Fluorescence labeling of mitochondria in living cells by the cationic photosensitizer ZnTM2,3PyPz, and the possible roles of redox processes and pseudobase formation in facilitating dye uptake.

The study of labeling selectivity and mechanisms of fluorescent organelle probes in living cells is of continuing interest in biomedical sciences. The tetracationic phthalocyanine-like ZnTM2,3PyPz photosensitizing dye induces a selective violet fluorescence in mitochondria of living HeLa cells under UV excitation that is due to co-localization of the red signal of the dye with NAD(P)H blue autofluorescence. Both red and blue signals co-localize with the green emission of the mitochondria probe, rhodamine 123. Microscopic observation of mitochondria was improved using image processing and analysis methods. High dye concentration and prolonged incubation time were required to achieve optimal mitochondrial labeling. ZnTM2,3PyPz is a highly cationic, hydrophilic dye, which makes ready entry into living cells unlikely. Redox color changes in solutions of the dye indicate that colorless products are formed by reduction. Spectroscopic studies of dye solutions showed that cycles of alkaline titration from pH 7 to 8.5 followed by acidification to pH 7 first lower, then restore the 640 nm absorption peak by approximately 90%, which can be explained by formation of pseudobases. Both reduction and pseudobase formation result in formation of less highly charged and more lipophilic (cell permeant) derivatives in equilibrium with the parent dye. Some of these are predicted to be lipophilic and therefore membrane-permeant; consequently, low concentrations of such species could be responsible for slow uptake and accumulation in mitochondria of living cells. We discuss the wider implications of such phenomena for uptake of hydrophilic fluorescent probes into living cells.

A simplified chromatin dispersion (nuclear halo) assay for detecting DNA breakage induced by ionizing radiation and chemical agents.

Methods for visualizing DNA damage at the microscopic level are based on treatment of cell nuclei with saline or alkaline solutions. These procedures for achieving chromatin dispersion produce halos that surround the nuclear remnants. We improved the fast halo assay for visualizing DNA breakage in cultured cells to create a simplified method for detection and quantitative evaluation of DNA breakage. Nucleated erythrocytes from chicken blood were selected as a model test system to analyze the production of nuclear halos after treatment with X-rays or H(2)O(2). After staining with ethidium bromide or Wright's methylene blue-eosin solution, nuclear halos were easily observed by fluorescence or bright-field microscopy, respectively, which permits rapid visualization of DNA breakage in damaged cells. By using image processing and analysis with the public domain ImageJ software, X-ray dose and H(2)O(2) concentration could be correlated well with the size of nuclear halos and the halo:nucleus ratio. Our results indicate that this simplified nuclear halo assay can be used as a rapid, reliable and inexpensive procedure to detect and quantify DNA breakage induced by ionizing radiation and chemical agents. A mechanistic model to explain the differences between the formation of saline or alkaline halos also is suggested.

The pesticide malathion induces alterations in actin cytoskeleton and in cell adhesion of cultured breast carcinoma cells.

We have studied the effects of the organophosphorous pesticide malathion on cell viability, actin cytoskeleton, cell adhesion complex E-cadherin/beta-catenin, and Rho and Rac1 GTPases from the human mammary carcinoma cell line MCF-7. Malathion induced cell lethality, determined by the MTT assay, depending on the treatment conditions. Cells incubated with low concentrations of malathion, 16-32 microg/ml, showed high survival rates (>95%) at any evaluated time (1-5 days), whereas complete cell lethality was found using 512 microg/ml and 5 days of treatment. Deep morphological changes were induced with high doses of 64 and 128 microg/ml, and long incubation time (5 days); cells showed perinuclear vacuoles, rounding, shrinkage, and a gradual loss of adhesion. These changes were related to a decrease in the expression of the adhesion molecules, E-cadherin and beta-catenin, and to the distribution and reactivity of actin microfilaments to TRITC-phalloidin. Disruption of microfilaments, accompanied by the collapse of actin to perinuclear region, were characteristic of cells with loss of adhesion. At lower concentrations, some cells presented deformations on the plasma membrane as lamellipodia-like structures, which were particularly evident from 32 to 128 microg/ml. Conversely, we observed an increase in the expression of Rho and Rac1 GTPases, modulators of actin cytoskeleton and cell adhesion.

Specific heterochromatic banding of metaphase chromosomes using nuclear yellow.

