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Dive into the research topics where Alfonso Blázquez-Castro is active.

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Featured researches published by Alfonso Blázquez-Castro.


Acta Histochemica | 2012

MTT assay for cell viability: Intracellular localization of the formazan product is in lipid droplets

Juan C. Stockert; Alfonso Blázquez-Castro; Magdalena Cañete; Richard W. Horobin; Angeles Villanueva

Although MTT is widely used to assess cytotoxicity and cell viability, the precise localization of its reduced formazan product is still unclear. In the present study the localization of MTT formazan was studied by direct microscopic observation of living HeLa cells and by colocalization analysis with organelle-selective fluorescent probes. MTT formazan granules did not colocalize with mitochondria as revealed by rhodamine 123 labeling or autofluorescence. Likewise, no colocalization was observed between MTT formazan granules and lysosomes labeled by neutral red. Taking into account the lipophilic character and lipid solubility of MTT formazan, an evaluation of the MTT reaction was performed after treatment of cells with sunflower oil emulsions to induce a massive occurrence of lipid droplets. Under this condition, lipid droplets revealed a large amount of MTT formazan deposits. Kinetic studies on the viability of MTT-treated cells showed no harmful effects at short times. Quantitative structure-activity relations (QSAR) models were used to predict and explain the localization of both the MTT tetrazolium salt and its formazan product. These predictions were in agreement with experimental observations on the accumulation of MTT formazan product in lipid droplets.


European Journal of Cell Biology | 2012

Protoporphyrin IX-dependent photodynamic production of endogenous ROS stimulates cell proliferation.

Alfonso Blázquez-Castro; Elisa Carrasco; Maria Calvo; Pedro Jaén; Juan C. Stockert; Angeles Juarranz; Francisco Sanz-Rodríguez; Jesús Espada

Photodynamic therapy using methyl 5-aminolevulinate (MAL) as a precursor of the photosensitizing agent protoporphyrin IX is widely used in clinical practice for the treatment of different pathologies, including cancer. In this therapeutic modality, MAL treatment promotes the forced accumulation of the endogenous photoactive compound protoporphyrin IX in target malignant cells. Subsequent irradiation of treated tissues with an appropriate visible light source induces the production of reactive oxygen species (ROS) that, once accumulated above a critical level, promote cell death. Here we demonstrate that a photodynamic treatment with low MAL concentrations can be used to promote a moderate production of endogenous ROS, which efficiently stimulates cell growth in human immortalized keratinocytes (HaCaT). We also show that this proliferative response requires Src kinase activity and is associated to a transient induction of cyclin D1 expression. Taken together, these results demonstrate for the first time that a combination of light and a photoactive compound can be used to modulate cell cycle progression through Src kinase activation and that a moderate intracellular increase of photogenerated ROS efficiently stimulates cell proliferation.


Optics Express | 2011

Photovoltaic versus optical tweezers

J. Villarroel; Héctor Burgos; A. García-Cabañes; M. Carrascosa; Alfonso Blázquez-Castro; F. Agulló-López

The operation of photovoltaic (PV) tweezers, using the evanescent light-induced PV fields to trap and pattern nano- and micro-meter particles on a LiNbO(3) crystal surface, is discussed. The case of a periodic light pattern is addressed in detail, including the role of particle shape and the modulation index of the light pattern. The use of a single Gaussian light beam is also considered. Illustrative experiments for the two situations are presented. The performance of such PV tweezers in comparison to the best established case of optical tweezers, using optical forces, is considered. Differential features between the two trapping approaches are remarked.


