHX + DMAP treatment was significantly low compared to the ANY treatment (p < 0.05) and did not differ significantly from the other treatments. In conclusion, oocyte activation by dual inhibition of protein synthesis and phosphorylation induces MPF inactivation without degradation of CCNB1, while MAPK inactivation occurs differentially between these inhibitors. Thus, although the combined use of these inhibitors does not affect early developmental competence in vitro, it positively impacts the expression of transcripts associated with embryonic quality.
Assuntos
Fator Promotor de Maturação , Partenogênese , Bovinos , Animais , Proteínas Quinases Ativadas por Mitógeno , Adenina/farmacologia , Oócitos , Cicloeximida/farmacologia , Blastocisto , Anisomicina/farmacologia , MamíferosRESUMO
Hypertension is associated with high circulating angiotensin II (Ang II). We have reported that autophagy regulates Ang II-induced vascular smooth muscle cell (VSMC) hypertrophy, but the mechanism mediating this effect is still unknown. Therefore, we studied how Ang II regulates LC3 levels in VSMCs and whether Bag3, a co-chaperone known to regulate LC3 total levels, may be involved in the effects elicited by Ang II. A7r5 cell line or rat aortic smooth muscle cell (RASMC) primary culture were stimulated with Ang II 100 nM for 24 h and LC3 I, LC3 II and Bag3 protein levels were determined by Western blot. MAP1LC3B mRNA levels were assessed by RT-qPCR. Ang II increased MAP1LC3B mRNA levels and protein levels of LC3 I, LC3 II and total LC3 (LC3 I + LC3 II). Cycloheximide, but not actinomycin D, abolished LC3 II and total LC3 increase elicited by Ang II in RASMCs. In A7r5 cells, cycloheximide prevented the Ang II-mediated increase of LC3 I and total LC3, but not LC3 II. Moreover, Ang II increased Bag3 levels, but this increase was not observed upon co-administration with either losartan 1 µM (AT1R antagonist) or Y-27632 10 µM (ROCK inhibitor). These results suggest that Ang II may regulate total LC3 content through transcriptional and translational mechanisms. Moreover, Bag3 is increased in response to Ang II by a AT1R/ROCK signalling pathway. These data provide preliminary evidence suggesting that Ang II may stimulate autophagy in VSMCs by increasing total LC3 content and LC3 processing.
Assuntos
Angiotensina II , Músculo Liso Vascular , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Angiotensina II/metabolismo , Angiotensina II/farmacologia , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Células Cultivadas , Cicloeximida/metabolismo , Cicloeximida/farmacologia , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , RNA Mensageiro/genética , RatosRESUMO
KEY MESSAGE: The moss Pseudocrossidium replicatum is a desiccation-tolerant species that uses an inducible system to withstand severe abiotic stress in both protonemal and gametophore tissues. Desiccation tolerance (DT) is the ability of cells to recover from an air-dried state. Here, the moss Pseudocrossidium replicatum was identified as a fully desiccation-tolerant (FDT) species. Its gametophores rapidly lost more than 90% of their water content when exposed to a low-humidity atmosphere [23% relative humidity (RH)], but abscisic acid (ABA) pretreatment diminished the final water loss after equilibrium was reached. P. replicatum gametophores maintained good maximum photosystem II (PSII) efficiency (Fv/Fm) for up to two hours during slow dehydration; however, ABA pretreatment induced a faster decrease in the Fv/Fm. ABA also induced a faster recovery of the Fv/Fm after rehydration. Protein synthesis inhibitor treatment before dehydration hampered the recovery of the Fv/Fm when the gametophores were rehydrated after desiccation, suggesting the presence of an inducible protective mechanism that is activated in response to abiotic stress. This observation was also supported by accumulation of soluble sugars in gametophores exposed to ABA or NaCl. Exogenous ABA treatment delayed the germination of P. replicatum spores and induced morphological changes in protonemal cells that resembled brachycytes. Transcriptome analyses revealed the presence of an inducible molecular mechanism in P. replicatum protonemata that was activated in response to dehydration. This study is the first RNA-Seq study of the protonemal tissues of an FDT moss. Our results suggest that P. replicatum is an FDT moss equipped with an inducible molecular response that prepares this species for severe abiotic stress and that ABA plays an important role in this response.