The bis-benzimidazole compound nuclear yellow (NY) belongs to the same chemical family as the DNA binding fluorochromes Hoechst 33258 and Hoechst 33342. Spectroscopic studies of NY alone and in the presence of calf thymus DNA show high DNA binding affinity and behavior similar to the Hoechst fluorochromes above. Mitotic metaphase chromosomes from Balb/c mice stained with NY show C-banding and weak G/Q-banding, both of them disappearing after distamycin A (DA) or methyl green (MG) counterstaining. The same staining of human metaphase chromosomes from lymphocyte cultures, however, reveal only faint G/Q-banding (NY) and a characteristic DA-DAPI-like banding (NY-DA, NY-MG). Image analysis of NY stained human chromosomes, confirms that NY is suitable for studying polymorphisms affecting size in the pericentromeric heterochromatin of pairs 1, 9 and 16, and shows significant enhancement of NY fluorescence induced by DA in DA-DAPI heterochromatin. Our spectroscopic and cytological results show that NY, either alone or counterstained with DA or MG, can be used for DNA cytochemistry and chromosome banding. Possible mechanisms for the banding patterns induced by NY are discussed.

Formation and microscopical application of a fluorescent 1,10-phenanthroline derivative of ruthenium red.

In this work we describe the formation and microscopical application of a fluorescent derivative of Ruthenium Red (RR) obtained by heating the dye in the presence of 1,10-phenanthroline (OP). The RR-OP reaction product showed absorption maxima at 416 and 444 nm and intense fluorescence emission at 578 nm under 440 nm exciting light. Neither RR nor OP solutions alone were fluorescent when excited at 440 nm. Using fluorescence microscopy, chicken blood cell smears stained 5 min with the RR-OP derivative showed the chromatin of erythrocyte nuclei with a bright orange fluorescence under violet-blue (436 nm) exciting light.

A simple method to detect sperm chromatin abnormalities: cytochemical mechanism and possible value in predicting semen quality in assisted reproductive procedures.

Protamine crosslinking by disulphide (-SS-) bonds in the main factor responsible for the stability of chromatin structure in mammalian spermatozoa. Sperm chromatin containing arginine/cysteine-rich protamines shows a deeply modified cytochemical reactivity (e.g. basophilia) when compared with somatic chromatic. After methanol or ethanol-acetic acid fixation and toluidine blue (TB) staining, most sperm heads in semen smear from human fertile donors exhibited a pale blue (orthochromatic) colour, while a few sperm heads exhibited violet-blue or violet (metachromatic) staining. Smears from infertile patients showed an increased amount of metachromatic sperm nuclei. After reduction of -SS- bonds by dithiothreitol, sperm heads from all smears were metachromatic, suggesting that DNA phosphates then become available for TB stacking. Micro- and macro-spectrophotometric studies confirmed the microscopic colour reaction of sperm nuclei. The ortho-/metachromatic ration seems a useful parameter for evaluation of altered chromatin structure in spermatozoa cells. Taking into account the current interest in complementary staining tests for evaluation of semen quality, the metachromatic TB reaction represents a simple cytochemical approach for detecting sperm chromatin abnormalities based on differences in -SS- crosslinking.

Cytochemical application of tris (2,2'-bipyridine) ruthenium (II): fluorescence reaction with sulfated polyanions of mast cell granules.

We describe the use of tris (2,2'-bipyridine) ruthenium (II) (Rubipy) as a cationic fluorochrome for cytochemical and histochemical studies. After staining with Rubipy, mast cell granules (MCGs) and lymphocyte nuclei (LN) from mouse peritoneal cavity and human breast carcinoma showed intense orange fluorescence and no fading under blue or blue-violet exciting light. Staining at low pH (< 2) or pre-treatment with Al3+ ions strongly diminished the fluorescence of LN, whereas that of MCG was less affected. Ca2+ and Ba2+ ions only diminished MCG fluorescence. Blots of DNA, pectic acid, heparin, and other sulfated polysaccharides stained with Rubipy showed high emission, which was reduced in DNA and pectic acid staining at low pH. Studies with chemically modified heparins suggested that O-sulfates were more important than N-sulfates in Rubipy-heparin interactions. These results are in agreement with an ionic binding mode between Rubipy and heparin. A very suitable method for mast cell detection was found with Mayer's hematoxylin before Rubipy staining, which could be of great value for histopathological studies. This procedure allowed visualization of the mast cells by fluorescence microscopy, and nuclei and tissue morphology were easily visualized under brightfield illumination.

Metachromatic staining of human sperm nuclei after reduction of disulphide bonds.

