Publications by authors named "Silvia Prado"

16 Publications

  • Page 1 of 1

Determination of minimum bactericidal concentration, in single or combination drugs, against .

Future Microbiol 2020 01 17;15:107-114. Epub 2020 Feb 17.

Postgraduate Program in Bioscience and Physiopathology, State University of Maringá, Paraná, Brazil.

To evaluate an assay to detect minimum bactericidal concentration (MBC) in , using as single model rifampicin, isoniazid, levofloxacin (LVX) and linezolid (LNZ) and in combination. MBCs were carried out directly from resazurin microtiter assay plate and 3D checkerboard in HRv and five resistant clinical isolates. The proposed MBC assay showed similar values to those determined by MGIT™, used as control. LVX and LNZ's MBC values were close to their MIC values. LNZ or LVX combined with isoniazid and rifampicin showed MBC value reduced in 63.7% of the assays. The proposed assay to determine MBCs of drugs can be applied to the study of new compounds with anti- activity to detect their bactericidal effect and also in laboratory routine for clinical dose adjustment of drugs according to the patient's profile.
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http://dx.doi.org/10.2217/fmb-2019-0050DOI Listing
January 2020

Measurement of Free Iodine in Different Formulations of Povidone-Iodine Eye Drops 5.

Int J Pharm Compd 2019 Sep-Oct;23(5):418-421

Pharmacy Services, Instituto Oftalmológico Fernández Vega, Oviedo, Spain.

There are multiple studies in the literature that support the use of povidone iodine in the preparation of the surgical field of cataract as the most effective means to reduce the bacteria present in the ocular surface and the risk of infection. The concentration of free iodine is related to the antiseptic activity of these compounds, being, therefore, a good indicator of its effectiveness. The objective of this study was to determine the amount of free iodine and the evolution of it in different formulations of povidone iodine eye drops. The povidone iodine 5% eye drops were prepared starting from Betadine 10% dermal or the active principle and using a solvent, phosphate-citrate buffer solution, and sodium chloride 0.9% or sterile water for injection. Aliquots of 5 mL were packed in low-absorption absorption eye drops, topaz glass vials, and polyethylene syringe. The determination of free iodine was made by volumetric titration. Titration was performed with 0.1 M sodium thiosulfate using a starch solution as an indicator. Of the 0.1 M sodium thiosulfate, 1 mL is equivalent to 12.69 mg of available iodine, and it is expressed as a percentage of free iodine in the iodized povidone (% free iodine). Eyewash titrations were performed by replacing the substance with 5 mL of eye drops and following the remaining steps. Valuations were made on days 0, 7, and 14, as well as the measurement of pH and osmolarity. The results show that there are no differences between the average results at the three measurements taken on days 0, 7, and 14. We conclude that the free iodine remains stable during the stability period of 14 days. Regarding the pH and osmolarity data, the authors believe that the best tolerated formula will be that elaborated with povidone iodine and a phosphate- citrate buffer solution.
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October 2019

A small-molecule inhibitor of HIV-1 Rev function detected by a diversity screen based on RRE-Rev interference.

Biochem Pharmacol 2018 10 31;156:68-77. Epub 2018 Jul 31.

Facultad de Medicina, Universidad Católica de Valencia, C/Quevedo 2, 46001 Valencia, Spain. Electronic address:

The Rev protein of HIV-1 binds to the Rev Recognition Element (RRE) in the virus RNA to promote nuclear export of unspliced and partially spliced transcripts, an essential step in the virus transmission cycle. Here, we describe the screening of a library of chemically diverse compounds with an assay based on monitoring the interaction between the RNA-binding α-helix of Rev and its high-affinity binding site in the RRE. This screen allowed the identification of a benzofluorenone compound that inhibited the formation of the full-length RRE-Rev ribonucleoprotein by associating to the RRE, and blocked HIV-1 transcription and Rev action in cells. This molecule, previously studied as a cytostatic agent, had substantial antiretroviral activity. Together with other screening hits, it provides a new chemical scaffold for the development of antiretroviral agents based on blockage of HIV-1 RNA biogenesis.
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http://dx.doi.org/10.1016/j.bcp.2018.07.040DOI Listing
October 2018

An Alternative Homodimerization Interface of MnmG Reveals a Conformational Dynamics that Is Essential for Its tRNA Modification Function.