Assuntos
Adaptação Fisiológica/genética , Bryopsida/genética , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica de Plantas/genética , Ácido Abscísico/farmacologia , Adaptação Fisiológica/efeitos dos fármacos , Alfa-Amanitina/farmacologia , Bryopsida/metabolismo , Cicloeximida/farmacologia , Desidratação , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Geografia , México , Inibidores da Síntese de Ácido Nucleico/farmacologia , Reguladores de Crescimento de Plantas/farmacologia , Inibidores da Síntese de Proteínas/farmacologia , RNA-Seq/métodos , Estresse Fisiológico , Fatores de TempoRESUMO
PURPOSE: The incidence of fungal infection after corneal transplant has increased significantly in recent years, especially Candida spp. This study aimed to evaluate the efficacy and safety of the addition of cycloheximide in Optisol-GS media in decreasing the growth of Candida spp. strains. METHODS: This in vitro laboratory efficacy study measured fungal colony growth in 24 vials of Optisol-GS that were divided into 6 groups of 4 vials each, as follows: (1) MIC/2 cycloheximide, (2) MIC cycloheximide, (3) MICx5 cycloheximide, (4) MICx10 cycloheximide, from MIC values obtained for each strain, (5) unsupplemented optisol-GS as a positive control (added inoculum), and (6) unsupplemented optisol-GS as a negative control (no inoculum). In each group was added Candida albicans, C. glabrata and C. parapsilosis, except in the negative control. The evaluated variables were fungal colony growth from the Optisol-GS vials, corneal endothelial cell density and endothelial cell viability at different concentrations of cycloheximide. RESULTS: In the efficacy study, all strains showed a reduction in fungal cell growth from the second day at all evaluated concentrations of optisol-GS supplemented with cycloheximide, even at subinhibitory concentrations (MIC/2). For C. glabrata, the colony count was reduced to 99%. No evidence of corneal endothelial toxicity was found at any concentration, in the safety study, compared with the paired control. CONCLUSION: The addition of cycloheximide to optisol-GS decreased the fungal growth, demonstrating fungicide action against C. glabrata and fungistatic action against C. albicans and C. parapsilosis. This drug did not demonstrate toxicity to the corneal endothelium at different concentrations.
Assuntos
Antifúngicos/farmacologia , Candida/efeitos dos fármacos , Sulfatos de Condroitina/farmacologia , Cicloeximida/farmacologia , Dextranos/farmacologia , Gentamicinas/farmacologia , Candida/crescimento & desenvolvimento , Misturas Complexas/farmacologia , Testes de Sensibilidade MicrobianaRESUMO
Memory formation depends upon several parametric training conditions. Among them, trial number and inter-trial interval (ITI) are key factors to induce long-term retention. However, it is still unclear how individual training trials contribute to mechanisms underlying memory formation and stabilization. Contextual conditioning in Neohelice granulata has traditionally elicited associative long-term memory (LTM) after 15 spaced (ITI = 3 min) trials. Here, we show that LTM in crabs can be induced after only two training trials by increasing the ITI to 45 min (2t-LTM) and maintaining the same training duration as in traditional protocols. This newly observed LTM was preserved for at least 96 h, exhibiting protein synthesis dependence during consolidation and reconsolidation as well as context-specificity. Moreover, we demonstrate that 2t-LTM depends on inter-trial and post-training ERK activation showing a faster phosphorylation after the second trial compared to the first one. In summary, we present a new training protocol in crabs through a reduced number of trials showing associative features similar to traditional spaced training. This novel protocol allows for intra-training manipulation and the assessment of individual trial contribution to LTM formation.
