Biomedical subjects

J C Stockert

Publications and source records attributed to J C Stockert.

At least 37 records · Page 2Linked to original sources

PubMed1997

New cationic fluorochromes from diaryloxazole scintillators: fluorescence of chromatin DNA induced by N-quaternary POPOP derivatives.

N-quaternary derivatives of the diaryloxazole scintillators POPOP and dimethyl-POPOP (dmPOPOP) in chloroform solution were obtained by methylation with dimethylsulfate. After drying, aqueous solutions of the corresponding oxazolium compounds (Q-POPOP and Q-dmPOPOP) revealed strong fluorescence (peaks at 485 and 493 nm, respectively). Under 365 nm excitation, both N-quaternary derivatives induced a bright greenish blue fluorescence in nuclei of chicken erythrocytes and human buccal cells, as well as in the kinetoplast DNA of Trypanosoma cruzi epimastigotes; mouse mast cell granules showed a green-yellow metachromatic emission. Chromatin fluorescence was dependent on the presence of DNA; it was abolished by washing with a 10 mM solution of the bisguanidine compound Phenformin, whereas 1 M NaCl or MgCl2 had no effect. The oxazolium derivatives were hydrophilic (log P: -6.409 and -5.373 for Q-POPOP and Q-dmPOPOP, respectively). Molecular modelling studies revealed that these cationic and non-rigid (cis) scintillator derivatives are well suited to locate along the convex floor of the narrow DNA minor groove from adenine-thymine regions.

Animals↗

PubMed1997

A new fluorescence reaction in DNA cytochemistry: microscopic and spectroscopic studies on the aromatic diamidino compound M&B 938.

We describe the fluorescence properties and cytochemical applications of the aromatic diamidine M&B 938. Treatment of cell smears (chicken blood, Ehrlich ascites tumor, rat bone marrow, mouse mast cells, and Trypanosoma cruzi epimastigotes) with aqueous solutions of M&B 938 (0.5-1 microgram/ml at pH 6-7; UV excitation) induced bright bluish-white fluorescence in DNA-containing structures (interphase and mitotic chromatin, AT-rich kinetoplast DNA of T. cruzi), which was abolished by previous DNA extraction. DNA was the unique fluorescent polyanion after staining with M&B 938 at neutral or alkaline pH, other polyanions such as RNA and heparin showing no emission. M&B 938-stained mouse metaphase chromosomes revealed high fluorescence of the AT-rich centromeric heterochromatin, and strong emission of heterochromatin in human chromosomes 1, 9, 15, 16, and Y was found after distamycin A counterstaining. On agarose gel electrophoresis, M&B 938-stained DNA markers appeared as fluorescent bands. The 1.635-KBP fragment from DNA ladder revealed a higher emission value than that expected from linear regression analysis. Spectroscopic studies showed bathochromic and hyperchromic shifts in the absorption spectrum of M&B 938 complexed with DNA, as well as strong enhancement of fluorescence at 420 nm. In the presence of poly(dA)-poly(dT), the emission of M&B 938 was 4.25-fold higher than with DNA; no fluorescence was observed with poly(dG)-poly(dC). Experimental results and considerations of the chemical structure suggest that the minor groove of AT regions of DNA could be the specific binding site for M&B 938, which shows interesting properties and useful applications as a new DNA fluorochrome.

Animals↗

PubMed1996

Photodynamic damage to HeLa cell microtubules induced by thiazine dyes.

The aim of this study was to analyze possible alterations of the microtubule cytoskeleton of cultured cells subjected to photodynamic treatments with the thiazine dyes methylene blue or toluidine blue. Indirect immunofluorescence labeling of alpha-tubulin was performed in HeLa cells after 1 or 18 h of incubation with thiazines followed by red-light irradiation for 15 min [leading to surviving fractions (SF) of about 65% (SF65) or 1% (SF1), respectively]. Untreated control cells showed the normal distribution of interphase microtubules, whereas considerable or severe disorganization of the microtubule network was observed after SF65 or SF1 photodynamic treatments, respectively. A great amount of blebs showing homogeneous fluorescence was also found on the cell surface after SF1 treatments. Possible mechanisms responsible for the photodamage to microtubules induced by thiazine dyes are briefly discussed.

