scholarly journals A New Fluorescence Reaction in DNA Cytochemistry: Microscopic and Spectroscopic Studies on the Aromatic Diamidino Compound M&B 938

1997 ◽  
Vol 45 (1) ◽  
pp. 97-105 ◽  
Author(s):  
Juan C. Stockert ◽  
Clara I. Trigoso ◽  
Teresa Cuéllar ◽  
José L. Bella ◽  
José A. Lisanti
Keyword(s):  
Specific Binding ◽  
Linear Regression Analysis ◽  
Spectroscopic Studies ◽  
Centromeric Heterochromatin ◽  
Dna Ladder ◽  
Metaphase Chromosomes ◽  
Distamycin A ◽  
Ehrlich Ascites ◽  
Fluorescence Reaction ◽  
High Fluorescence

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 μg/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.

Fluorescence Patterns of Chromatin and Cytoplasm by Hematoxylin Solutions

1983 ◽  
Vol 38 (1-2) ◽  
pp. 153-155 ◽  
Author(s):  
J. C. Stockert ◽  
M. L. Molero ◽  
R. H. Espelosin
Keyword(s):  
Spectral Analysis ◽  
Fluorescence Emission ◽  
Ehrlich Ascites Tumor ◽  
Ascites Tumor ◽  
Ehrlich Ascites ◽  
Fluorescence Reaction ◽  
Chicken Blood

Hematoxylin, Fluorescence, Fluorochrom es Treatments o f chicken blood and Ehrlich ascites tumor smears with hematoxylin solutions give a fluorescence reaction in chromatin, basophilic cytoplasm and leukocyte granules. In these structures the fluorescence emission in­ creases upon dye aging and prolonged staining times. We present a preliminar spectral analysis suggesting the possibility to employ hematoxylin as a fluorochrome.

scholarly journals Mouse satellite DNA, centromere structure, and sister chromatid pairing.

1986 ◽  
Vol 103 (4) ◽  
pp. 1145-1151 ◽  
Author(s):  
L M Lica ◽  
S Narayanswami ◽  
B A Hamkalo
Keyword(s):  
Electron Microscopy ◽  
Sister Chromatid ◽  
Satellite Dna ◽  
Marker Chromosome ◽  
Restriction Enzymes ◽  
Centromeric Heterochromatin ◽  
Mitotic Chromosomes ◽  
Metaphase Chromosomes ◽  
Sister Chromatids ◽  
Mouse Satellite Dna

The experiments described were directed toward understanding relationships between mouse satellite DNA, sister chromatid pairing, and centromere function. Electron microscopy of a large mouse L929 marker chromosome shows that each of its multiple constrictions is coincident with a site of sister chromatid contact and the presence of mouse satellite DNA. However, only one of these sites, the central one, possesses kinetochores. This observation suggests either that satellite DNA alone is not sufficient for kinetochore formation or that when one kinetochore forms, other potential sites are suppressed. In the second set of experiments, we show that highly extended chromosomes from Hoechst 33258-treated cells (Hilwig, I., and A. Gropp, 1973, Exp. Cell Res., 81:474-477) lack kinetochores. Kinetochores are not seen in Miller spreads of these chromosomes, and at least one kinetochore antigen is not associated with these chromosomes when they were subjected to immunofluorescent analysis using anti-kinetochore scleroderma serum. These data suggest that kinetochore formation at centromeric heterochromatin may require a higher order chromatin structure which is altered by Hoechst binding. Finally, when metaphase chromosomes are subjected to digestion by restriction enzymes that degrade the bulk of mouse satellite DNA, contact between sister chromatids appears to be disrupted. Electron microscopy of digested chromosomes shows that there is a significant loss of heterochromatin between the sister chromatids at paired sites. In addition, fluorescence microscopy using anti-kinetochore serum reveals a greater inter-kinetochore distance than in controls or chromosomes digested with enzymes that spare satellite. We conclude that the presence of mouse satellite DNA in these regions is necessary for maintenance of contact between the sister chromatids of mouse mitotic chromosomes.

Estrogen and Progesterone Measurement and its Quality Control in Breast Cancer: A Reappraisal

1986 ◽  
Vol 1 (1) ◽  
pp. 15-28 ◽  
Author(s):  
Adriano Piffanelli ◽  
Gloria Giovannini ◽  
Dario Pelizzola ◽  
Michele De Bortoli
Keyword(s):  
Quality Control ◽  
Specific Binding ◽  
Low Cost ◽  
Estrogen Receptor Status ◽  
Linear Regression Analysis ◽  
Advanced Technique ◽  
Sensitivity Assessment ◽  
Tumor Tissues ◽  
Immuno Assay ◽  
Enzyme Immuno Assay

This article illustrates the two main methods for routine measurement of cytoplasmic estrogen receptor status in neoplastic biopsy. The first is the Dextran Coated Charcoal Technique (D.C.C. Assay) which is still the method of choice in the majority of clinical laboratories for its simplicity, reproducibility and low cost. The second is a more advanced technique based on the specific binding, enzimatically displayed, of commercially available antiestrogen monoclonal antibodies (Enzyme Immuno Assay - ABBOTT). The sui generis characteristics of endocrine sensitivity assessment on tumor tissues and the importance of decision-making connected with the assay justify rigorous quality assurance schemes. The quality control design proposed by the Italian Committee concerned the evaluation of several lyophilized preparations with scalar receptor content; this permits the identification through linear regression analysis of systematic and non-systematic errors. The Italian Committee has currently connected 50 labs from most regions of the country.