Assuntos
Comportamento Animal/fisiologia , Braquiúros/fisiologia , Consolidação da Memória/fisiologia , Memória de Longo Prazo/fisiologia , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Prática Psicológica , Inibidores de Proteínas Quinases/farmacologia , Inibidores da Síntese de Proteínas/farmacologia , Animais , Cicloeximida/farmacologia , Dimetil Sulfóxido/administração & dosagem , Flavonoides/farmacologia , Masculino , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores da Síntese de Proteínas/administração & dosagemRESUMO
The NMDA receptor is crucial to several functions in CNS physiology and some of its effects are mediated by promoting nitric oxide production from L-arginine and activation of signaling pathways and the transcription factor CREB. Our previous work demonstrated in retinal cells that increasing intracellular free L-arginine levels directly correlates to nitric oxide (NO) generation and can be promoted by protein synthesis inhibition and increase of free L-arginine concentration. Eukaryotic elongation factor 2 kinase (eEF2K), a calcium/calmodulin-dependent kinase, is also known to be activated by NMDA receptors leading to protein synthesis inhibition. Here we explored how does eEF2K participate in NMDA-induced NO signaling. We found that when this enzyme is inhibited, NMDA loses its ability to promote NO synthesis. On the other hand, when NO synthesis is increased by protein synthesis inhibition with cycloheximide or addition of exogenous L-arginine, eEF2K has no participation, showcasing a specific link between this enzyme and NMDA-induced NO signaling. We have previously shown that inhibition of the canonical NO signaling pathway (guanylyl cyclase/cGMP/cGK) blocks CREB activation by glutamate in retinal cells. Interestingly, pharmacological inhibition of eEF2K fully prevents CREB activation by NMDA, once again demonstrating the importance of eEF2K in NMDA receptor signaling. In summary, we demonstrated here a new role for eEF2K, directly controlling NMDA-dependent nitrergic signaling and modulating L-arginine availability in neurons, which can potentially be a new target for the study of physiological and pathological processes involving NMDA receptors in the central nervous system.
Assuntos
Sistema Nervoso Central/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Quinase do Fator 2 de Elongação/metabolismo , N-Metilaspartato/farmacologia , Óxido Nítrico/biossíntese , Animais , Arginina/farmacologia , Galinhas , Cicloeximida/farmacologia , Quinase do Fator 2 de Elongação/antagonistas & inibidores , Indazóis/farmacologia , Masculino , Fosforilação/efeitos dos fármacos , Piridinas/farmacologia , Pirimidinas/farmacologia , RatosRESUMO
Extinction is a process that involves new learning that inhibits the expression of previously acquired memories. Although temporarily effective, extinction does not erase an original fear association. Since the extinction trace tends to fade over time, the original memory can resurge. On the other hand, strengthening effects have been described in several reconsolidation studies using different behavioral and pharmacological manipulations. In order to know whether an extinction memory can be strengthened by reactivation-based interventions in the contextual fear conditioning task, we began by replicating the classic phenomenon of spontaneous recovery to show that brief reexposure sessions can prevent the decay of the extinction trace over time in a long-lasting way. This fear attenuation was shown to depend both on L-type calcium channels and protein synthesis, which suggests a reconsolidation process behind the reactivation-induced strengthening effect. The extinction trace was also susceptible to enhancement by a post-reactivation infusion of a memory-enhancing drug (NaB), which was also able to prevent rapid fear reacquisition (savings). These findings point to new reactivation-based approaches able to strengthen an extinction memory to promote its persistence. The constructive interactions between extinction and reconsolidation may represent a promising novel approach in the realm of fear-related disorder treatments.
Assuntos
Condicionamento Clássico , Extinção Psicológica/efeitos dos fármacos , Memória/efeitos dos fármacos , Animais , Ácido Butírico/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Cicloeximida/farmacologia , Extinção Psicológica/fisiologia , Medo , Inibidores de Histona Desacetilases/farmacologia , Masculino , Memória/fisiologia , Nimodipina/farmacologia , Inibidores da Síntese de Proteínas/farmacologia , Ratos , Ratos WistarRESUMO
Early leaf yellowing in cut alstroemeria (Alstroemeria aurantiaca) flowers before flower development and petal abscission is an important limiting postharvest quality and vase life factors. Early leaf senescence reduces postharvest longevity of cut flowers and promotes petal's wilting. A study was made to evaluate the response of cut alstroemeria flowers at varying concentrations of cycloheximide (CHI) (50, 100 and 200 mg l-1), coconut water (5, 10 and 20%) and 6-benzyladenine (BA) (50, 100 and 200 mg l-1). CHI, coconut water and BA extended the vase life at all concentrations compared to the control, but coconut water at 5% concentration (with 17.39 days) was the most effective treatment. Control cut flowers showed the least vase life (10.76 days). Ethylene production in cut flowers promoted flower senescence. All concentrations of CHI, coconut water and BA delayed ethylene production compared to the control. Treatment of cut flowers with coconut water at concentration of 5% maintained the highest fresh weight of flowers and increased the content of water uptake. The chlorophyll degradation was significantly reduced by the application of CHI, coconut water and BA. The maximum content of membrane's lipid peroxidation and antioxidant enzymes activity (super oxide dismutase and peroxidase) was obtained in control cut flowers. Thus, 5% fresh coconut water has the potential to be applied as vase solution (preservative medium) due to prolongs of cut alstroemeria flowers.