Intra- and interprotamine cross-linking by disulphide bonds are the main factors responsible for the highly compact and stable structure of chromatin in mammalian spermatozoa. Unfixed or methanol fixed smears of human sperm and sperm suspensions from fertile donors and oligospermic patients were subjected to a reductive cleavage of disulphide bonds by using 2-mercaptoethanol (ME) or dithiothreitol (DTT). Untreated (control) and ME or DTT treated samples were stained with toluidine blue (TB) and examined in light microscopy; spectral characteristics of TB stained sperm nuclei were also analyzed. Untreated smears from fertile donors showed an orthochromatic (pale blue) staining of most sperm heads, while a variable proportion of metachromatic nuclei was found in spermatozoa from patients with oligospermia. After treatment with DTT followed by TB staining, fixed and unfixed smears showed metachromatic sperm heads. ME treatment only induced a scarce colour shift, whereas a striking metachromatic reaction and variable nuclear swelling were observed in DTT treated sperm suspensions. These results indicate that after cleavage of disulphide bonds, phosphate groups from chromatin DNA are unmasked and available for TB binding and metachromatic staining.

9-fenil-2,3,7-trihidroxi-6-fluorona como colorante y fluorocromo / 9-fenil-2,3,7-trihidroxy-6-fluorone as stain and fluorochrome

Por aplicación de disoluciones de fenil fluorona (compuesto derivado del xanteno), a distintas concentraciones, sobre frotis de sangre circulante humana, de pollo y de caballo, y cortes histológicos de materiales incluidos em parafina y Epon, se comprobó su alta afinidad por estructura típicamente acidófilas visualizadas en microscopias óptica y de fluorescencia en función de la concentración. El trabajo se centró en el estudio de la reacción de fenil fluorona con los componentes de los eosinófilos sanguíneos, caracterizándose citoespectralmente por una absorción a 505-510 nm, similar a la presentada por soluciones de fenil fluorona 10-5M y 5 x 10-5M, que a su vez muestran una emisión de fluorescencia típica a 540 nm, bajo luz de excitación de 470 nm, que corroboran las observaciones microscópicas. La fenil fluorona se considera, por tanto, de gran utilidad como colorante y fluorocromo para la visualización fácil, rápida y selectiva de estructuras básicas y, en particular, granulaciones acidófilas, cuya caracterización es de importancia en el diagnóstico hematológico (AU)

9-fenil-2,3,7-trihidroxi-6-fluorona como colorante y fluorocromo / 9-fenil-2,3,7-trihidroxy-6-fluorone as stain and fluorochrome

Por aplicación de disoluciones de fenil fluorona (compuesto derivado del xanteno), a distintas concentraciones, sobre frotis de sangre circulante humana, de pollo y de caballo, y cortes histológicos de materiales incluidos em parafina y Epon, se comprobó su alta afinidad por estructura típicamente acidófilas visualizadas en microscopias óptica y de fluorescencia en función de la concentración. El trabajo se centró en el estudio de la reacción de fenil fluorona con los componentes de los eosinófilos sanguíneos, caracterizándose citoespectralmente por una absorción a 505-510 nm, similar a la presentada por soluciones de fenil fluorona 10-5M y 5 x 10-5M, que a su vez muestran una emisión de fluorescencia típica a 540 nm, bajo luz de excitación de 470 nm, que corroboran las observaciones microscópicas. La fenil fluorona se considera, por tanto, de gran utilidad como colorante y fluorocromo para la visualización fácil, rápida y selectiva de estructuras básicas y, en particular, granulaciones acidófilas, cuya caracterización es de importancia en el diagnóstico hematológico

Citoquimica de la union de colorantes y fluorocromos con DNA y cromatina. / Cytochemistry of the binding of dyes and flurochromes with DNA and chromatin

El estudio de la interaccion entre colorantes y fluorocromos basicos y acidos nucleicos ha llegado a adquirir una gran importancia en la biologia molecular actual, asi como indudables repercusiones sobre las ciencias biomedicas. El analisis citoquimico de la tincion y/o fluorescencia de la cromatina por colorantes y fluorocromos derivados del xanteno, azina, tiazina, isoquinolina, bezotiazol y ftalocianina, permite obtener indicaciones sobre especificidades y mecanismos de union con el DNA, asi como correlacionar los resultados microscopicos con las caracteristicas fisicoquimicas de los colorantes y de su interaccion con acidos nucleicos

Citoquimica de la union de colorantes y fluorocromos con DNA y cromatina. / Cytochemistry of the binding of dyes and flurochromes with DNA and chromatin

El estudio de la interaccion entre colorantes y fluorocromos basicos y acidos nucleicos ha llegado a adquirir una gran importancia en la biologia molecular actual, asi como indudables repercusiones sobre las ciencias biomedicas. El analisis citoquimico de la tincion y/o fluorescencia de la cromatina por colorantes y fluorocromos derivados del xanteno, azina, tiazina, isoquinolina, bezotiazol y ftalocianina, permite obtener indicaciones sobre especificidades y mecanismos de union con el DNA, asi como correlacionar los resultados microscopicos con las caracteristicas fisicoquimicas de los colorantes y de su interaccion con acidos nucleicos