J Mol Biol 2018 08 2;430(17):2822-2842. Epub 2018 Jun 2.

Centro de Investigación Príncipe Felipe, Valencia 46012, Spain; Biomedical Research Networking Centre for Rare Diseases (CIBERER, Node 721), Valencia, Spain. Electronic address:

The Escherichia coli homodimeric proteins MnmE and MnmG form a functional complex, MnmEG, that modifies tRNAs using GTP, methylene-tetrahydrofolate, FAD, and glycine or ammonium. MnmE is a tetrahydrofolate- and GTP-binding protein, whereas MnmG is a FAD-binding protein with each protomer composed of the FAD-binding domain, two insertion domains, and the helical C-terminal domain. The detailed mechanism of the MnmEG-mediated reaction remains unclear partially due to incomplete structural information on the free- and substrate-bound forms of the complex. In this study, we show that MnmG can adopt in solution a dimer arrangement (form I) different from that currently considered as the only biologically active (form II). Normal mode analysis indicates that form I can oscillate in a range of open and closed conformations. Using isothermal titration calorimetry and native red electrophoresis, we show that a form-I open conformation, which can be stabilized in vitro by the formation of an interprotomer disulfide bond between the catalytic C277 residues, appears to be involved in the assembly of the MnmEG catalytic center. We also show that residues R196, D253, R436, R554 and E585 are important for the stabilization of form I and the tRNA modification function. We propose that the form I dynamics regulates the alternative access of MnmE and tRNA to the MnmG FAD active site. Finally, we show that the C-terminal region of MnmG contains a sterile alpha motif domain responsible for tRNA-protein and protein-protein interactions.
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http://dx.doi.org/10.1016/j.jmb.2018.05.035DOI Listing
August 2018

Bioavailable inhibitors of HIV-1 RNA biogenesis identified through a Rev-based screen.

Biochem Pharmacol 2016 May 17;107:14-28. Epub 2016 Feb 17.

Facultad de Medicina, Universidad Católica de Valencia, C/Quevedo 2, 46001 Valencia, Spain. Electronic address:

New antiretroviral agents with alternative mechanisms are needed to complement the combination therapies used to treat HIV-1 infections. Here we report the identification of bioavailable molecules that interfere with the gene expression processes of HIV-1. The compounds were detected by screening a small library of FDA-approved drugs with an assay based on measuring the displacement of Rev, and essential virus-encoded protein, from its high-affinity RNA binding site. The antiretroviral activity of two hits was based on interference with post-integration steps of the HIV-1 cycle. Both hits inhibited RRE-Rev complex formation in vitro, and blocked LTR-dependent gene expression and viral transcription in cellular assays. The best compound altered the splicing pattern of HIV-1 transcripts in a manner consistent with Rev inhibition. This mechanism of action is different from those used by current antiretroviral agents. The screening hits recognized the Rev binding site in the viral RNA, and the best compound did so with substantial selectivity, allowing the identification of a new RNA-binding scaffold. These results may be used for developing novel antiretroviral drugs.
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http://dx.doi.org/10.1016/j.bcp.2016.02.007DOI Listing
May 2016

Modification of the wobble uridine in bacterial and mitochondrial tRNAs reading NNA/NNG triplets of 2-codon boxes.

RNA Biol 2014 ;11(12):1495-507

a Laboratory of RNA Modification and Mitochondrial Diseases ; Centro de Investigación Príncipe Felipe ; Valencia , Spain.

