Center for Biological Research

Madrid, Spain

Center for Biological Research

Madrid, Spain
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Vasl J.,Slovenian National Institute of Chemistry | Oblak A.,Slovenian National Institute of Chemistry | Peternelj T.T.,Slovenian National Institute of Chemistry | Klett J.,Center for Biological Research | And 5 more authors.
Journal of Immunology | Year: 2016

Myeloid differentiation factor 2 (MD-2) is an extracellular protein, associated with the ectodomain of TLR4, that plays a critical role in the recognition of bacterial LPS. Despite high overall structural and functional similarity, human (h) and murine (m) MD-2 exhibit several species-related differences. hMD-2 is capable of binding LPS in the absence of TLR4, whereas mMD-2 supports LPS responsiveness only when mMD-2 and mTLR4 are coexpressed in the same cell. Previously, charged residues at the edge of the LPS binding pocket have been attributed to this difference. In this study, site-directed mutagenesis was used to explore the hydrophobic residues within the MD-2 binding pocket as the source of functional differences between hMD-2 and mMD-2. Whereas decreased hydrophobicity of residues 61 and 63 in the hMD-2 binding pocket retained the characteristics of wild-type hMD-2, a relatively minor change of valine to alanine at position 135 completely abolished the binding of LPS to the hMD-2 mutant. The mutant, however, retained the LPS binding in complex with TLR4 and also cell activation, resulting in a murine-like phenotype. ©2016 by The American Association of Immunologists, Inc.


Alvarez M.,Central University of Venezuela | Villanueva A.,Autonomous University of Madrid | Acedo P.,Autonomous University of Madrid | Canete M.,Autonomous University of Madrid | And 2 more authors.
Acta Histochemica | Year: 2011

When cultured cells are treated with fluorescent organelle probes or photosensitizer agents, a characteristic redistribution of fluorescence in cell structures occurs frequently after light irradiation. It is currently assumed that such changes, referred to as relocalizations of the fluorescent compounds, represent an important aspect of the photodynamic process, which is based on the excitation of photosensitizers by light in the presence of oxygen. As cell damage and death result from the oxidative stress induced by photodynamic treatments, we have studied here the redistribution of acridine orange (AO) and 3,3'-dimethyl-oxacarbocyanine (DiOC 1(3)) fluorescence after incubation of HeLa cell cultures with these compounds followed by blue light irradiation to achieve lethal effects. The relocalization of dyes from their original labeling sites (AO: lysosomes, DiOC 1(3): mitochondria) to nucleic acid-containing structures (cytoplasm, nuclei and nucleoli) appeared clearly associated with cell death. Therefore, the relocalization phenomenon simply reflects fluorescence changes due to the different affinity of these dyes for living and damaged or dead cells. As fluorescent probes are often photosensitizers, prolonged light exposures using fluorescence microscopy will produce lethal photodynamic effects with relocalization of the fluorescent signal and changes in the cell morphology. © 2010 Elsevier GmbH.


Del Castillo P.,Autonomous University of Madrid | Horobin R.W.,University of Glasgow | Blazquez-Castro A.,Autonomous University of Madrid | Stockert J.C.,Autonomous University of Madrid | Stockert J.C.,Center for Biological Research
Biotechnic and Histochemistry | Year: 2010

Simple methods for predicting intercalation or groove binding of dyes and analogous compounds with double stranded DNA are described. The methods are based on a quantitative assessment of the aspect (width to length) ratio of the dyes. The procedures were validated using a set of 38 cationic dyes of varied chemical structures binding to well oriented DNA fibers and assessing binding orientation by linear dichroism and polarized fluorescence. We demonstrated that low aspect ratio dyes bound by intercalation, whereas more rod-like dyes were groove binders. Some problems that result and possible applications are discussed briefly. © 2010 Biological Stain Commission.


Stockert J.C.,Autonomous University of Madrid | Stockert J.C.,Center for Biological Research | Abasolo M.I.,University of Buenos Aires
Biotechnic and Histochemistry | Year: 2011

Representations of the chemical structures of dyes and fluorochromes often are used to illustrate staining mechanisms and histochemical reactions. Unfortunately, inaccurate chemical structures sometimes are used, which results in problems for teaching and research in histochemistry. We comment here on published examples of inadequate chemical drawing and modeling. In particular, omission of hydrogen atoms can lead to misleading hydrogen-bonding interactions, and inaccurate drawing and modeling procedures result in a variety of implausible molecular structures. The examples and arguments given here are easily intelligible for non-chemists and could be used as part of a training approach to help avoid publication of misleading or puzzling dye structures and molecular models for illustrating biological staining and histochemical studies. © 2011 The Biological Stain Commission.