O amarelecimento precoce das folhas em flores de alstroemeria (Alstroemeria aurantiaca) cortadas antes do desenvolvimento floral e da abscisão de pétalas é um importante limitante da qualidade pós-colheita e dos fatores de vida do vaso. A senescência precoce da folha reduz a longevidade pós-colheita das flores cortadas e promove o murchamento da pétala. Um estudo foi realizado para avaliar a resposta de flores de alstroemeria cortadas em diferentes concentrações de cicloheximida (CHI) (50, 100 e 200 mg l-1), água de coco (5, 10 e 20%) e 6-benziladenina (BA) 50, 100 e 200 mg l-1). CHI, água de coco e BA prolongou a vida do vaso em todas as concentrações em comparação com o controle, mas a água de coco a 5% de concentração (com 17,39 dias) foi o tratamento mais eficaz. As flores cortadas de controlo mostraram a menor vida útil do vaso (10,76 dias). A produção de etileno em flores cortadas promoveu a senescência da flor. Todas as concentrações de CHI, água de coco e BA atrasaram a produção de etileno em comparação com o controle. O tratamento de flores cortadas com água de coco a uma concentração de 5% manteve o maior peso fresco de flores e aumentou o conteúdo de absorção de água. A degradação da clorofila foi significativamente reduzida pela aplicação de CHI, água de coco e BA. O teor máximo de atividade de enzimas antioxidantes e de peroxidação lipídica da membrana (super óxido dismutase e peroxidase) foi obtido em flores cortadas de controle. Assim, 5% de água de coco fresca tem potencial para ser aplicada como solução de vaso (meio de conservação) devido a prolongamentos das flores de alstroemeria cortadas.
Assuntos
Reguladores de Crescimento de Plantas , Cicloeximida , Alstroemeria , Alimentos de CocoRESUMO
Cystic fibrosis (CF) is caused by mutations in the CFTR gene, which encodes a cAMP-regulated chloride channel. Several cellular functions are altered in CF cells. However, it is not clear how the CFTR failure induces those alterations. We have found previously several genes differentially expressed in CF cells, including c-Src, MUC1, MTND4, and CISD1 (CFTR-dependent genes). Recently, we also reported the existence of several chloride-dependent genes, among them GLRX5 and RPS27. Here, varying the intracellular chloride concentration [Cl- ]i of IB3-1 CF bronchial epithelial cells, we show that IL-1ß mRNA expression and secretion are also under Cl- modulation. The response to Cl- is biphasic, with maximal effects at 75 mM Cl- . The regulation of the IL-1ß mRNA expression involves an IL-1ß autocrine effect, since in the presence of the IL-1ß receptor antagonist IL1RN or anti-IL-1ß blocking antibody, the mRNA response to Cl- disappeared. Similar effects were obtained with the JNK inhibitor SP600125, the c-Src inhibitor PP2 and the IKK inhibitor III (BMS-345541). On the other hand, the IL-1ß secretion is still modulated by Cl- in the presence of IL-1RN, IL-1ß blocking antibody, or cycloheximide, suggesting that Cl- is affecting the IL-1ß maturation/secretion, which in turn starts an autocrine positive feedback loop. In conclusion, the Cl- anion acts as a second messenger for CFTR, modulating the IL-1ß maturation/secretion. The results also imply that, depending on its intracellular concentration, Cl- could be a pro-inflammatory mediator. J. Cell. Biochem. 118: 2131-2140, 2017. © 2016 Wiley Periodicals, Inc.
