Ligands with affinity to specific sequence of DNA base pairs. XII. The synthesis and cytological studies of dimeric Hoechst 33258 molecules

We synthesized dimeric Hoechst dye molecules composed of two moieties of the Hoechst 33258 fluorescent dye phenolic hydroxy groups of which were tethered via pentamethylene, heptamethylene, or triethylene oxide linkers. A characteristic pattern of differential staining of chromosome preparations from human premonocytic leukemia HL60 cells was observed for all the three fluorescent dyes. The most contrast pattern was obtained for the bis-Hoechst analogue with the heptamethylene linker; its quality was comparable with the picture obtained in the case of chromosome staining with 4',6-diamidino-2-phenylindole. The ability to penetrate into the live human fibroblasts was studied for the three bis-Hoechst compounds. The fluorescence intensity of nuclei of live and fixed cells stained with the penta- and heptamethylene-linked bis-Hoechst analogues was found to differ only slightly, whereas the fluorescence of the nuclei of live cells stained with triethylene oxide-linked bis-Hoechst was considerably weaker than that of the fixed cells. The bis-Hoechst molecules are new promising fluorescent dyes that can both differentially stain chromosome preparations and penetrate through cell and nuclear membranes and effectively stain cell nuclei.     

Modifications of Kohn's chlorazol black E staining and Wheatley's trichrome staining for temporary wet mount and permanent preparation of Entamoeba histolytica.

Preparation of stained smears of Entamoeba histolytica has several drawbacks. We therefore tried to simplify the staining procedures by modifing Kohn's chlorazol black E staining and Wheatley's trichrome staining techniques. Trophozoites and cysts of axenically cultured E. histolytica and Entamoeba invadens, respectively, and trophozoites and cysts of E. histolytica in stools of patients were used. Karyosomes and peripheral chromatin of nuclei and chromatoid bodies became distinctly visible after amoebae were suspended in the basic solution of Kohn's stain. Amoebae fixed in suspension with either basic solution or Bouin's fixative were clearly stained with Kohn's and trichrome preparations, both as wet mounts directly and as permanent slides after processing for mounting. These procedures were easier when the basic solution was used as a fixative and trichrome stain was employed. Erythrocytes ingested by trophozoites, however, were not stained with either of these preparations after fixation in the basic solution but were clearly stained when Bouin's fixative was used. Cysts of E. histolytica in stools concentrated using basic solution (instead of formalin) and ether were also stained with these stains. Consequently, without employing highly toxic mercuric chloride, wet mounts and permanent smears can be prepared with permanent stains, and preserved cysts can be stained after concentration.     

Method of determining the concentration of glycogen, DNA, 3H-thymidine label and dry weight in one and the same cell]

A method is proposed for determination of glycogen, DNA, 3H-thymidine incorporation and dry weight in the same cell, the technique being based on successive discovery and measuring of each of these indices. Cells are obtained from animals, previously injected with 3H-thymidine, to be charted on preparation, made pictures and measured in square units. Then on preparations embedded into glycerine or vaseline oil, the optical path difference of rays for the nucleus and cytoplasm of selected cells is measured with the interferencial microscope. This is followed by the fluorescent PAS reaction and the content of glycogen is registered microfluorimetrically in the same cells. Preparations after that are treated with a freshly prepared water solution of 0.025% borohydride sodium, stained with the routine or fluorescent Feulgen reaction, and DNA content is determined in the same cells in which glycogen and delta delta were previously measured. The stained nuclei are photographed, their areas are measured and the dry weight of the nucleus and cytoplasm of marked cells is calculated from the values of the nuclear areas and of delta delta. Eventually the preparations are covered by emulsion and exposed, and 3H-thymidine-containing nuclei are determined, the index of marked nuclei and the marking intensity over the nucleus are calculated. As a result, a precise and reliable determination of glycogen, DNA, dry weight and 3H- or 14C-thymidine incorporation is made in either of the marked cell.     