Posttranscriptional modification of the uridine located at the wobble position (U34) of tRNAs is crucial for optimization of translation. Defects in the U34 modification of mitochondrial-tRNAs are associated with a group of rare diseases collectively characterized by the impairment of the oxidative phosphorylation system. Retrograde signaling pathways from mitochondria to nucleus are involved in the pathophysiology of these diseases. These pathways may be triggered by not only the disturbance of the mitochondrial (mt) translation caused by hypomodification of tRNAs, but also as a result of nonconventional roles of mt-tRNAs and mt-tRNA-modifying enzymes. The evolutionary conservation of these enzymes supports their importance for cell and organismal functions. Interestingly, bacterial and eukaryotic cells respond to stress by altering the expression or activity of these tRNA-modifying enzymes, which leads to changes in the modification status of tRNAs. This review summarizes recent findings about these enzymes and sets them within the previous data context.
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http://dx.doi.org/10.4161/15476286.2014.992269DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4615368PMC
October 2015

The tRNA-modifying function of MnmE is controlled by post-hydrolysis steps of its GTPase cycle.

Nucleic Acids Res 2013 Jul 28;41(12):6190-208. Epub 2013 Apr 28.

RNA Modification and Mitochondrial Diseases Laboratory, Centro de Investigación Príncipe Felipe, 46012-Valencia, Spain.

MnmE is a homodimeric multi-domain GTPase involved in tRNA modification. This protein differs from Ras-like GTPases in its low affinity for guanine nucleotides and mechanism of activation, which occurs by a cis, nucleotide- and potassium-dependent dimerization of its G-domains. Moreover, MnmE requires GTP hydrolysis to be functionally active. However, how GTP hydrolysis drives tRNA modification and how the MnmE GTPase cycle is regulated remains unresolved. Here, the kinetics of the MnmE GTPase cycle was studied under single-turnover conditions using stopped- and quench-flow techniques. We found that the G-domain dissociation is the rate-limiting step of the overall reaction. Mutational analysis and fast kinetics assays revealed that GTP hydrolysis, G-domain dissociation and Pi release can be uncoupled and that G-domain dissociation is directly responsible for the 'ON' state of MnmE. Thus, MnmE provides a new paradigm of how the ON/OFF cycling of GTPases may regulate a cellular process. We also demonstrate that the MnmE GTPase cycle is negatively controlled by the reaction products GDP and Pi. This feedback mechanism may prevent inefficacious GTP hydrolysis in vivo. We propose a biological model whereby a conformational change triggered by tRNA binding is required to remove product inhibition and initiate a new GTPase/tRNA-modification cycle.
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http://dx.doi.org/10.1093/nar/gkt320DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3695501PMC
July 2013

Enzymology of tRNA modification in the bacterial MnmEG pathway.

Biochimie 2012 Jul 28;94(7):1510-20. Epub 2012 Feb 28.

Laboratorio de Genética Molecular, Centro de Investigación Príncipe Felipe, Molecular Genetics, Avenida Autopista del Saler, 16-3, 46012-Valencia, Spain.

Among all RNAs, tRNA exhibits the largest number and the widest variety of post-transcriptional modifications. Modifications within the anticodon stem loop, mainly at the wobble position and purine-37, collectively contribute to stabilize the codon-anticodon pairing, maintain the translational reading frame, facilitate the engagement of the ribosomal decoding site and enable translocation of tRNA from the A-site to the P-site of the ribosome. Modifications at the wobble uridine (U34) of tRNAs reading two degenerate codons ending in purine are complex and result from the activity of two multi-enzyme pathways, the IscS-MnmA and MnmEG pathways, which independently work on positions 2 and 5 of the U34 pyrimidine ring, respectively, and from a third pathway, controlled by TrmL (YibK), that modifies the 2'-hydroxyl group of the ribose. MnmEG is the only common pathway to all the mentioned tRNAs, and involves the GTP- and FAD-dependent activity of the MnmEG complex and, in some cases, the activity of the bifunctional enzyme MnmC. The Escherichia coli MnmEG complex catalyzes the incorporation of an aminomethyl group into the C5 atom of U34 using methylene-tetrahydrofolate and glycine or ammonium as donors. The reaction requires GTP hydrolysis, probably to assemble the active site of the enzyme or to carry out substrate recognition. Inactivation of the evolutionarily conserved MnmEG pathway produces a pleiotropic phenotype in bacteria and mitochondrial dysfunction in human cell lines. While the IscS-MnmA pathway and the MnmA-mediated thiouridylation reaction are relatively well understood, we have limited information on the reactions mediated by the MnmEG, MnmC and TrmL enzymes and on the precise role of proteins MnmE and MnmG in the MnmEG complex activity. This review summarizes the present state of knowledge on these pathways and what we still need to know, with special emphasis on the MnmEG pathway.
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http://dx.doi.org/10.1016/j.biochi.2012.02.019DOI Listing
July 2012

