Structure and variability of human chromosomes analyzed by recent techniques

Summary Besides the AT-specific fluorochromes, GC-specific fluorescent antibiotics are now available for chromosomal analysis. Chromosomal bands represent large accumulation of DNA sequences with similar AT:GC ratio. These uniform differences from the mean AT:GC ratio in the bands can be explained only by at least partial repetition of short DNA sequences in these regions. By comparison of various staining techniques more information also on the constitutive heterochromatin in man becomes available. The human NOR region exhibits a complex organization when studied by various basespecific fluorochromes and silver staining. The DNA-specific fluorochromes are also useful tools in cytophotometric DNA measurements.     

Assay for apoptosis using the mitochondrial probes, Rhodamine123 and 10-N-nonyl acridine orange

Apoptosis plays a pivotal role in the regulation of cell turnover, and a defect or an excess of apoptosis has been implicated in several human diseases. Apoptosis is activated from an extracellular death signal, or from an internal pathway starting from the endoplasmatic reticulum or the mitochondria. To investigate the mitochondrial compartment during apoptosis, we have established a protocol using fluorochromes and flow cytometry to probe the structure and function of mitochondria kinetically. The protocol could be applied to whole cells or to isolated mitochondria. In the first case, cells are counterstained with ethidium bromide (EB) to evaluate plasma membrane function. The presence of the electrochemical gradient in the mitochondria is probed with Rhodamine123 (Rh123), whereas the structure and the integrity of mitochondria are assessed using 10-N-nonyl-acridine orange (NAO). Not considering the time requested for cell/mitochondria preparation and the activation of apoptosis, the protocol lasts <1 h.     

Ethidium bromide- and propidium iodide-PTA staining of nucleic acids at the electron microscopic level

Ultra-thin sections of various tissues were stained with ethidium bromide or propidium iodide, two fluorescent markers widely used for quantitation of nucleic acids. The fluorochromes, tested at different concentrations, were then revealed by incubation of the sections with neutralized phosphotungstic acid. We showed that at the electron microscopic level only nucleic acid-containing structures are revealed. Chromatin, nucleolus, and ribosomes appear to be stained by the end-product of the reaction. Furthermore, controls with proteases and nucleases showed that the staining is related to the binding of the fluorochromes to DNA and RNA and to the subsequent detection of the dyes by neutralized PTA.     

The use of base pair specific DNA binding agents as affinity labels for the study of mammalian chromosomes

The fluorochromes Hoechst 33258 and olivomycin are base pair specific DNA binding agents. The fluorescence enhancement of Hoechst 33258 and olivomycin in the presence of DNA can be directly related to the A-T and G-C content of the interacting DNA respectively. Cytological observations of metaphase chromosomes treated with these two compounds suggest that the fluorescent banding patterns produced are the reverse of one another. —Non-fluorescent base pair specific DNA binding agents have been used as counterstains in chromosome preparations to enhance the contrast of the banding patterns produced by the base specific fluorochromes. The non-fluorescent G-C specific antibiotic actinomycin-D enhanced the resolution of fluorescent bands produced by the A-T specific fluorochrome Hoechst 33258. Similarly the non-fluorescent A-T specific antibiotic netropsin was found to enhance resolution of the bands produced by the G-C specific fluorochrome olivomycin. Netropsin was also found to increase the differential fluorescent enhancement of complexes of olivomycin with DNAs of various base composition in solution. These findings suggest that counterstaining agents act through a base sequence dependent inhibition of subsequent binding by base pair specific fluorochromes.—The base specific DNA binding agents have been used to differentiate different types of constitutive heterochromatin in mammalian species, and to facilitate chromosome identification in somatic cell hybrids.     

Cellular retinoic acid binding protein is associated with mitochondria

We report that immunohistochemical staining for cellular retinoic acid-binding protein (CRABP) was restricted to the cytoplasm of cortical cells in bovine adrenal. In contrast, staining for the similar protein, cellular retinol-binding protein (CRBP), was found throughout these cells. After transfections of CRABP and CRBP into cultured cells, immunofluorescence analyses again revealed cytoplasmic restriction only for CRABP, with a pronounced punctate appearance. Use of organelle-specific fluorochromes indicated that CRABP immunofluorescence overlaid exactly with the pattern of the mitochondrial-specific fluorochrome. Confirmation of this association came with subcellular fractionation of the adrenal cortex. CRABP, but not CRBP, co-sedimented with the mitochondria, a novel finding for a member of this superfamily of cellular lipid-binding proteins.     

