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Stockert J.C.,Autonomous University of Madrid | Stockert J.C.,Center for Biological Research | Del Castillo P.,Autonomous University of Madrid | Blazquez-Castro A.,Autonomous University of Madrid
Acta Histochemica | Year: 2011

The use of simple model substrates to analyze conditions and mechanisms of metachromatic reactions is an important strategy in histochemical studies. In this study we show that in the presence of the three-layered silicate clay montmorillonite, diluted solutions of thionine and toluidine blue develop an immediate metachromatic shift (from blue to reddish violet). Likewise, hypoemission and red shift in the fluorescence spectra of acridine orange, pyronine Y and ethidium bromide appear when montmorillonite is added to the dye solution. Cationic dyes could insert as stacked structures into the negatively charged interlamellar spaces of the clay. These spectral results indicate that on account of its strong aggregating capacity, montmorillonite is a suitable model substrate to study metachromatic reactions. © 2010 Elsevier GmbH.

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.

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.

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.

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