Assuntos
Brônquios/citologia , Cloretos/farmacologia , Células Epiteliais/metabolismo , Interleucina-1beta/metabolismo , Antracenos/farmacologia , Western Blotting , Linhagem Celular , Cicloeximida/farmacologia , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células Epiteliais/efeitos dos fármacos , Humanos , Proteína Antagonista do Receptor de Interleucina 1/metabolismo , Interleucina-1beta/antagonistas & inibidores , Interleucina-1beta/genética , Interleucina-6/metabolismo , Pirimidinas/farmacologia , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Tumor necrosis factor alpha (TNF-α) is a potent proinflammatory cytokine that plays a significant role in the pathogenesis of asthma by inducing hyperresponsiveness and airway remodeling. TNF-α diminishes the L-type voltage dependent Ca(2+) channel (L-VDCC) current in cardiac myocytes, an observation that seems paradoxical. In guinea pig sensitized tracheas KCl responses were lower than in control tissues. Serum from sensitized animals (Ser-S) induced the same phenomenon. In tracheal myocytes from nonsensitized (NS) and sensitized (S) guinea pigs, an L-VDCC current (ICa) was observed and diminished by Ser-S. The same decrease was detected in NS myocytes incubated with TNF-α, pointing out that this cytokine might be present in Ser-S. We observed that a small-molecule inhibitor of TNF-α (SMI-TNF) and a TNF-α receptor 1 (TNFR1) antagonist (WP9QY) reversed ICa decrease induced by Ser-S in NS myocytes, confirming the former hypothesis. U0126 (a blocker of ERK 1/2 kinase) also reverted the decrease in ICa. Neither cycloheximide (a protein synthesis inhibitor) nor actinomycin D (a transcription inhibitor) showed any effect on the TNF-α-induced ICa reduction. We found that CaV1.2 and CaV1.3 mRNA and proteins were expressed in tracheal myocytes and that sensitization did not modify them. In cardiac myocytes, ERK 1/2 phosphorylates two sites of the L-VDCC, augmenting or decreasing ICa; we postulate that, in guinea pig tracheal smooth muscle, TNF-α diminishes ICa probably by phosphorylating the L-VDCC site that reduces its activity through the ERK1/2 MAP kinase pathway.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Músculo Liso/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Animais , Butadienos/farmacologia , Células Cultivadas , Cicloeximida/farmacologia , Dactinomicina/farmacologia , Cobaias , Células Musculares/efeitos dos fármacos , Células Musculares/metabolismo , Nitrilas/farmacologia , Peptídeos Cíclicos/farmacologia , Traqueia/citologia , Fator de Necrose Tumoral alfa/antagonistas & inibidoresRESUMO
Increasing the efficiency of intracytoplasmic sperm injection (ICSI) in domestic animals has been attempted by many researchers, however embryonic development to the blastocyst stage remains low compared with that of in vitro fertilization (IVF) embryos. One of the main problems observed in cattle is inadequate oocyte activation after ICSI. The present study compared the effect of cycloheximide (CHX), 6-dimethylaminopurine (DMAP), and anisomycin (ANY) on the fertilization rate, development, ploidy and quality of bovine embryos generated by ICSI. Although no differences were observed between treatments in terms of cleavage, higher blastocyst rates were observed for ANY (37.3%) compared with CHX (21.8%, P 0.05) treatments. No differences were observed in the quality of embryos as assessed by the total number of cells, their distribution to the different embryo compartments [inner cell mass (ICM) and trophectoderm (TE)], the proportion of ICM cells to the total cell numbers and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL)-positive cells. Similarly, no differences were observed in the normal ploidy of embryos (56, 67, and 55%) for ANY, CHX and DMAP, respectively. However, higher fertilization rates were observed for ANY (75%) and CHX (87%) treatments compared with DMAP (35%). In conclusion, ANY showed a superior developmental rate compared with CHX treatment. Although no significant differences were observed compared with an improved protocol of DMAP (2Io-DMAP), the lower fertilization rate recorded with DMAP strongly suggests that ANY could be a better alternative for oocyte activation than traditional chemical compounds used currently in ICSI.
Assuntos
Anisomicina/farmacologia , Blastocisto/efeitos dos fármacos , Ploidias , Inibidores da Síntese de Proteínas/farmacologia , Injeções de Esperma Intracitoplásmicas/veterinária , Adenina/análogos & derivados , Adenina/farmacologia , Animais , Blastocisto/citologia , Blastocisto/fisiologia , Bovinos , Cicloeximida/farmacologia , Técnicas de Cultura Embrionária , Feminino , Masculino , Injeções de Esperma Intracitoplásmicas/métodosRESUMO
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
Leishmania contains two phosphoglycerate kinase (PGK) genes, PGKB and PGKC, which code for the cytosolic and glycosomal isoforms of the enzyme, respectively. Although differences in PGKB and PGKC transcript and protein levels and isoform activities have been well documented, the mechanisms of control of both transcript and protein abundance have not been described to date. To better understand the regulation of Leishmania PGK expression, we investigated the stabilities of both PGK transcripts using reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) in combination with transcription and trans-splicing inhibitors. Cells were treated with sinefungin and actinomycin D, and RNA decay kinetics were assessed. In addition, immunoblotting and protein synthesis inhibition by cycloheximide were employed to evaluate protein steady states and degradation. We observed increased stabilities of both PGKB mRNA and protein compared with the glycosomal isoform (PGKC). Our results indicate that both post-transcriptional and post-translational events contribute to the distinct expression levels of the PGKB and PGKC isoforms in Leishmania major.