Reactions of seven basic fluorochromes with unfixed cells obtained from the salivary glands of the dipteran fly Megaselia scalaris Loew (Phoridae)

Seven basic fluorochromes with varying specificities were used to stain the large squamous epithelial cells isolated from the larval salivary glands of Megaselia scalaris (Phoridae). Although the EDTA-based method selected for isolating the cells produced permeabilization and a loss of viability of the cells, consistent results were obtained with the various fluorochromes. The "classical" pattern of green nuclear and red cytoplasmic fluorescence observed in cells stained with acridine orange could be changed to green cytoplasmic and red nuclear fluorescence by pretreatment with RNase. The predominantly cytoplasmic and nucleolar fluorescence obtained with pyronine Y could be changed to mainly nuclear fluorescence by RNase pretreatment. The other five fluorochromes tested were not affected appreciably by extraction with RNase. Quinacrine mustard, dicarbocyanine (DiOC3(3)), and rhodamine 123 produced primarily cytoplasmic and nucleolar fluorescence, while nile red revealed mainly cytoplasmic lipid droplets. Phosphine 3R initially stained lipid droplets but very rapidly redistributed throughout the cytoplasm and nucleus. Because of their large size, flatness, and content of histochemically demonstrable components, the cells of Megaselia are especially appropriate for use as "optical objects" or controls in various studies. New methods of isolating the cells, however, will be needed to prevent permeabilization and loss of viability of the cells.     

Novel near-infrared cyanine fluorochromes: synthesis,properties, and bioconjugation

Recently, near-infrared (NIR) fluorescence light has been applied to image various biological events in vivo, because it penetrates tissue more efficiently than light in the visible spectrum. Compounds exhibiting fluorescent properties in the NIR range are key elements for this upcoming optical imaging technology. In this paper, we report the synthesis of four new, water-soluble NIR cyanine fluorochromes which have superior chemical stability and optical properties. Each fluorochrome was designed with a monoreactive carboxyl group for labeling purposes. When multiple fluorochromes were attached to a single macromolecule, fluorescence quenching was observed. On the basis of this property, a novel autoquenched enzyme sensitive NIR fluorescence probe was prepared.     

Oxidation-reduction ratio studies of mitochondria in freeze-trapped samples. NADH and flavoprotein fluorescence signals.

The recording of oxidation-reduction-related fluorescence signals of oxidized flavoprotein (Fp) and reduced pyridine nucleotide (PN) from isolated mitochondria at temperatures below -80 degrees C can be accompanished with a high degree of accuracy and a wide dynamic range. The specific low temperature enhancement of the fluorescence signals due to increased quantum yield and to multiple scattering affords increased accuracy and less interference due to screening pigments such as hemoglobin and myoglobin. Since the metabolic processes are arrested and the recording speed can be greatly diminished, the technique can operate with a much smaller concentration of mitochondria than is needed at room temperature, and the method is suitable for localized oxidation-reduction measurements. The Fp and PN signals originate from the mitochondrial matrix space in which they represent the major fluorochromes. Since Fp and PN are near oxidation-reduction equilibrium, the ratio of the two fluorescence intensities, suitably normalized, approximates the oxidation-reduction ratio of oxidized flavoprotein/reduced pyridine nucleotide. Thus, this technique affords a foundation for the resolution of oxidation-reduction states in two and three dimensions.     

Age-related changes in the mitochondrial depolarization induced by oxidative injury in human peripheral blood leukocytes

Aging is associated with impaired immunity and reduced host defenses. Mitochondrial bioenergetic dysfunctions and reduced antioxidative ability of immunocompetent cells may contribute to this phenomenon. In this study, 60 healthy volunteers of different age groups donated their blood after overnight fasting. Leukocytes were subjected to oxidative injuries by exposure to t-butylhydroperoxide, and were labeled with fluorochromes for measuring mitochondria transmembrane potential (Δω_m), membrane peroxidation and mitochondrial oxidant formation. Δω_m declined after t-butylhydroperoxide exposure, and the change was more prominent in leukocytes from older individuals. Cyclosporin A partly restored Δω_m, implying the contributing role of mitochondrial permeability transition pores. The mitochondrial depolarization was accompanied by increased oxidant formation and oxidation of pyridine nucleotides, which were more prominent in older subjects. The results support the view that the bioenergetic functions of mitochondria are more susceptible to oxidative injury in aged individuals. The decreased ability of leukocytes to resist oxidative stress may contribute to immunosenescence in humans.     