Pre-historic eating patterns in Latin America and protective effects of plant-based diets on cardiovascular risk factors.

Clinics (Sao Paulo) 2010 ;65(10):1049-54

Heart Institute, Faculdade de Medicina, Universidade de São Paulo, São Paulo, SP, Brazil.

In this review, we present the contributions to nutrition science from Latin American native peoples and scientists, appreciated from a historic point of view since pre-historic times to the modern age. Additionally, we present epidemiological and clinical studies on the area of plant-based diets and their relation with the prevention and treatment of cardiovascular diseases conducted in recent decades, and we discuss challenges and perspectives regarding aspects of nutrition in the region.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2972603PMC
http://dx.doi.org/10.1590/s1807-59322010001000022DOI Listing
August 2011

Evaluation of COX-2, EGFR, and p53 as biomarkers of non-dysplastic oral leukoplakias.

Exp Mol Pathol 2010 Oct 1;89(2):197-203. Epub 2010 Jul 1.

Medicine Department, University of La Coruña, La Coruña, Spain.

Objective: Identify candidate SEBs (surrogate endpoint biomarkers) for premalignant trends in head and neck mucosa.

Study Design: Study, by qPCR (quantitative real-time polymerase chain reaction), the expression of COX-2, EGFR and p53 in 24 biopsies of non-dysplastic oral leukoplakia and contra-lateral normal-appearing mucosa.

Results: COX-2 was up-regulated in leukoplakia (79.2%); whereas EGFR and p53 were up-regulated (p>0.05) in oral contra-lateral normal-appearing mucosa (60% and 46% respectively). Also, p53 expression was correlated with tobacco smoke habits and Spearman's rank correlation coefficient showed a positive linear correlation between p53 and EGFR mRNA expression levels.

Conclusions: COX-2 would serve as SEB of oral leukoplakia. The results suggest that p53 appears to be one of the molecular targets of tobacco-related carcinogens in leukoplakia and that the co-expression of p53 and EGFR may play a role in this kind of oral pre-cancerous lesion. More detailed studies of EGFR and p53 should be continued in the future.
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http://dx.doi.org/10.1016/j.yexmp.2010.06.004DOI Listing
October 2010

Structure-function analysis of Escherichia coli MnmG (GidA), a highly conserved tRNA-modifying enzyme.

J Bacteriol 2009 Dec 2;191(24):7614-9. Epub 2009 Oct 2.

Department of Biochemistry, McGill University, Montreal, Quebec, Canada.

The MnmE-MnmG complex is involved in tRNA modification. We have determined the crystal structure of Escherichia coli MnmG at 2.4-A resolution, mutated highly conserved residues with putative roles in flavin adenine dinucleotide (FAD) or tRNA binding and MnmE interaction, and analyzed the effects of these mutations in vivo and in vitro. Limited trypsinolysis of MnmG suggests significant conformational changes upon FAD binding.
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http://dx.doi.org/10.1128/JB.00650-09DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2786596PMC
December 2009

Evolutionarily conserved proteins MnmE and GidA catalyze the formation of two methyluridine derivatives at tRNA wobble positions.