Assuntos
Leishmania major/enzimologia , Fosfoglicerato Quinase/genética , Adenosina/análogos & derivados , Adenosina/farmacologia , Antiprotozoários/farmacologia , Cicloeximida/farmacologia , Citosol/enzimologia , Dactinomicina/farmacologia , Regulação da Expressão Gênica , Meia-Vida , Immunoblotting , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Leishmania major/efeitos dos fármacos , Leishmania major/genética , Microcorpos/enzimologia , Peso Molecular , Fosfoglicerato Quinase/química , Fosfoglicerato Quinase/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , RNA Mensageiro/metabolismo , RNA de Protozoário/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição GênicaRESUMO
E2F transcription factors regulate a wide range of biological processes, including the cellular response to DNA damage. In the present study, we examined whether E2F family members are transcriptionally induced following treatment with several genotoxic agents, and have a role on the cell DNA damage response. We show a novel mechanism, conserved among diverse species, in which E2F1 and E2F2, the latter specifically in neuronal cells, are transcriptionally induced after DNA damage. This upregulation leads to increased E2F1 and E2F2 protein levels as a consequence of de novo protein synthesis. Ectopic expression of these E2Fs in neuronal cells reduces the level of DNA damage following genotoxic treatment, while ablation of E2F1 and E2F2 leads to the accumulation of DNA lesions and increased apoptotic response. Cell viability and DNA repair capability in response to DNA damage induction are also reduced by the E2F1 and E2F2 deficiencies. Finally, E2F1 and E2F2 accumulate at sites of oxidative and UV-induced DNA damage, and interact with γH2AX DNA repair factor. As previously reported for E2F1, E2F2 promotes Rad51 foci formation, interacts with GCN5 acetyltransferase and induces histone acetylation following genotoxic insult. The results presented here unveil a new mechanism involving E2F1 and E2F2 in the maintenance of genomic stability in response to DNA damage in neuronal cells.
Assuntos
Dano ao DNA , Fator de Transcrição E2F1/metabolismo , Fator de Transcrição E2F2/metabolismo , Instabilidade Genômica , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Cicloeximida/toxicidade , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/efeitos da radiação , Reparo do DNA/efeitos dos fármacos , Dactinomicina/toxicidade , Fator de Transcrição E2F1/genética , Fator de Transcrição E2F2/genética , Instabilidade Genômica/efeitos dos fármacos , Instabilidade Genômica/efeitos da radiação , Células HEK293 , Histonas/metabolismo , Humanos , Peróxido de Hidrogênio/toxicidade , MAP Quinase Quinase Quinases/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Inibidores da Síntese de Proteínas/toxicidade , Rad51 Recombinase/metabolismo , Raios Ultravioleta , Regulação para Cima/efeitos dos fármacos , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
Long-term memory of active avoidance in mice is not disturbed by administration of protein synthesis inhibitors (PSIs) when relatively high levels of training are used, whereas a detrimental effect is produced with lower levels of training. PSIs also disrupt extinction of avoidance behaviors in rodents, but it is not clear whether PSIs also affect this form of learning when the behavior to be extinguished was produced by a high level of training. Experiment 1 demonstrated that rats treated with the PSI cycloheximide (CXM) 30 min before training developed normal acquisition after training with either high or low foot-shock stimulation, but that memory consolidation was hindered only after low foot-shock training. Experiment 2 demonstrated that CXM disrupted extinction when administered before the first of a series of extinction sessions when low foot-shock intensity was used during training; in contrast, after training with a higher foot-shock, the PSI treatment only interfered transiently with extinction. These results indicate that acquisition, consolidation, and extinction of active avoidance learning produced by high aversive stimulation are not dependent on protein synthesis and that these processes are governed by mechanisms different from those underlying moderate forms of learning.