Specific binding of hoechst 33258 to the d(CGCAAATTTGCG)2 duplex: calorimetric and spectroscopic studies

1997 ◽  
Vol 271 (2) ◽  
pp. 244-257 ◽  
Author(s):  
Ihtshamul Haq ◽  
John E Ladbury ◽  
Babur Z Chowdhry ◽  
Terence C Jenkins ◽  
Jonathan B Chaires
Keyword(s):  
Specific Binding ◽  
Spectroscopic Studies ◽  
Hoechst 33258

Levels of conservation and variation of heterochromatin and nucleolus organizers in the Bovidae

1985 ◽  
Vol 27 (6) ◽  
pp. 665-682 ◽  
Author(s):  
B. Mayr ◽  
D. Schweizer ◽  
M. Mendelak ◽  
J. Krutzler ◽  
W. Schleger ◽  
...  
Keyword(s):  
Nucleolus Organizer ◽  
Centromeric Heterochromatin ◽  
Banding Patterns ◽  
X Chromosomes ◽  
Distamycin A ◽  
Nucleolus Organizer Regions ◽  
Standard Karyotype ◽  
Swamp Buffalo ◽  
High Degree ◽  
Nucleolus Organizers

Chromomycin A3 banding of the mitotic sets of 10 species of Bovidac (cattle, wisent, yak, banteng, gaur, red buffalo, swamp buffalo, sheep, mufflon, and goat) serves to demarcate both centromeric constitutive heterochromatin and R-banding patterns capable of identifying all the chromosomes within a given complement. In all species significant amounts of chromomycin-bright heterochromatin are present at the centromeres of all autosomes, though there was a high degree of intra- and inter-individual variation in the size of the heterochromatic blocks. Marked interspecies differences in the centromeric patterns were evident. The X chromosomes contained appreciable amounts of centromeric heterochromatin only in the two buffaloes. All the animals studied lacked distamycin A – diamidinophenylindole type heterochromatin. AgNO3 staining was applied sequentially to detect the location of active nucleolus organizer regions (NORs). The distribution of NORs was reasonably conservative in most of the species. An exceptional situation was found in the two buffaloes, where only one NOR pair matched with the standard karyotype of the Bovidae.Key words: heterochromatin, chromomycin A3 fluorescence, nucleolus organizers, Bovidae.

scholarly journals Changes in Chromosomal Localization of Heterochromatin-binding Proteins during the Cell Cycle in Drosophila

1998 ◽  
Vol 140 (6) ◽  
pp. 1297-1306 ◽  
Author(s):  
J. Suso Platero ◽  
Amy K. Csink ◽  
Adrian Quintanilla ◽  
Steven Henikoff
Keyword(s):  
Cell Cycle ◽  
Self Assembly ◽  
Chromosomal Localization ◽  
Specific Binding ◽  
Pericentric Heterochromatin ◽  
Gaga Factor ◽  
Metaphase Chromosomes ◽  
Satellite Sequence ◽  
High Affinity ◽  
Target Satellite

We examined the heterochromatic binding of GAGA factor and proliferation disrupter (Prod) proteins during the cell cycle in Drosophila melanogaster and sibling species. GAGA factor binding to the brownDominant AG-rich satellite sequence insertion was seen at metaphase, however, no binding of GAGA factor to AG-rich sequences was observed at interphase in polytene or diploid nuclei. Comparable mitosis-specific binding was found for Prod protein to its target satellite in pericentric heterochromatin. At interphase, these proteins bind numerous dispersed sites in euchromatin, indicating that they move from euchromatin to heterochromatin and back every cell cycle. The presence of Prod in heterochromatin for a longer portion of the cell cycle than GAGA factor suggests that they cycle between euchromatin and heterochromatin independently. We propose that movement of GAGA factor and Prod from high affinity sites in euchromatin occurs upon condensation of metaphase chromosomes. Upon decondensation, GAGA factor and Prod shift from low affinity sites within satellite DNA back to euchromatic sites as a self-assembly process.

A quantitative assay for the cytoplasmic androgen receptor using [3H]dihydrotestosterone in the presence of NAD+-nucleosidase

1983 ◽  
Vol 102 (1) ◽  
pp. 153-160 ◽  
Author(s):  
Hans Moeller ◽  
Günter Oettling ◽  
Bernhard Fiederer ◽  
Gerd Brügmann
Keyword(s):  
Binding Sites ◽  
Androgen Receptors ◽  
Binding Capacity ◽  
Specific Binding ◽  
Linear Regression Analysis ◽  
Receptor Complex ◽  
Agar Gel ◽  
Progestin Receptors ◽  
Agar Gel Electrophoresis ◽  
Kinetics Of

Abstract. In prostatic cytosol DHT1 is metabolized to 5α-androstane-3α (or β), 17α-diols with a half life of 2 h even at 4°C. Thus, [3H]DHT appears to be a poor marker for a quantitative assessment of androgen receptors (AR). Methyltrienolone (R1881) seems to be advantageous as it is not metabolized. However, because of considerable binding to progestin receptors, assays using [3H]R1881 are not specific for AR in tissues containing progestin receptors. We, therefore, developed a specific assay for AR using [3H]DHT (14 nm) as marker, where metabolism of DHT is prevented by pre-incubation with NAD+-nucleosidase. The [3H]DHT-receptor complex is separated from free, SHBG-bound and unspecifically bound [3H]DHT by agar gel electrophoresis. The binding sites of high affinity and low capacity are characterized by suppression with unlabelled R1881 (2 μm) in a parallel assay. Under these conditions DHT and R1881 appear to have the same kinetics of association and dissociation. Weighted non-linear regression analysis of specific binding capacity at various ligand concentrations reveals that in rat prostatic cytosol the affinity of DHT (Kd = 0.405 ± 0.0839 nm) is significantly higher (P < 0.01) than that of R1881 (Kd = 1.25 ± 0.271 nm).

Sign in / Sign up

Export Citation Format

Share Document