RESUMEN
OBJECTIVE: The aim: of the work was to study the antiviral activity of the metabolites of the probiotic strain Lactobacillus rhamnosus GG (LGG or ATCC 53103) regarding clinical strains of enteroviruses (Coxsackie B-5, ECNO21) isolated from the feces of intestinal infections. PATIENTS AND METHODS: Materials and methods: The object of the study was substrate-dependent cell cultures of HeLa, Vero, Hep-2 lines. The titer of the virus was determined by the presence of a clear cytopathic action (CPA) in the monolayer infected cells of the virus. RESULTS: Results: Determination of the enteric virus infections activity in the culture fluid showed that in samples with the LGG metabolites, the infections activity of the clinical strains of enteroviruses decreased after 24 hours, at 1.5-1.7 (p <0.05) times, and after 96 hours in 3, 6 - 5,7 times (p <0,01). the processing of cell cultures by metabolites in the amount of 0.3 mg / ml contributed to a decrease in the titer of viruses by 2.77 ± 0.11 lg TCDD50 / cm3, 2.83 ± 0.11 lg TCD50 / cm3 and 2.94 ± 0.13 lg TCD50 / cm3 for Vero, HeLa and Hep-2 line cells in 24 hours. CONCLUSION: Conclusions: It has been experimentally determined that the maximum tolerated dose (MTD) of L. rhamnosus GG metabolites was 0.3 µg / ml for all cultures of cell lines. Determination of the antiviral activity of L. rhamnosus GG metabolites in clinical viruses of enteroviruses (Coxsackie B-5 and ECNO-21) showed a decrease in infection activity in 1.5-1.7 times, (p <0.05) of clinical trials in clinical trials enteroviruses.
Asunto(s)
Infecciones por Enterovirus , Lacticaseibacillus rhamnosus , Probióticos , Antivirales , Heces , HumanosRESUMEN
Programmed cell death 4 (Pdcd4) is frequently suppressed in tumors of various origins and its suppression correlates with tumor progression. Pdcd4 inhibits cap-dependent translation from mRNAs with highly structured 5'-regions through interaction with the eukaryotic translation initiation factor 4A (eIF4A) helicase and a target transcript. Decrease in Pdcd4 protein is believed to provide a relief of otherwise suppressed eIF4A-dependent translation of proteins facilitating tumor progression. However, it remains unknown if lowered Pdcd4 levels in cells suffices to cause a relief in translation inhibition through appearance of the Pdcd4-free translation-competent eIF4A protein, or more complex and selective mechanisms are involved. Here we showed that eIF4A1, the eIF4A isoform involved in translation, significantly over-represents Pdcd4 both in cancerous and normal cells. This observation excludes the possibility that cytoplasmic Pdcd4 can efficiently exert its translation suppression function owing to excess of eIF4A, with Pdcd4-free eIF4A being in excess over Pdcd4-bound translation-incompetent eIF4A, thus leaving translation from Pdcd4 mRNA targets unaffected. This contradiction is resumed in the proposed model, which supposes initial complexing between Pdcd4 and its target mRNAs in the nucleus, with subsequent transport of translation-incompetent, Pdcd4-bound target mRNAs into the cytoplasm. Noteworthy, loss of nuclear Pdcd4 in cancer cells was reported to correlate with tumor progression, which supports the proposed model of Pdcd4 functioning.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/genética , Factor 4A Eucariótico de Iniciación/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias/genética , ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Transporte Biológico , Muerte Celular/genética , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Factor 4A Eucariótico de Iniciación/metabolismo , Humanos , Modelos Genéticos , Neoplasias/metabolismo , Neoplasias/patología , Unión Proteica , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Transducción de SeñalRESUMEN
Agarose gel electrophoresis with subsequent DNA extraction from gel is routinely used for DNA fragment isolation after plasmid DNA digestion. We describe a gel-less method for DNA fragment isolation after plasmid DNA digestion which is based on in-solution negative selection through depletion of vector backbone bearing LoxP sites by sorption on solid phase-immobilized mutated Cre recombinase. The method might be especially useful in preparation of DNA fragments for transgenic animal generation where residual agarose presence is a concern, and DNA fragments are frequently large in size and thus might be mechanically damaged during purification with conventional affinity-based gel extraction methods.
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ADN/aislamiento & purificación , Vectores Genéticos , Integrasas/genética , Plásmidos/aislamiento & purificación , Animales , Animales Modificados Genéticamente , ADN/genética , Plásmidos/genéticaRESUMEN
MAK-V/Hunk is a scantily characterized AMPK-like protein kinase. Recent findings identified MAK-V as a pro-survival and anti-apoptotic protein and revealed its role in embryonic development as well as in tumorigenesis and metastasis. However molecular mechanisms of MAK-V action and regulation of its activity remain largely unknown. We identified Nedd4 as an interaction partner for MAK-V protein kinase. However, this HECT-type E3 ubiquitin ligase is not involved in the control of MAK-V degradation by the ubiquitin-proteasome system that regulates MAK-V abundance in cells. However, Nedd4 in an ubiquitin ligase-independent manner rescued developmental defects in Xenopus embryos induced by MAK-V overexpression, suggesting physiological relevance of interaction between MAK-V and Nedd4. This identifies Nedd4 as the first known regulator of MAK-V function.
Asunto(s)
Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Proteínas Quinasas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Western Blotting , Cicloheximida/farmacología , Electroforesis en Gel de Poliacrilamida , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Humanos , Ratones , Ubiquitina-Proteína Ligasas Nedd4 , Células PC12 , Unión Proteica , Proteínas Quinasas/genética , Ratas , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación/efectos de los fármacos , Proteínas de XenopusRESUMEN
Pdcd4 (programmed cell death 4) gene is tumor suppressor which expression is frequently down-regulated in tumors, which is considered as a diagnostic and prognostic marker as well as promising target for anti-cancer therapy. Pdcd4 protein is a target for post-translational regulation by phosphorylation marking Pdcd4 for degradation. We questioned if Pdcd4 mRNA decline in human lung tumors is accompanied by proportional depletion of Pdcd4 protein. We found that Pdcd4 protein-to-mRNA ratio varies greatly in human lung cancer cell lines. In squamous cell carcinoma samples where Pdcd4 mRNA suppression was found to be a typical event, Pdcd4 protein level frequently remained unchanged or even up-regulated. Our studies demonstrate that at least in squamous cell carcinoma, alterations in Pdcd4 mRNA and protein levels are not directly linked, and this fact should be taken into consideration when developing Pdcd4-based anti-cancer therapeutic approaches.