DNA Sequence-Specific Ligands: XII. Synthesis and Cytological Studies of Dimeric Hoechst 33258 Molecules

We synthesized dimeric Hoechst dye molecules composed of two moieties of Hoechst 33258 fluorescent dye with the phenolic hydroxy groups tethered via pentamethylene, heptamethylene, or triethylene oxide linkers. A characteristic pattern of differential staining of chromosome preparations from human HL60 premonocytic leukemia cells was observed for all the three fluorescent dyes. The most contrasting pattern was obtained for the bisHoechst analogue with the heptamethylene linker; its quality was comparable with the picture obtained in the case of chromosome staining with 4′,6-diamidino-2-phenylindole. The ability to penetrate into live human fibroblasts was studied for the three bisHoechst compounds. The fluorescence intensity of nuclei of live and fixed cells stained with the penta- and heptamethylene-linked bisHoechst analogues was found to differ only slightly, whereas the fluorescence of the nuclei of live cells stained with triethylene oxide-linked bisHoechst was considerably weaker than that of the fixed cells. The bisHoechst molecules are new promising fluorescent dyes that can both differentially stain chromosome preparations and penetrate through cell and nuclear membranes and effectively stain cell nuclei.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 4, 2005, pp. 385–393.Original Russian Text Copyright © 2005 by Gromyko, Popov, Mosoleva, Streltsov, Grokhovsky, Oleinikov, Zhuze.     

A Permanent Mounting Procedure for Bacteria

Bacterial preparations, stained by the DeLamater (1951) method, are mounted in a fluid medium (Jensen, 1921) which consists of 75 parts heavy mineral oil and 25 parts alphabromonaphthalene. In these permanent preparations the bacteria appear to be undistorted and the nuclei can be seen clearly and in detail.     

Über die Struktur und Teilung der Kerne von Geotrichum candidum und Geotrichum magnusii

Summary The nuclei in germinating spores and growing hyphae ofGeotrichum magnusii andG. candidum have been examined during life and in fixed and stained preparations.The spores and the cells of the hyphae are multinucleate. The nuclei consist of a dense Feulgen-negative nucleolus surrounded by a less dense shell of Feulgen-positive particles. No membrane was seen at the margin of either living or fixed and stained nuclei. The mass of chromatin and the nucleolus divide at the same time by elongation followed by constriction. Chromosomes could not be detected in either resting or dividing nuclei.

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Mit 5 Textabbildungen     

A Nile Blue Staining Technic for the Differentiation of Melanin and Lipofuscins

When paraffin sections are stained in 0.05-.01% Nile blue in 1 % sulfuric acid, washed thoroughly in water and mounted in aqueous media, lipofuscins color deep blue, melanins dark green, myelin and red cells lighter greens and background pale green. If, immediately after staining, the preparations are at once extracted with acetone with or without a 1% sulfuric acid rinse, melanins remain dark green, mast cells color purple, lipofuscins and background decolorize and nuclei may stain light green.     

A comparison of four quantitative cytochemical methods directed toward demonstration of DNA.

Populations of nuclei isolated from mouse brain tissue were stained by the following cytochemical methods considered stoichiometric for DNA: (1) the Feulgen reaction; (2) gallocyanin-chromalum after RNase; (3) pH 4.0 methylene blue after RNase; and (4) methyl green used in the presence of 2M magnesium chloride. Replicate preparations to be stained with gallocyanin-chromalum, methylene blue, and methyl green were acetylated prior to staining. All of these groups were examined by high-resolution scanning microspectrophotometry. The results indicated that of the methods examined, the Feulgen reaction, gallocyanin-chromalum used without prior acetylation, and methylene blue used with prior acetylation were the most useful in revealing differences attributable to variability in chromatin organization. The greatest variability in total extinction measurements was observed in acetylated, methylene blue-stained nuclei, while the least variability was observed in nuclei stained with methyl green in the presence of 2 M magnesium chloride. Acetylation produced different effects on dye-binding in different groups. It greatly increased binding in nuclei stained with methylene blue; it reduced binding in the methyl green-2 M magnesium chloride series.     