Stockert J.C.,Autonomous University of Madrid | Stockert J.C.,Center for Biological Research | Abasolo M.I.,University of Buenos Aires | Blzquez-Castro A.,Autonomous University of Madrid | And 3 more authors.
Biotechnic and Histochemistry | Year: 2010

We evaluated a number of lipophilic dyes and fluorochromes, including oxazone and thiazone derivatives of oxazine and thiazine dyes, scintillator agents, a carotenoid and a metal-porphyrin complex for visualization of lipid droplets within aldehyde fixed cultured HeLa and BGC-1 cells. Observation under ultraviolet, blue or green exciting light revealed selective fluorescence of lipid droplets, particularly after treatment with aqueous solutions of Nile blue and brilliant cresyl blue oxazones, toluidine blue thiazone, or propylene glycol solutions of canthaxanthin, ethyl-BAO, and ZnTPyP. Mounting in water was required to maintain the fluorescence of lipids; the use of glycerol, Mowiol or Vectashield was not adequate. The effect of dye structure on staining intensity was assessed with the aid of numerical structure parameters modeling lipophilicity (HI and log P), overall size (MW) and the size of the conjugated system (conjugated bond number; CBN). The best stains for lipid droplets were relatively lipophilic (HI > 4.0, log P > 5.0), of small size overall (MW < 370), with small conjugated systems (CBN < 24), and not significantly amphiphilic. The two hydrophobic-hydrophilic parameters (the classic log P and the hydrophobic index, HI; values calculated by molecular modeling software) were highly correlated; however, HI was a more suitable hydrophobicity index for the dyes studied here. © 2010 The Biological Stain Commission.


Horobin R.,University of Glasgow | Stockert J.C.,Autonomous University of Madrid | Stockert J.C.,Center for Biological Research
Biotechnic and Histochemistry | Year: 2011

We outline the factors involved in precise targeting of lipids and membranes by probes, namely, lipid and probe chemistry, geometry/topography of probe delivery, and probe uptake kinetics. The special case of probe orientation within membranes also is considered. The varieties of commercially available fluorophores are described, and an overview of probe physicochemical properties (amphiphilicity, conjugated system size, electrical properties, head group size, lipophilicity and solubility) is provided together with notes on their parameterization. Probe-lipid physicochemical interactions, and their relations to parameterization, then are discussed including the nature and derivation of decision-rule QSAR models, partitioning and insertion of probes into bulk lipids and complications of this, partitioning and insertion of probes into membranes, and flip-flop of probes across membrane leaflets. A general QSAR algorithm for understanding lipid probe application then is set out. Problems and limitations are outlined. Biological issues include varied biomembrane composition, cell line effects and toxicity of fluorescent probes. Methodological issues include difficulties of estimating certain numerical structure parameters, the impure character of many fluorochromes and dyes, and the perturbation of biomembrane structure by fluorescent probes. © 2011 The Biological Stain Commission.


PubMed | Slovenian National Institute of Chemistry, Center for Biological Research, National Institute of Chemistry, Slovenia and University of Iowa
Type: Journal Article | Journal: Journal of immunology (Baltimore, Md. : 1950) | Year: 2016

Myeloid differentiation factor 2 (MD-2) is an extracellular protein, associated with the ectodomain of TLR4, that plays a critical role in the recognition of bacterial LPS. Despite high overall structural and functional similarity, human (h) and murine (m) MD-2 exhibit several species-related differences. hMD-2 is capable of binding LPS in the absence of TLR4, whereas mMD-2 supports LPS responsiveness only when mMD-2 and mTLR4 are coexpressed in the same cell. Previously, charged residues at the edge of the LPS binding pocket have been attributed to this difference. In this study, site-directed mutagenesis was used to explore the hydrophobic residues within the MD-2 binding pocket as the source of functional differences between hMD-2 and mMD-2. Whereas decreased hydrophobicity of residues 61 and 63 in the hMD-2 binding pocket retained the characteristics of wild-type hMD-2, a relatively minor change of valine to alanine at position 135 completely abolished the binding of LPS to the hMD-2 mutant. The mutant, however, retained the LPS binding in complex with TLR4 and also cell activation, resulting in a murine-like phenotype. These results were supported by the molecular dynamics simulation. We propose that the residue at position 135 of MD-2 governs the dynamics of the binding pocket and its ability to accommodate lipid A, which is allosterically affected by bound TLR4.


Garcia-Garcia I.,Center for Biological Research | Gonzalez-Delgado C.A.,University of Habana | Valenzuela-Silva C.M.,Center for Biological Research | Diaz-Machado A.,University of Habana | And 9 more authors.
BMC Pharmacology | Year: 2010