Assuntos
Aprendizagem da Esquiva/fisiologia , Cicloeximida/farmacologia , Extinção Psicológica/fisiologia , Consolidação da Memória/fisiologia , Inibidores da Síntese de Proteínas/farmacologia , Animais , Aprendizagem da Esquiva/efeitos dos fármacos , Eletrochoque , Extinção Psicológica/efeitos dos fármacos , Masculino , Consolidação da Memória/efeitos dos fármacos , Ratos , Ratos WistarRESUMO
The compounds 6-dimethylaminopurine (6-DMAP) and cyclohexamide (CHX) are currently used to stimulate the development of embryos produced by nuclear transfer in the production of cloned mammals with a great deal success. This study investigated the effects of 6-DMAP and CHX in vivo using biological assays to evaluate reproductive performance in females, teratogenesis, and mutagenesis. The results of this study demonstrated that the activating agents of oocyte cytoplasm, 6-DMAP and CHX, altered the reproductive performance of the experimental animals, as well as increased the rate malformations. In addition to these adverse effects, the administration of these compounds in pregnant females resulted in genotoxic and mutagenic toxicity, as determined by comet and micronucleus assays carried out in peripheral blood samples, respectively. Based on these findings and that alterations in DNA are important, morpho-functional teratogenesis and diminished embryonic viability, suggesting that 6-DMAP and CHX, which are utilized during the cloning of mammals, are responsible for the fact that embryos produced by nuclear transfer show low viability after implantation in utero or after birth because of congenital malformations and/or alterations in their DNA.
Assuntos
Adenina/análogos & derivados , Clonagem de Organismos , Cicloeximida/efeitos adversos , Mutagênicos/efeitos adversos , Reprodução/efeitos dos fármacos , Adenina/efeitos adversos , Animais , Transferência Embrionária , Feminino , Camundongos , Gravidez , Reprodução/genética , Teratogênese/efeitos dos fármacosRESUMO
Human prolactin (hPRL) is a polypeptide hormone occurring in the non-glycosylated (NG-hPRL) and glycosylated (G-hPRL) forms, with MM of approximately 23 and 25kDa, respectively. It has a single, partially occupied N-glycosylation site located at Asn-31, which makes it a particularly simple and interesting model for glycosylation studies. The bioactivity of G-hPRL is lower than that of NG-hPRL (by ca. 4-fold) and its physiological function is not clear. However, carbohydrate moieties generally play important roles in the biosynthesis, secretion, biological activity, and plasma survival of glycohormones and can vary depending on the host cell. The main objective of this study was to determine the N-glycan structures present in native, pituitary G-hPRL and compare them with those present in the recombinant hormone. To obtain recombinant G-hPRL, genetically modified Chinese hamster ovary cells (CHO), adapted to growth in suspension, were treated with cycloheximide, thus increasing the glycosylation site occupancy from 5.5% to 38.3%, thereby facilitating G-hPRL purification. CHO cell-derived G-hPRL (CHO-G-hPRL) was compared to pituitary G-hPRL (pit-G-hPRL) especially with regard to N-glycoprofiling. Among the main differences found in the pituitary sample were an extremely low presence of sialylated (1.7%) and a high percentage of sulfated (74.0%) and of fucosylated (90.5%) glycans. A â¼6-fold lower in vitro bioactivity and a higher clearance rate in mice were also found for pit-G-hPRL versus CHO-G-hPRL. N-Glycan profiling proved to be a useful and accurate methodology also for MM and carbohydrate content determination for the two G-hPRL preparations, in good agreement with the values obtained directly via MALDI-TOF-MS.
Assuntos
Polissacarídeos/química , Prolactina/química , Prolactina/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Cicloeximida/farmacologia , Citoproteção/efeitos dos fármacos , Glicosilação , Humanos , Camundongos , Prolactina/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Kinins are pro-inflammatory peptides that mimic the cardinal features of inflammation. We examined the concept that expression levels of endothelial intercellular adhesion molecule-1 (ICAM-1) and neutrophil integrins Mac-1 and LFA-1 are modulated by the kinin B1 receptor (B1R) agonist, Lys-des[Arg(9)]bradykinin (LDBK). Stimulation of endothelial cells with LDBK increased the levels of ICAM-1 mRNA transcripts/protein, and also of E-selectin and platelet endothelial adhesion molecule-1. ICAM-1 levels increased in a magnitude comparable with that produced by TNF-α. This stimulatory effect was reduced when endothelial cells, which had been previously transfected with a B1R small interfering RNA, were stimulated with LDBK, under comparable conditions. Similarly, LDBK produced a significant increase in protein levels of LFA-1 and Mac-1 integrins in human neutrophils, an effect that was reversed by pretreatment of cells with 10 µg/ml cycloheximide or a B1R antagonist. Functional experiments performed with post-confluent monolayers of endothelial cells stimulated with LDBK and neutrophils primed with TNF-α, and vice versa, resulted in enhanced adhesiveness between both cells. Neutralizing Abs to ICAM-1 and Mac-1 reduced the adhesion between them. Our results indicate that kinin B1R is a novel modulator that promotes adhesion of leukocytes to endothelial cells, critically enhancing the movement of neutrophils from the circulation to sites of inflammation.