Journal of Cellular Physiology | 2009

Oncogenic H-Ras and PI3K signaling can inhibit E-cadherin-dependent apoptosis and promote cell survival after photodynamic therapy in mouse keratinocytes

Jesús Espada; Sergio Galaz; Francisco Sanz-Rodríguez; Alfonso Blázquez-Castro; Juan C. Stockert; Lorea Bagazgoitia; Pedro Jaén; Salvador González; Amparo Cano; Angeles Juarranz

Maintenance of E‐cadherin mediated cell–cell contacts is often required for the survival of epithelial cells and tissues. Here we report that oncogenic activation of H‐Ras in murine keratinocytes can prevent cell death induced by immunological disruption of E‐cadherin adhesion. A similar situation was observed in cells showing constitutive activation of the p110α catalytic subunit of class IA PI3K. This protective effect is associated with β‐catenin‐dependent transcription and with activation of survival factor Akt/PKB. In addition, we induced cell death by employing photodynamic therapy, using Zn‐phthalocyanine as a photosensitizer that targets E‐cadherin adhesion complexes. We have found that cell death based on this photodynamic action is also bypassed in cells showing constitutive activation of H‐Ras and p110α. Taken together, these results indicate that H‐Ras/PI3K/Akt signaling plays a key role in cell survival mediated by E‐cadherin cell–cell contacts. J. Cell. Physiol. 219: 84–93, 2009.


Biotechnic & Histochemistry | 2009

Binding of cationic dyes to DNA: distinguishing intercalation and groove binding mechanisms using simple experimental and numerical models

P. Del Castillo; Richard W. Horobin; Alfonso Blázquez-Castro; Juan C. Stockert

Abstract 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.


Journal of Investigative Dermatology | 2015

Photoactivation of ROS Production In Situ Transiently Activates Cell Proliferation in Mouse Skin and in the Hair Follicle Stem Cell Niche Promoting Hair Growth and Wound Healing.

Elisa Carrasco; Maria Calvo; Alfonso Blázquez-Castro; Daniela Vecchio; Alicia Zamarrón; Irma Joyce Dias de Almeida; Juan C. Stockert; Michael R. Hamblin; Angeles Juarranz; Jesús Espada

The role of reactive oxygen species (ROS) in the regulation of hair follicle (HF) cycle and skin homeostasis is poorly characterized. ROS have been traditionally linked to human disease and aging, but recent findings suggest that they can also have beneficial physiological functions in vivo in mammals. To test this hypothesis, we transiently switched on in situ ROS production in mouse skin. This process activated cell proliferation in the tissue and, interestingly, in the bulge region of the HF, a major reservoir of epidermal stem cells, promoting hair growth, as well as stimulating tissue repair after severe burn injury. We further show that these effects were associated with a transient Src kinase phosphorylation at Tyr416 and with a strong transcriptional activation of the prolactin family 2 subfamily c of growth factors. Our results point to potentially relevant modes of skin homeostasis regulation and demonstrate that a local and transient ROS production can regulate stem cell and tissue function in the whole organism.


Methods | 2016

Exerting better control and specificity with singlet oxygen experiments in live mammalian cells.

Michael Westberg; Mikkel Bregnhøj; Chiranjib Banerjee; Alfonso Blázquez-Castro; Thomas Breitenbach; Peter R. Ogilby

Singlet molecular oxygen, O2(a1Δg), is a Reactive Oxygen Species, ROS, that acts as a signaling and/or perturbing agent in mammalian cells, influencing processes that range from cell proliferation to cell death. Although the importance of O2(a1Δg) in this regard is acknowledged, an understanding of the targets and mechanisms of O2(a1Δg) action is inadequate. Thus, methods that better facilitate studies of O2(a1Δg) in mammalian cells are highly desired. This is particularly important because, as a consequence of its chemistry in a cell, O2(a1Δg) can spawn the generation of other ROS (e.g., the hydroxyl radical) that, in turn, can have a unique influence on cell behavior and function. Therefore, exerting better control and specificity in O2(a1Δg) experiments ultimately reduces the number of variables in general studies to unravel the details of ROS-dependent cell dynamics. In this article, we summarize our recent efforts to produce O2(a1Δg) with increased control and selectivity in microscope-based single-cell experiments. The topics addressed include (1) two-photon excitation of a photosensitizer using a focused laser to create a spatially-localized volume of O2(a1Δg) with sub-cellular dimensions, (2) protein-encapsulated photosensitizers that can be localized in a specific cellular domain using genetic engineering, and (3) direct excitation of dissolved oxygen in sensitizer-free experiments to selectively produce O2(a1Δg) at the expense of other ROS. We also comment on our recent efforts to monitor O2(a1Δg) in cells and to monitor the cells response to O2(a1Δg).