Background: Interferon (IFN) alpha conjugation to polyethylene glycol (PEG) results in a better pharmacokinetic profile and efficacy. The aim of this study was to compare the pharmacokinetic, pharmacodynamic and safety properties of a new, locally developed, 40-kDa PEG-IFN alpha-2b preparation with a reference, commercially available PEG-IFN alpha-2a in healthy male volunteers.Methods: A randomized, crossover, double-blind study with a 3-weeks washout period, was done. A single 180 micrograms PEG-IFN alpha-2 dose was administered subcutaneously in both groups. Sixteen apparently healthy male subjects were included. Serum PEG-IFN concentration was measured during 336 hours by an enzyme immunoassay (EIA). Other clinical and laboratory variables were used as pharmacodynamic and safety criteria.Results: The pharmacokinetic comparison by EIA yielded a high similitude between the formulations. In spite of a high subject variability, the parameters' mean were very close (in all cases p > 0.05): AUC: 53623 vs. 44311 pg.h/mL; Cmax: 333 vs. 271 pg/mL; Tmax: 54 vs. 55 h; half-life (t1/2): 72.4 vs. 64.8 h; terminal elimination rate (lambda): 0.011 vs. 0.014 h-1; mean residence time (MRT): 135 vs. 123 h for reference and study preparations, respectively. There were no significant differences with respect to the pharmacodynamic variables either: serum neopterin and beta-2 microglobulin levels, stimulation of 2'5' oligoadenylate synthetase expression, and serum IFN antiviral activity. A strong Spearman's rank order correlation (p < 0.01) between the pharmacokinetic and pharmacodynamic concentration-time curves was observed. Both products caused similar leukocyte counts diminution and had similar safety profiles. The most frequent adverse reactions were leukopenia, fever, thrombocytopenia, transaminases increase and asthenia, mostly mild.Conclusions: Both formulations are fully comparable from the pharmacokinetic, pharmacodynamic, and safety profiles. Efficacy trials can be carried out to confirm clinical similarity.Trial registration: Registro Público Cubano de Ensayos Clínicos RPCEC00000039. © 2010 García-García et al; licensee BioMed Central Ltd.


Begay O.,Center for Biological Research | Jean-Pierre N.,Center for Biological Research | Abraham C.J.,Center for Biological Research | Chudolij A.,Center for Biological Research | And 8 more authors.
AIDS Research and Human Retroviruses | Year: 2011

Over-the-counter personal lubricants are used frequently during vaginal and anal intercourse, but they have not been extensively tested for biological effects that might influence HIV transmission. We evaluated the in vitro toxicity anti-HIV-1 activity and osmolality of popular lubricants. A total of 41 lubricants were examined and compared to Gynol II and Carraguard as positive and negative controls for toxicity, respectively. Cytotoxicity was assessed using the XTT assay. The MAGI assay with R5 and X4 HIV-1 laboratory strains was used to evaluate antiviral activity. The effect of the lubricants on differentiated Caco-2 cell monolayers (transepithelial electrical resistance, TEER) was also measured. None of the lubricants tested showed significant activity against HIV-1. Surprisingly, four of them, Astroglide Liquid, Astroglide Warming Liquid, Astroglide Glycerin & Paraben-Free Liquid, and Astroglide Silken Secret, significantly enhanced HIV-1 replication (p<0.0001). A common ingredient in three of these preparations is polyquaternium-15. In vitro testing of a chemically related compound (MADQUAT) confirmed that this similarly augmented HIV-1 replication. Most of the lubricants were found to be hyperosmolar and the TEER value dropped approximately 60% 2 h after exposure to all lubricants tested. Cells treated with Carraguard, saline, and cell controls maintained about 100% initial TEER value after 2-6 h. We have identified four lubricants that significantly increase HIV-1 replication in vitro. In addition, the epithelial damage caused by these and many other lubricants may have implications for enhancing HIV transmission in vivo. These data emphasize the importance of performing more rigorous safety testing on these products. © Copyright 2011, Mary Ann Liebert, Inc.


Interferon (IFN) alpha conjugation to polyethylene glycol (PEG) results in a better pharmacokinetic profile and efficacy. The aim of this study was to compare the pharmacokinetic, pharmacodynamic and safety properties of a new, locally developed, 40-kDa PEG-IFN alpha-2b preparation with a reference, commercially available PEG-IFN alpha-2a in healthy male volunteers.A randomized, crossover, double-blind study with a 3-weeks washout period, was done. A single 180 micrograms PEG-IFN alpha-2 dose was administered subcutaneously in both groups. Sixteen apparently healthy male subjects were included. Serum PEG-IFN concentration was measured during 336 hours by an enzyme immunoassay (EIA). Other clinical and laboratory variables were used as pharmacodynamic and safety criteria.The pharmacokinetic comparison by EIA yielded a high similitude between the formulations. In spite of a high subject variability, the parameters mean were very close (in all cases p > 0.05): AUC: 53623 vs. 44311 pg.h/mL; Cmax: 333 vs. 271 pg/mL; Tmax: 54 vs. 55 h; half-life (t1/2): 72.4 vs. 64.8 h; terminal elimination rate (lambda): 0.011 vs. 0.014 h(-1); mean residence time (MRT): 135 vs. 123 h for reference and study preparations, respectively. There were no significant differences with respect to the pharmacodynamic variables either: serum neopterin and beta-2 microglobulin levels, stimulation of 25 oligoadenylate synthetase expression, and serum IFN antiviral activity. A strong Spearmans rank order correlation (p < 0.01) between the pharmacokinetic and pharmacodynamic concentration-time curves was observed. Both products caused similar leukocyte counts diminution and had similar safety profiles. The most frequent adverse reactions were leukopenia, fever, thrombocytopenia, transaminases increase and asthenia, mostly mild.Both formulations are fully comparable from the pharmacokinetic, pharmacodynamic, and safety profiles. Efficacy trials can be carried out to confirm clinical similarity.

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