Nucleic Acids Res 2009 Nov;37(21):7177-93

Laboratorio de Genética Molecular, Centro de Investigación Príncipe Felipe, 46012-Valencia, Spain.

The wobble uridine of certain bacterial and mitochondrial tRNAs is modified, at position 5, through an unknown reaction pathway that utilizes the evolutionarily conserved MnmE and GidA proteins. The resulting modification (a methyluridine derivative) plays a critical role in decoding NNG/A codons and reading frame maintenance during mRNA translation. The lack of this tRNA modification produces a pleiotropic phenotype in bacteria and has been associated with mitochondrial encephalomyopathies in humans. In this work, we use in vitro and in vivo approaches to characterize the enzymatic pathway controlled by the Escherichia coli MnmE*GidA complex. Surprisingly, this complex catalyzes two different GTP- and FAD-dependent reactions, which produce 5-aminomethyluridine and 5-carboxymethylamino-methyluridine using ammonium and glycine, respectively, as substrates. In both reactions, methylene-tetrahydrofolate is the most probable source to form the C5-methylene moiety, whereas NADH is dispensable in vitro unless FAD levels are limiting. Our results allow us to reformulate the bacterial MnmE*GidA dependent pathway and propose a novel mechanism for the modification reactions performed by the MnmE and GidA family proteins.
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http://dx.doi.org/10.1093/nar/gkp762DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2790889PMC
November 2009

Characterization of human GTPBP3, a GTP-binding protein involved in mitochondrial tRNA modification.

Mol Cell Biol 2008 Dec 13;28(24):7514-31. Epub 2008 Oct 13.

Laboratorio de Genética Molecular, Centro de Investigación Príncipe Felipe, Avenida Autopista del Saler, 16-3, 46012 Valencia, Spain.

Human GTPBP3 is an evolutionarily conserved, multidomain protein involved in mitochondrial tRNA modification. Characterization of its biochemical properties and the phenotype conferred by GTPBP3 inactivation is crucial to understanding the role of this protein in tRNA maturation and its effects on mitochondrial respiration. We show that the two most abundant GTPBP3 isoforms exhibit moderate affinity for guanine nucleotides like their bacterial homologue, MnmE, although they hydrolyze GTP at a 100-fold lower rate. This suggests that regulation of the GTPase activity, essential for the tRNA modification function of MnmE, is different in GTPBP3. In fact, potassium-induced dimerization of the G domain leads to stimulation of the GTPase activity in MnmE but not in GTPBP3. The GTPBP3 N-terminal domain mediates a potassium-independent dimerization, which appears as an evolutionarily conserved property of the protein family, probably related to the construction of the binding site for the one-carbon-unit donor in the modification reaction. Partial inactivation of GTPBP3 by small interfering RNA reduces oxygen consumption, ATP production, and mitochondrial protein synthesis, while the degradation of these proteins slightly increases. It also results in mitochondria with defective membrane potential and increased superoxide levels. These phenotypic traits suggest that GTPBP3 defects contribute to the pathogenesis of some oxidative phosphorylation diseases.
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http://dx.doi.org/10.1128/MCB.00946-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2593442PMC
December 2008

Structural insights into the GTPase domain of Escherichia coli MnmE protein.

Proteins 2007 Feb;66(3):726-39

Department of Physical Chemistry, University of Valencia, C/Dr. Moliner, 50, Burjassot 46100 Valencia, Spain.