Indirect immunofluorescence modified to display two antigens with one light filter

Summary We present a modification of double indirect immunofluorescence in which we used four antibodies raised in three species to visualize two different antigens. The procedure, which relies on dual recognition of a secondary antibody, requires that one primary antibody and one of the secondary antibodies be raised in the same species. As the two secondary antibodies are conjugated to two different fluorochromes, both of the antigens studied are visualized with one light filter while only one antigen is displayed with another filter. This, in turn, allows more efficient comparison of the distribution of the two antigens in a single field or photograph than is possible by comparing two fields or photographs by conventional double staining. The method is especially useful for determining possible co-localization of two cellular structures. We illustrate the method in adrenal cells in which mitochondria and intermediate filaments are seen to be co-localized.     

UV lasers for flow cytometric analysis: HeCd versus argon laser excitation.

Applying flow cytometric single cell analysis, we compared the performance of UV excitation from argon ion and HeCd lasers using various UV-excitable fluorochromes of cell kinetic and cell physiological relevance. The AT-specific DNA fluorochromes DAPI, Hoechst 33258, and Hoechst 33342 showed no significant differences of G1-phase resolution and cell cycle distribution. With the HeCd laser, high-resolution cell kinetic analysis applying the novel BrdU/Hoechst-PI quenching technique showed superior resolution and an almost normalized G2M/G1 channel ratio of the first cell cycle. Indo-1 analysis for detection of intracellular free calcium gave similar results for both excitation sources, although the indo-1 ratio of activated cells was lower for HeCd excitation. Monochlorobimane as an indicator fluorochrome of glutathione content could not be excited sufficiently with the 325-nm line of the HeCd laser and exhibited poor resolution between positive and negative cells. However, the second glutathione-specific fluorochrome o-phtalaldehyde gave even better results with the HeCd laser. Our data indicate that air-cooled HeCd lasers are cheap and reliable UV-excitation sources for most UV-excitable fluorochromes, and might be an alternative to the expensive water-cooled argon and krypton laser.     

Estimating percentage constitutive heterochromatin by flow cytometry.

Flow cytometry is a powerful method for the assessment of both plant and animal genomes. One of the most interesting aspects is the analysis of chromatin structure. By using intercalating and base pair-specific fluorochromes, the chromatin structure in various cell cultures and microorganisms has been determined. In this study, several maize lines of known heterochromatic composition were analyzed. The nuclei of each line were isolated and stained with DAPI (base pair specific) and PI (intercalator) separately. For each maize line, the PI/DAPI ratio was determined. A significant negative correlation was observed between C-band number and PI/DAPI ratio (r = 0.920) and between percentage heterochromatin and PI/DAPI ratio (r = 0.997). Flow cytometry with use of the fluorochromes DAPI and PI was found to be a rapid and efficient method of determining heterochromatin amount in maize.     

Linear dichroism and polarized fluorescence of dye-complexed DNA fibers

Summary A simple method to obtain well orientated DNA fibers for studying the ordered binding of dyes and fluorochromes by linear dichroism and polarized fluorescence is described. The metachromatic dye toluidine blue and the intercalating fluorochromes ethidium bromide and acridine orange showed a perpendicular alignement to DNA; the minor groove binding fluorochromes 33258 Hoechst and DAPI appeared parallel. Thus, DNA fibers represent a suitable cytochemical test substrate for studying the orientation of bound dyes by polarization methods.     

Further use of fluorochromes in the cytochemical characterization of phytoplankton

Summary The rapid, specific effects of 25 fluorochromes at low concentration and physiological conditions of pH and temperature were investigated on live cells of five phytoplankton species (Prorocentrum micans, Amphidinium carterae, Dunaliella tertiolecta, Chlamydomonas moewusii andFragilaria crotonensis). They allowed the identification of cellular components such as the plasma membrane, endoplasmic reticulum, Golgi apparatus, thecal plates, nucleus, mitochondria, trichocysts, vacuoles/lysosomes, polyphosphate and starch granules, lipid bodies and hydrolytic enzymes. Morphological alterations of some of these constituents were examined in cells at different metabolic states. It was found that the thickness ofProrocentrum thecal plates increases during cell development while surface pores appear to be formed in the early stages of thecal formation. The number and size of mitochondria varies among cells at different stages of growth. The number of trichocysts, the size of vacuoles and the quantity of polyphosphates, starch or lipid inclusions increases in nitrogen-depleted cells. Photodegradation and photoenhancement phenomena are described. Some important factors helping to avoid quenching and some applications of the fluorochroming technique are presented.     