Biotechnic & Histochemistry | 2009

Selective labeling of lipid droplets in aldehyde fixed cell monolayers by lipophilic fluorochromes

Juan C. Stockert; Mi Abasolo; Alfonso Blázquez-Castro; Richard W. Horobin; M Revilla; Dm Lombardo

Abstract 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.


Methods | 2016

Switching on a transient endogenous ROS production in mammalian cells and tissues

Elisa Carrasco; Alfonso Blázquez-Castro; Maria Calvo; Angeles Juarranz; Jesús Espada

There is a growing interest in the physiological roles of reactive oxygen species (ROS) as essential components of molecular mechanisms regulating key cellular processes, including proliferation, differentiation and apoptosis. This interest has fostered the development of new molecular tools to localize and quantify ROS production in cultured cells and in whole living organisms. An equally important but often neglected aspect in the study of ROS biology is the development of accurate procedures to introduce a ROS source in the biological system under study. At present, this experimental requirement is solved in most cases by an external and systemic administration of ROS, usually hydrogen peroxide. We have previously shown that a photodynamic treatment based on the endogenous photosensitizer protoporphyrin IX and further irradiation of the target with adequate light source can be used to transiently switch on an in situ ROS production in human cultured keratinocytes and in mouse skin in vivo. Using this approach we reported that qualitatively low levels of ROS can activate cell proliferation in cultured cells and promote a transient and reversible hyperproliferative response in the skin, particularly, in the hair follicle stem cell niche, promoting physiological responses like acceleration of hair growth and supporting the notion that a local and transient ROS production can regulate stem cell function and tissue homeostasis in a whole organism. Our principal aim here is to provide a detailed description of this experimental methodology as a useful tool to investigate physiological roles for ROS in vivo in different experimental systems.


PLOS ONE | 2015

Reliable screening of dye phototoxicity by using a Caenorhabditis elegans fast bioassay

Javier Ignacio Bianchi; Juan C. Stockert; Lucila Ines Buzz; Alfonso Blázquez-Castro; Sergio H. Simonetta

Phototoxicity consists in the capability of certain innocuous molecules to become toxic when subjected to suitable illumination. In order to discover new photoactive drugs or characterize phototoxic pollutants, it would be advantageous to use simple biological tests of phototoxicy. In this work, we present a pilot screening of 37 dyes to test for phototoxic effects in the roundworm Caenorhabditis elegans. Populations of this nematode were treated with different dyes, and subsequently exposed to 30 min of white light. Behavioral outcomes were quantified by recording the global motility using an infrared tracking device (WMicrotracker). Of the tested compounds, 17 dyes were classified as photoactive, being phloxine B, primuline, eosin Y, acridine orange and rose Bengal the most phototoxic. To assess photoactivity after uptake, compounds were retested after washing them out of the medium before light irradiation. Dye uptake into the worms was also analyzed by staining or fluorescence. All the positive drugs were incorporated by animals and produced phototoxic effects after washing. We also tested the stress response being triggered by the treatments through reporter strains. Endoplasmic reticulum stress response (hsp-4::GFP strain) was activated by 22% of phototoxic dyes, and mitochondrial stress response (hsp-6::GFP strain) was induced by 16% of phototoxic dyes. These results point to a phototoxic perturbation of the protein functionality and an oxidative stress similar to that reported in cell cultures. Our work shows for the first time the feasibility of C. elegans for running phototoxic screenings and underscores its application on photoactive drugs and environmental pollutants assessment.

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Juan C. Stockert

Autonomous University of Madrid

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Angeles Juarranz

Autonomous University of Madrid

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Jesús Espada

Spanish National Research Council

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A. García-Cabañes

Autonomous University of Madrid

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Alicia Zamarrón

Autonomous University of Madrid

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Cerro Elisa Carrasco

Autonomous University of Madrid

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