The Escherichia coli MnmE protein is a 50-kDa multidomain GTPase involved in tRNA modification. Its homologues in eukaryotes are crucial for mitochondrial respiration and, thus, it is thought that the human protein might be involved in mitochondrial diseases. Unlike Ras, MnmE shows a high intrinsic GTPase activity and requires effective GTP hydrolysis, and not simply GTP binding, to be functionally active. The isolated MnmE G-domain (165 residues) conserves the GTPase activity of the entire protein, suggesting that it contains the catalytic residues for GTP hydrolysis. To explore the GTP hydrolysis mechanism of MnmE, we analyzed the effect of low pH on binding and hydrolysis of GTP, as well as on the formation of a MnmE transition state mimic. GTP hydrolysis by MnmE, but not GTP binding or formation of a complex with mant-GDP and aluminium fluoride, is impaired at acidic pH, suggesting that the chemistry of the transition state mimic is different to that of the true transition state, and that some residue(s), critical for GTP hydrolysis, is severely affected by low pH. We use a nuclear magnetic resonance (NMR)-based approach to get insights into the MnmE structure and properties. The combined use of NMR restraints and homology structural information allowed the determination of the MnmE G-domain structure in its free form. Chemical shift structure-based prediction provided a good basis for structure refinement and validation. Our data support that MnmE, unlike other GTPases, does not use an arginine finger to drive catalysis, although Arg252 may play a role in stabilization of the transition state.
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http://dx.doi.org/10.1002/prot.21186DOI Listing
February 2007

Biological evaluation of some selected cyclic imides: mitochondrial effects and in vitro cytotoxicity.

Z Naturforsch C J Biosci 2004 Sep-Oct;59(9-10):663-72

Department of Biochemistry and Molecular Biology, Federal University of Parana, Curitiba, Paraná, Brazil.

Cyclic imides such as succinimides, maleimides, glutarimides, phthalimides and their derivatives contain an imide ring and a general structure -CO-N(R)-CO- that confers hydrophobicity and neutral characteristic. A diversity of biological activities and pharmaceutical uses have been attributed to them, such as antibacterial, antifungal, antinociceptive, anticonvulsant, antitumor. In spite of these activities, much of their action mechanisms at molecular and cellular levels remain to be elucidated. We now show the effects of several related cyclic imides: maleimides (S2, S2.1, S2.2, S3), glutarimides (S4, S5, S6), 4-aminoantipyrine derivatives (L1, F1, AL1, F1.14, F1.2) and sulfonated succinimides (RO1, FA, FE, FD, MC, DMC) on isolated rat liver mitochondria, B16-F10 melanoma cell line, peritoneal macrophages and different bacterial streams. The effects on mitochondrial respiratory parameters, cell viability and antibacterial activity were also evaluated. The results indicated that S3, S5 and S6 caused an increased oxygen consumption in the presence of ADP (state III) or its absence (state IV), while all other compounds decreased those parameters at different degrees of inhibition. All the compounds decreased the respiratory control coefficient (RCC). Loss of cell viability of peritoneal macrophages and the B16-F10 cell line was observed, L1 and S2.1 being more effective. S1, S2, S3, L1 and F1 compounds showed antibacterial activity at experimental concentrations.
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http://dx.doi.org/10.1515/znc-2004-9-1010DOI Listing
December 2004

Isolation and transcriptional regulation of the Kluyveromyces lactis FBA1 (fructose-1,6-bisphosphate aldolase) gene.

Can J Microbiol 2004 Aug;50(8):645-52

Department of Molecular and Cell Biology, University of A Coruña, Campus da Zapateira s/n, 15071- A Coruña, Spain.

Cloning and transcriptional regulation of the KlFBA1 gene that codes for the class II fructose-1,6-bisphosphate aldolase of the yeast Kluyveromyces lactis are described. KlFBA1 mRNA diminishes transiently during the shift from hypoxic to fully aerobic conditions and increases in the reversal shift. This regulation is mediated by heme since expression was higher in a mutant defective in heme biosynthesis. KlFBA1 transcription is not induced by calcium-shortage, low temperature, or at stationary phase. These data suggest that KlFBA1 plays a role in the balance between oxidative and fermentative metabolism and that this gene is differentially regulated in K. lactis and Saccharomyces cerevisiae, i.e., a respiratory vs. fermentative yeast.
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http://dx.doi.org/10.1139/w04-038DOI Listing
August 2004