Assuntos
Células Endoteliais/imunologia , Inflamação/imunologia , Calidina/análogos & derivados , Neutrófilos/efeitos dos fármacos , Receptor B1 da Bradicinina/metabolismo , Adesão Celular/efeitos dos fármacos , Comunicação Celular , Movimento Celular , Células Cultivadas , Cicloeximida/farmacologia , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Calidina/farmacologia , Antígeno-1 Associado à Função Linfocitária/metabolismo , Antígeno de Macrófago 1/metabolismo , Neutrófilos/imunologia , RNA Interferente Pequeno/genética , Receptor B1 da Bradicinina/agonistas , Receptor B1 da Bradicinina/genéticaRESUMO
OBJECTIVE: to relate neck circumference with metabolic syndrome and its criteria among college students. METHOD: cross-sectional study conducted with 702 college students in Fortaleza, CE, Brazil from September 2010 to June 2011. Socio-demographic data, waist circumference and neck circumference were collected together with blood pressure, fasting blood sugar, triglyceride levels, and HDL-C. RESULTS: 1.7% of the studied sample presented metabolic syndrome. Of these, 58.3% presented altered neck circumference (p<0.006). As neck circumference decreases, pressure levels improve (p<0.001). Additionally, college students with high fasting blood sugar (p=0.003) and high triglyceride levels (p<0.001) presented higher values of neck circumference. CONCLUSION: neck circumference is a potential predictive marker in the detection of metabolic syndrome and its components among college students. .
OBJETIVO: relacionar a circunferência do pescoço com a síndrome metabólica e seus critérios em universitários. MÉTODO: estudo transversal, realizado com 702 universitários de Fortaleza, CE, Brasil, no período de setembro de 2010 a junho de 2011. Coletaram-se dados sociodemográficos, circunferência da cintura, circunferência do pescoço, níveis de pressão arterial e glicemia plasmática de jejum, triglicerídeos e lipoproteína de alta densidade. RESULTADOS: 1,7% da amostra investigada tinha a síndrome metabólica. Desses, 58,3% apresentaram circunferência do pescoço alterada (p<0,006). Na medida em que decresce a circunferência do pescoço, os valores pressóricos dos universitários melhoram (p<0,001). Também, observou-se que universitários com valores de glicemia de jejum plasmática (p=0,003) e triglicerídeos (p<0,001) elevados apresentaram maiores valores de circunferência do pescoço. CONCLUSÃO: a circunferência do pescoço mostrou-se um possível marcador preditivo para detecção da síndrome metabólica e seus componentes em universitários. .
OBJETIVO: relacionar la circunferencia del cuello con el síndrome metabólico y sus criterios en universitarios. MÉTODO: estudio transversal realizado con 702 universitarios de Fortaleza-CE, Brasil, en el período de septiembre de 2010 a junio de 2011. Se recolectaron datos sociodemográficos, circunferencia de la cintura, circunferencia del cuello, niveles de presión arterial y glucemia plasmática de ayuno, triglicéridos y HDL-C. RESULTADOS: 1,7% de la muestra investigada tenían el síndrome metabólico. De estos, 58,3% presentaron circunferencia del cuello alterada (p<0,006). A medida que decrece la circunferencia del cuello mejoran los valores de la presión de los universitarios (p<0,001). También, se observó que los universitarios con valores de glucemia de ayuno plasmática (p=0,003) y triglicéridos (p<0,001) elevados presentaron mayores valores de circunferencia del cuello. CONCLUSIÓN: la circunferencia del cuello se mostró un posible indicador de predicción para la detección del síndrome metabólico y sus componentes, en universitarios. .