Determination of restriction enzyme activity when cutting DNA labeled with the TOTO dye family

Optical mapping, a single DNA molecule genome analysis platform that can determine methylation profiles, uses fluorescently labeled DNA molecules that are elongated on the surface and digested with a restriction enzyme to produce a barcode of that molecule. Understanding how the cyanine fluorochromes affect enzyme activity can lead to other fluorochromes used in the optical mapping system. The effects of restriction digestion on fluorochrome labeled DNA (Ethidium Bromide, DAPI, H33258, EthD-1, TOTO-1) have been analyzed previously. However, TOTO-1 is a part of a family of cyanine fluorochromes (YOYO-1, TOTO-1, BOBO-1, POPO-1, YOYO-3, TOTO-3, BOBO-3, and POPO-3) and the rest of the fluorochromes have not been examined in terms of their effects on restriction digestion. In order to determine if the other dyes in the TOTO-1 family inhibit restriction enzymes in the same way as TOTO-1, lambda DNA was stained with a dye from the TOTO family and digested. The restriction enzyme activity in regards to each dye, as well as each restriction enzyme, was compared to determine the extent of digestion. YOYO-1, TOTO-1, and POPO-1 fluorochromes inhibited ScaI-HF, PmlI, and EcoRI restriction enzymes. Additionally, the mobility of labeled DNA fragments in an agarose gel changed depending on which dye was intercalated.     

Unstainable DNA in cell nuclei. Comparison of ten different fluorochromes

Ten fluorochromes with specificity for DNA were used to compare the stainability of nuclei of exponentially growing, nondifferentiated Friend leukemia (FL) cells with that of dimethylsulfoxide-induced, fully differentiated FL cell nuclei. Decreased accessibility of DNA to several dyes, particularly pronounced in the case of some intercalators, was observed in differentiated cells. Dye binding was also compared for both sets of nuclei following extraction of nuclear proteins, mostly histones, with 0.1-N HCl. Acid extraction of nuclear proteins increased the accessibility of DNA to varying degrees, depending upon the fluorochrome. In most cases, the differences in fluorescence between differentiated and nondifferentiated nuclei stained with most intercalating dyes was abolished by acid treatment. The results are discussed in terms of the mode of interaction between DNA and the various fluorochromes and the factors associated with chromatin structure, which may affect or be associated with different degrees of proliferative activity.     

Microfluorometric investigations of chromatin structure. I. Evaluation of nine DNA-specific fluorochromes as probes of chromatin organization

The ability of the highly condensed chromatin of small thymocyte nuclei and the more loosely organized chromatin of hepatocyte nuclei to interact with nine DNA-specific fluorochromes was assessed by microfluorometry. Although the results obtained with five of the fluorochromes - mithramycin, 7-aminoactinomycin D, Hoechst 33258, DAPI, and propidium iodide - were found to be virtually unaffected by differences in the degree of condensation of the chromatin, the values obtained with the remaining fluorochromes - proflavine, quinacrine mustard, berberine sulfate, and pyronin Y - appeared to be affected significantly by organizational differences of the chromatin. All of the latter "structural probes," except quinacrine mustard, produced fluorescence values which were higher in the 2c nuclei of hepatocytes than in the nuclei of small thymocytes. Quinacrine mustard yielded higher values in thymocyte nuclei; and in the hepatocyte polyploid series (2, 4, and 8c), it did not produce the expected multiples of the 2c value. Pretreatment of the two types of nuclei with RNase affected their total fluorescence in unpredictable ways. While RNase extraction lessened the differences between thymocyte and 2c hepatocyte nuclei stained with propidium iodide, Hoechst 33258, proflavine, and berberine sulfate, it increased the differences between nuclei stained with mithramycin, quinacrine mustard, pyronin Y, and 7-aminoactinomycin D. The ability of RNA-depleted chromatin to interact with various types of fluorochromes might be a useful parameter in subsequent studies of chromatin organization.     