Scanning Feulgen-deoxyribonucleic acid cytophotometry of Papanicolaou destained preparations.

Feulgen deoxyribonucleic acid cytophotometry of Papanicolaou destained specimens revealed a differential loss in Feulgen reactivity among human buccal and cervical smears, cultured embryonic lung fibroblasts and invasive cervical carcinoma cells. Loss in Feulgen reactivity in Papanicolaou destained fibroblasts and polyploid nuclei of malignant lesions was observed to result in underestimates of relative Feulgen deoxyribonucleic acid and nuclear area values using scanning integrating microdensitometry. Thus, Papanicolaou stained preparations may not be suitable for deoxyribonucleic acid quantification of high ploidy lesions since distributional absorption error is unpredictably influenced by such factors as ploidy level, nuclear size, chromatin dispersion and differential aldehyde loss during destaining. Feulgen deoxyribonucleic acid cytophotometry of Papanicolaou stained preparations can be useful for differentiating benign from malignant lesions if extent of aneuploidy (as reflected in abnormal deoxyribonucleic acid frequency distribution profile) is used as a diagnostic indicator.     

A Nile Blue Staining Technic for the Differentiation of Melanin and Lipofuscins

When paraffin sections are stained in 0.05-.01% Nile blue in 1 % sulfuric acid, washed thoroughly in water and mounted in aqueous media, lipofuscins color deep blue, melanins dark green, myelin and red cells lighter greens and background pale green. If, immediately after staining, the preparations are at once extracted with acetone with or without a 1% sulfuric acid rinse, melanins remain dark green, mast cells color purple, lipofuscins and background decolorize and nuclei may stain light green.     

The microfilaria of Brugia timori (Partono et al. 1977 = Timor microfilaria, David and Edeson, 1964): morphologic description with comparison to Brugia malayi of Indonesia.

The microfilaria of Brugia timori was compared with microfilariae of Indonesian strains of periodic and subperiodic Brugia malayi using alcohol-fixed (stained) and formalin-fixed (unstained) preparations. As noted by other observers of the Timor microfilaria, the absence of a stained sheath in Giemsa preparations, a long cephalic space with a length-to-width ratio of about 3:1, and a great overall body length are features which most readily distinguish this parasite. Additionally, B. timori has greater numbers of single row nuclei in the terminal column of body cells and a lesser bulge of the cuticle surrounding nuclei in the distal portion of the tail than does B. malayi. About 60% of B. timori microfilariae were exsheathed in haemalum-stained thick blood films. Brugia timori microfilariae were found to be distinct from microfilariae of B. malayi by comparing percentages of total body length included between the cephalic tip and major internal anatomic markers.     

Improved confocal characterization of targets in nuclei of cytogenetic preparations

OBJECTIVE: To show that cellular preparations requiring depth analysis of different domains stained by molecular cytogenetic methods (fluorescence in situ hybridization and primed in situ) can be improved by regularized factor analysis of medical image sequences (FAMIS) to isolate fluorescent probes by means of intensity depth profiles of fluorochromes, to track relevant DNA sequences (cosmids and centromeres) in cell nuclei during interphase and to improve the use of cytogenetic techniques resulting in flat preparations of whole cells that are assumed to preserve probe access to their targets. STUDY DESIGN: 3D sequences of images obtained by depth displacement in a confocal microscope were first analyzed by the FAMIS algorithm, which provides factor curves. Factor images then resulted from regularization methods that improve signal/noise ratio while preserving target contours. RESULTS: Factor curves and regularized factor images helped analyze targets inside nuclei. CONCLUSION: It is possible to process preparations containing numerous spots (even when they are on different planes) to differentiate stained targets, to investigate depth differences and to improve visualization and detection.     