Coloring Agents↗

PubMed1996

Fluorescent porphyrin counterstaining of chromatin DNA in conjunction with immunofluorescence methods using FITC-labelled antibodies.

This work describes the use of two cationic porphyrins for fluorescent DNA counterstaining of HeLa cells also stained by means of indirect immunofluorescence with fluorescein isothiocyanate (FITC)-conjugated secondary antibodies. Staining HeLa cells with meso-tetra(4-N-methylpyridyl)porphine (T4MPyP) and meso-tetra(p-N-trimethylanilinium)-porphine (TMAP), both used at 5 x 10(-6) M, gives rise to a deep red emission of chromatin from interphase nuclei and mitotic chromosomes when the cells are excited with blue (490 nm) light. The red-fluorescing chromatin contrasted very well with the yellowish-green emission from FITC-immunofluorescent staining. No significant difference in chromatin fluorescence found with either T4MPyP or TMAP was detected. Counterstaining with the porphyrins could be carried out as a separate step after immunolabeling or, more simply, by their inclusion in the mounting medium. Spectral analysis demonstrated that the fluorescent emission maximum of T4MPyP was at 665 nm and that the intensity of the fluorescent emission showed a considerable increase in the presence of DNA.

Aniline Compounds↗

PubMed1996

The osmium tetroxide-p-phenylenediamine procedure reveals the chromatid cores and kinetochores of meiotic chromosomes by light and electron microscopy.

We analyzed first-metaphase meiotic chromosomes of the grasshopper Chorthippus jucundus by two different methods, i.e., a silver impregnation technique and the osmium tetroxide-p-phenylenediamine (Os-PPD) procedure. The former was applied on squashed testes previously fixed in ethanol-acetic acid, whereas for Os-PPD the material was not subjected to any previous extraction treatment but was fixed in OsO4, treated with PPD, and embedded in Epon 812. Both techniques revealed chromatid cores and kinetochores regardless of the processing of the material (squashed or sectioned). Unstained Os-PPD sections were analyzed by light microscopy and transmission electron microscopy (TEM). The Os-PPD technique provided a high contrast of chromatid cores and kinetochores in relation to the chromatin, which revealed a low electron density. To determine the Os-PPD reaction mechanism, the PAS procedure, as well as scanning electron microscopy (SEM) backscattering and SEM X-ray microanalysis, was performed on sections. By use of the Os-PPD-PAS procedure, glycol groups formed by oxidation of osmium bound to aromatic substrates were detected in chromatid cores and kinetochores by brightfield and fluorescence microscopy. A high Z contrast was detected in these structures by backscattered electron imaging. SEM X-ray microanalysis showed osmium and phosphorus to be the main elements present on the chromatid cores. Taking into account the known reactivity of OsO4 and the present results, the possible participation of nucleic acids as well as proteins in the Os-PPD reaction mechanism and in the composition of chromatid cores and kinetochores is discussed.

Animals↗

PubMed1995

Fluorescence of mast cell granules in paraffin sections and cell smears induced by an N-quaternary oxazole scintillator.

The N-quaternized derivative of dimethyl-POPOP (termed Q4) induces a bluish-green fluorescent reaction in mast cell granules from paraffin sections and cell smears, in addition to a previously described bluish-white fluorescent reaction in chromatin DNA. The chromatin reaction was abolished by staining the samples either with Mayer's Haematoxylin before Q4 treatment or by Q4 treatment at pH 1.5. The reaction in mast cell granules was absent after substrate methylation. The staining sequence Haematoxylin-Eosin-Q4 also worked well in paraffin sections, allowing the observation of the current histological image under bright-field illumination as well as double-colour emission under fluorescence microscopy. The sequence is proposed as a new diagnostic procedure for demonstrating mast cell granules.

Animals↗

PubMed1995

Fluorescence of eosinophil leucocyte granules induced by 1-hydroxy-3,6,8-pyrenetrisulfonate. Visualization of differences in protein isoelectric points.