Chromosome banding in Amphibia. XXV. Karyotype evolution and heterochromatin characterization in Australian Mixophyes (Anura,Myobatrachidae)

The mitotic chromosomes of the Australian ground frogs Mixophyes fasciolatus and M. schevilli were analyzed by means of banding techniques and restriction endonuclease digestions. Chromosomal differentiation in these two species occurred exclusively by considerable changes in the amount of telomeric and centromeric heterochromatin, whereas the sizes and locations of interstitial heterochromatic regions, the sizes of all euchromatic segments as well as the positions of centromeres remained nearly identical during karyotype evolution. The major heterochromatic regions in the karyotypes of M. fasciolatus and M. schevilli amount to 30.2% and 20.7%, respectively. They consist of AT base pair-rich repetitive DNA sequences that are brightly labeled by AT-specific fluorochromes and display quenched fluorescence after staining with GC-specific fluorochromes. The heterochromatic regions can be differentiated by treatment of metaphase chromosomes and interphase cell nuclei with various restriction enzymes which either disclose the complete set of C-band patterns in the karyotypes of both species, or else reveal several subsets of these C-bands.     

Microscopy with Fluorescent Light

The principles of fluorescent microscopy are discussed, together with the apparatus necessary for its study. Specially necessary is a lamp giving radiation in the ultraviolet, with filters to remove most of the visible light. Some histological structures have a natural fluorescence and may be studied directly. In other instances fluorescence is induced by the addition of various activating substances (usually dyes) known as fluorochromes. A list of commercial preparations of this sort is given, together with the type of fluorescence which they induce in various histological structures.     

Assessment of fluorochromes for two-photon laser scanning microscopy of biofilms

A major limitation for the use of two-proton laser scanning microscopy (2P-LSM) in biofilm and other studies is the lack of a thorough understanding of the excitation-emission responses of potential fluorochromes. In order to use 2P-LSM, the utility of various fluorochromes and probes specific for a range of biofilm constituents must be evaluated. The fluorochromes tested in this study included classical nucleic acid-specific stains, such as acridine orange (AO) and 4",6"-diamidino-2-phenylindole (DAPI), as well as recently developed stains. In addition, stains specific for biofilm extracellular polymeric substances (EPS matrix components) were tested. Two-photon excitation with a Ti/Sapphire laser was carried out at wavelengths from 760 to 900 nm in 10-nm steps. It was found that autofluorescence of phototrophic organisms (cyanobacteria and green algae) resulted in strong signals for the entire excitation range. In addition, the coenzyme F(420)-related autofluorescence of methanogenic bacteria could be used to obtain images of dense aggregates (excitation wavelength, 780 nm). The intensities of the emission signals for the nucleic acid-specific fluorochromes varied. For example, the intensities were similar for excitation wavelengths ranging from 780 to 900 nm for AO but were higher for a narrower range, 780 to 810 nm, for DAPI. In selective excitation, fading, multiple staining, and combined single-photon-two-photon studies, the recently developed nucleic acid-specific fluorochromes proved to be more suitable regardless of whether they are intended for living or fixed samples. Probes specific for proteins and glycoconjugates allowed two-photon imaging of polymeric biofilm constituents. Selective excitation-emission was observed for Calcofluor White M2R (780 to 800 nm) and SyproOrange (880 to 900 nm). In addition, fluor-conjugated concanavalin A lectins were examined and provided acceptable two-photon emission signals at wavelengths ranging from 780 to 800 nm. Finally, CellTracker, a fluorochrome suitable for long-term labeling of microbial eucaryote cells, was found to give strong emission at wavelengths ranging from 770 to 810 nm. If fluorochromes have the same two-photon excitation cross section, they are suitable for multiple staining and multichannel recording. Generally, if an appropriate excitation wavelength and fluorochrome were used, it was possible to obtain more highly resolved images for thick biofilm samples with two-photon laser microscopy than with conventional single-photon laser microscopy. Due to its potential for higher resolution in light-scattering tissue-like material, such as biofilms, and extremely localized excitation, 2P-LSM is a valuable addition to conventional confocal laser scanning microscopy with single-photon excitation. However, further development of the method and basic research are necessary to take full advantage of nonlinear excitation in studies of interfacial microbial ecology.