123 Permanent Fluorescent Nuclear Staining of Binucleate Dinoflagellates

Typically, fluorescent microscopy of dinoflagellate nuclei is of poor resolution, due mainly to visual obstruction of the nuclei by plastids, pigment granules, and thecal plates. Moreover, the usual slide mounts using buffered glycerol are temporary, and fade after a week or so. We have developed a procedure to clear pigments from dinoflagellates, followed by fluorescent staining of the nuclei. The cells are then prepared as permanent mounts using an ultraviolet light-catalyzed resin to produce stained samples which may be kept for at least three years with little loss of fluorescence. This procedure can also be used to prepare plastic embedded dinoflagellate cells which can then be sectioned at 1–2 nm, fluorescent stained, and permanently mounted. Suitable nuclear stains are DAPI, Hoechst 33258, ethidium bromide and acridine orange. The dinoflagellate (dinokaryotic), and endosymbiont (eukaryotic) nuclei are clearly visualized, revealing individual chromosomes in the dinoflagellate nucleus, and a highly lobed morphology of the endosymbiont nucleus.     

Flow cytometric discrimination of mitotic cells: resolution of M, as well as G1, S, and G2 phase nuclei with mithramycin, propidium iodide, and ethidium bromide after fixation with formaldehyde

Cells in mitosis can be flow cytometrically discriminated from G1, S, and G2 cells by analysis of a nuclear suspension prepared with nonionic detergent, fixed with formaldehyde, and stained with mithramycin, propidium iodide, or ethidium bromide. With these DNA-fluorochromes, the fluorescence is quenched by formaldehyde less in mitotic nuclei than in interphase nuclei. Mitotic nuclei have a 20-40% increased mithramycin fluorescence and 30-60% decreased light scatter in comparison to those of G2 nuclei. There is a high correlation (r = 0.95; P less than 0.001) between microscope counts of mitotic figures in smear preparations of the initial cell suspension and the flow cytometrically estimated fraction of nuclei with increased mithramycin fluorescence. Flow sorting (FACS) demonstrates that the mitotic nuclei are confined to the peak of increased mithramycin fluorescence and decreased light scatter. The method has been applied to cultures of Yoshida ascites tumor cells, JB-1 reticulosarcoma cells, and PHA-stimulated human lymphocytes, incubated in the presence or absence of vinblastine for mitotic arrest. In a heteroploid mixture of fixed Yoshida (near-diploid) and JB-1 (hypotetraploid) nuclei, the mitotic fractions of the two cell lines could be estimated separately when analyzed with mithramycin fluorescence versus light scatter or with mithramycin fluorescence versus propidium iodide fluorescence.     

Four fluorochromes for the demonstration and microfluorometric estimation of RNA

Microfluorometric estimates of total RNA were made in selected test material stained with berberine sulfate, acridine orange, and Hoechst 33258. These measurements were compared with those obtained with propidium iodide, which is known to interact only with double-stranded nucleic acids. It was observed that all of the fluorochromes, including propidium iodide, yielded very similar patterns of fluorescence in the various types of material tested. In isolated thymocyte and hepatocyte nuclei stained with either propidium iodide or Hoechst 33258 at pH 2, it was evident that RNA could be estimated only indirectly by measuring the amount of fluorescence before and after extraction with RNase. It was apparent that the total fluorescence of small thymocyte nuclei was affected much less by RNase extraction than that of 2c hepatocyte nuclei. Attempts to obtain direct estimates of RNA by exposing the preparations to DNase were not successful: the fluorescence of thymocyte nuclei dropped almost to zero, and hepatocyte nuclei could no longer be assigned to distinct ploidy classes. In addition, since the highly condensed chromatin of thymocyte nuclei was stained much more prominently than the looser chromatin of hepatocyte nuclei with Hoechst 33258, it was apparent that this fluorochrome - when used at pH 2 - has potential usefulness as a "probe" of organizational differences in chromatin.     