After treatment of horse, rat and human blood smears with alkaline solutions of 1-hydroxy-3,6,8-pyrenetrisulfonate (HPTS), eosinophil leucocyte granules were the unique cell components which showed a bright green fluorescence. When stained with HPTS at pH 10, the whole granule of horse eosinophils showed high emission which strongly diminished after washing or staining in salt solutions or by using blocking methods for amino groups. Using HPTS at pH 12, the fluorescence reaction of horse granules was specifically located in the peripheral region, appearing as fluorescent rings. These microscopic observations, which indicate differences in the isoelectric point of proteins within the eosinophil granule, were also confirmed by HPTS staining of protein blots as model substrates. Spectral analysis of HPTS at pH 10 and 12 showed practically identical absorption and emission spectra with peaks at 450 nm and 510 nm, respectively. Our results indicate that mainly ionic binding occurs between cationic proteins and HPTS in alkaline solution, and that the most cationic proteins (with isoelectric points at pH higher than 12) are located in the peripheral annular region of horse eosinophil granules.

Animals↗

PubMed1995

Fluorescence of the natural dye saffron: selective reaction with eosinophil leucocyte granules.

Treatment of methanol-fixed chicken, rat, horse and human blood smears with saturated solutions of saffron in borate buffer at pH 10 results in a bright yellow-green fluorescence reaction of the acidophilic cytoplasm granules in mammalian eosinophils and chicken heterophils under violet-blue exciting light. Spectral characteristics of saffron (emission peak at 543 nm under 436 nm excitation) and its selective fluorescence with acidophilic structures support the possibility of employing this old microscopic stain as a new fluorochrome.

Animals↗

PubMed1995

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.

Absorption↗

PubMed1995

Simple beta-carboline alkaloids as nucleic acids fluorochromes.

Treatment of cell smears (chicken blood, Trypanosoma cruzi epimastigotes, Ehrlich ascites tumor cells and mouse spleen) with simple beta-carboline alkaloids induced a strong bluish white fluorescence emission of condensed chromatin and basophilic cytoplasm under ultraviolet excitation. The compounds used were harmine, harmol, harman, norharman, harmalol and harmaline (25-50 micrograms/ml aqueous solutions for 2-3 min). Spectrofluorometric studies on harmine solutions in vitro (emission peak at 420 nm) showed fluorescence quenching at high concentrations as well as in the presence of DNA. Microscopic fluorescence features of these new fluorochromes and possible binding modes to nucleic acids are discussed.

Alkaloids↗

PubMed1995

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.

Chromatin↗

PubMed1995

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.

2,2'-Dipyridyl↗

PubMed1995

Differential reactivity of structural components of eosinophil leucocyte granules as revealed by ethanolic phosphotungstic acid.

After glutaraldehyde fixation and ethanolic phosphotungstic acid (E-PTA) treatment before embedding, thin sections of rat bone marrow and large intestine showed a characteristic pattern of electron opacity in eosinophil leucocyte granules. In both mature eosinophils and precursor cells, the matrix appeared highly contrasted while the crystalline core revealed no electron density. Additional treatment of sections with uranyl acetate did not modify the contrasting pattern of eosinophil granules. The absence of electron dense reaction in the crystalline core after E-PTA treatment seems to originate from removal of core components. The selective reactivity of the matrix toward E-PTA could be a valuable ultrastructural marker for studies on the differentiation of specific granules along the maturation of eosinophil leucocytes.

Animals↗

PubMed1994

Sister chromatid differentiation in 5-bromo-2'-deoxyuridine-substituted chromosomes: a study with DNA-specific ligands and monoclonal antibody to histone H2B.

Human and Chinese hamster chromosomes were obtained from cells grown in the presence of 5-bromodeoxyuridine (BrdU) for (a) one replicative round, (b) two replicative rounds, (c) one replicative round followed by another round in thymidine and (d) the last period of synthetic phase. Untreated chromosomes and chromosomes treated with UV radiation after previous staining with 33258 Hoechst as photosensitizer were studied in order to investigate the mechanism(s) responsible for BrdU-induced sister chromatid differentiation (SCD). Metaphases were prepared by (1) standard methanol-acetic acid fixative for subsequent investigation with Giemsa or DNA-specific dyes such as ethidium bromide, acridine orange and monoclonal antibodies to double- or single-stranded DNA; (2) the procedure for observation under phase-contrast or electron microscopy; and (3) the cytospin method for subsequent immunoreaction with a monoclonal antibody to histone H2B. Our data exclude the possibility that the presence/absence of BrdU in the template strand might affect chromatin organization and thus resistance, while confirming that UV-induced DNA alteration is not sufficient, by itself, to explain SCD mechanism(s). That molecules other than DNA play a role in explaining SCD production is indicated by the fact that BrdU incorporation induces alterations in DNA-histone H2B interactions which, in turn, seem to produce structural variations in chromatin, possibly at the level of condensation, as monitored by phase-contrast and electron microscopy.

Animals↗

PubMed1994

Selective fluorescence reaction of indigocarmine stained eosinophil leucocyte granules induced by alkaline reduction of the bound dye to its leuco derivative.

After staining of mammalian blood smears with indigocarmine, eosinophil granules were the unique cell components which stained deeply blue under bright field illumination. Treatment of stained smears with the reducing agent sodium borohydride completely abolished this colour reaction, while under violet-blue exciting light eosinophil granules appeared with bright green fluorescence. Other reducing agents proved less suitable. Spectral analysis of indigocarmine solutions reduced by sodium borohydride showed an emission peak at lambda = 528 nm and confirmed the microscopic observations. These results indicate that the treatment of indigocarmine stained structures with alkaline solutions of strong reducing agents converts the bound dye into its reduced and highly fluorescent leuco derivative.

Animals↗

PubMed1994

Fluorescence of bisazo dye reaction products from the coupled tetrazonium method for proteins.

The coupled tetrazonium reaction is a well known histochemical method for proteins. This method, using Fast Blue B salt and 1-naphthol, has been applied on paraffin sections of grasshopper testis and rabbit trachea, as well as on horse blood smears. Microscopic observation under bright field illumination revealed the expected purple staining of protein-rich cell and tissue structures, which also showed a strong red fluorescence under ultraviolet, violet, violet-blue and green exciting light. Some weakly stained cell components (e.g., meiotic spindles) were easily visualized by fluorescence microscopy. Control preparations lacking either the tetrazonium or naphthol treatment, and spectroscopic studies on the bisazo dye produced in vitro (showing an emission peak at 660 nm) confirmed that the red fluorescence of stained structures arises from the protein-tetrazonium-naphthol reaction product formed in situ.

Animals↗

PubMed1994

A new fluorescence reaction in protein cytochemistry: formation of naphthalimide fluorophores from primary amino groups and 1,8-naphthalic anhydride derivatives.

In this work we describe the formation of fluorescent naphthalimide derivatives as a new cytochemical method for revealing protein amino groups. The reaction is based on the condensation of 1,8-naphthalic anhydrides in organic solvents with primary aliphatic amines. Under optimal violet-blue (436 nm) excitation, a strong yellow-green emission is observed in specific cell components from blood smears treated with 3-amino-1,8-naphthalic anhydride in N,N-dimethylformamide, which were the most suitable reagent and solvent for microscopic studies. Cytoplasmic granules of mammalian eosinophils and avian heterophils showed the highest fluorescence reaction, which was abolished by blocking procedures for amino groups. Spectrofluorometric analysis confirmed the emission characteristics of the naphthalimides produced from n-butylamine and gelatin. Taking into account the chemical reactivity of 1,8-naphthalic anhydrides and present results, the reaction can be considered selective for lysine and arginine residues of proteins.

Animals↗

PubMed1993

Voltammetric behavior of berenil.

Berenil is reduced on mercury drops electrode in buffered aqueous media. The reduction of -N=N- group is controlled by diffusion. Polarographic waves are of analytical usefulness. The hydrogen discharge is favored by the basic centers of the molecule in Co(II)/ammonia-buffered media.

Diminazene↗