HETEROCHROMATIN IN HUMAN MALE LEUKOCYTES

Tritiated thymidine was added to peripheral blood cultures containing phytohemagglutinin so that DNA synthesis in interphase nuclei of white blood cells in the human male could be studied. After 57 hours in culture, a large heterochromatic body with a central position is seen in unlabeled Feulgen-stained nuclei. In labeled nuclei in which DNA synthesis was taking place in both the eu- and heterochromatin at the time the thymidine became available, the heterochromatin shows a higher number of silver grains per unit area, accompanied by a stronger Feulgen reaction, an indication of its higher DNA content. The time of DNA synthesis in the heterochromatin blocks is different from that in the surrounding euchromatin. The large heterochromatic block is composed of chromosome segments gathered together around the nucleolus but it is not part of this organelle. In preparations stained with azure A and acid fuchsin for demonstrating both the nucleolus and the chromosomes, six distinctly heteropyenotic chromosome segments can be seen associated with the nucleolus. Cells of all size categories were found to incorporate tritiated thymidine. The distinct appearance of autosomal heterochromatin in white blood cells may be the result of the new physiological conditions to which the cells are subjected in the medium containing phytohemagglutinin.     

Fine needle aspiration cytology of seromas of the breast from irradiated lumpectomy sites

OBJECTIVE: To study the cytologic features of cells in breast aspirates of seromas in women who had undergone irradiation at lumpectomy sites. STUDY DESIGN: Cytologic material from 38 females with an age range of 39-72 years who had undergone aspiration of seromas were retrieved to determine tumor recurrence or other atypia. The material was obtained using the conventional method of needle aspiration with a 22-gauge needle and 10-mL syringe, maintaining negative pressure. The syringe and needle contents were washed in a cytology container containing 30% ethyl alcohol in physiologic saline. From half the washings filter preparations were made and stained by the Papanicolaou method, while from the other half cell blocks were made and sections cut after processing and stained with hematoxylineosin. RESULTS: In 2 of 38 patients, filter preparations and cell blocks showed malignant cells of breast carcinoma that were similar to the primary. This was confirmed on core biopsy and subsequent mastectomy. In 21 of 38 cases mildly atypical cells with degenerative effects, mild hyperchromasia, liberal cytoplasm, macrophages, inflammatory cells and a few apocrine cells were seen, while in 11 of 38 patients scanty debris, few inflammatory cells, rare apocrine cells and a few macrophages were found. In 4 of 38 cases highly atypical cells with hyperchromatic nuclei and nucleoli were noted. However, a core biopsy in all 4 patients revealed no evidence of residual tumor. CONCLUSION: Cell atypia in aspirates from seromas should be interpreted with caution to avoid unnecessary, invasive surgery, keeping in mind that radiation can cause misleading changes. However, patients with clinical indications of recurrent tumor or with severe atypia should undergo biopsy to permit appropriate management.     

A Simple Fluorescence Staining Technique for the Differentiation of Human Tissue Transplanted Into Nude Mice

Human and mouse nuclei can be distinguished by differences in the constitutive heterochromatin when stained with quinacrine dihydrochloride. With the staining method described, mouse heterochromatin during inter phase appears as brilliant fluorescent chromo-centers. By replacing the commonly used aqueous buffer mounting medium with a xylene-diluted synthetic resin, the haziness of the nuclear fluorescence is eliminated thus allowing identification of the heterochromatin pattern in histological preparations. A requirement for the definite identification of cells of human or murine origin in the nude mouse is the knowledge that the heterochromatin arrangement changes according to the stage of differentiation of the cell or the position of a particular nucleus within the cell cycle.     

A simple fluorescence staining technique for the differentiation of human tissue transplanted into nude mice.

Human and mouse nuclei can be distinguished by differences in the constitutive heterochromatin when stained with quinacrine dihydrochloride. With the staining method described, mouse heterochromatin during interphase appears as brilliant fluorescent chromocenters. By replacing the commonly used aqueous buffer mounting medium with a xylene-diluted synthetic resin, the haziness of the nuclear fluorescence is eliminated thus allowing identification of the heterochromatin pattern in histological preparations. A requirement for the definite identification of cells of human or murine origin in the nude mouse is the knowledge that the heterochromatin arrangements changes according to the stage of differentiation of the cell of the position of a particular nucleus within the cell cycle.